Characterization of Novel Glucagon Receptor Interactors
that Modify Receptor Activity
by
Sean Froese
A thesis submitted in conformity with the requirements for the degree of
Master of Science
Department of Physiology
University of Toronto
© Copyright by Sean Froese 2015
ii
Characterization of Novel Glucagon Receptor Interactors that
Modify Receptor Activity
Sean Froese
Master of Science
Department of Physiology
University of Toronto
2015
Abstract
Glucagon helps maintain blood glucose homeostasis by stimulating gluconeogenesis and
glycogenolysis. Elevated glucagon levels have been reported in type 2 diabetics, and may be in
part responsible for their abnormally high blood glucose. A comprehensive understanding of
glucagon receptor regulators is currently unavailable. Members of my lab performed a mass
spectrometry screen to detect glucagon receptor interactors, 5 of which were validated to be true
interactors by co-immunoprecipitation and Western blotting. The project outlined in this thesis
began with the functional characterization of these interactors. Two interactors, low-density
lipoprotein receptor (LDLR) and transmembrane emp24 domain trafficking protein 2 (TMED2)
significantly enhanced glucagon stimulated glucose production, while tyrosine 3-
monooxygenase/tryptophan 5-monooxygenase activation protein, beta (YWHAB) lowered
glucagon stimulated glucose production in primary hepatocytes. Because of YWHAB’s ability to
lower glucose production, the underlying mechanism was explored and evidence suggests
YWHAB enhances endocytosis of the glucagon receptor into the cell following glucagon
stimulation.
iii
Acknowledgments
This thesis would not have been possible on my own. The support and guidance from everyone in
the lab, my family, and my friends, has been invaluable throughout these past two years.
I would like to thank first and foremost Dr. Wheeler for taking a chance on an inexperienced
undergraduate two and a half years ago, for his guidance and insights, and his constant patience
during my time in the lab. I have learned more about science, and myself, in these past two years
than any other time and my life, and this is in no small part due to Dr. Wheeler.
Thank you to my committee members, Drs. Adeli, Brubaker and Heximer. Your patience with
planning committee meetings and willingness to accommodate my schedule was incredibly
appreciated. More importantly, your insights and challenges were highly motivating and incredibly
valuable to my education.
I would like to thank all of my co-authors, particularly Junfeng Han and Ming Zhang whose work
formed the basis for this thesis. I would also like to thank my fellow lab members for all of their
amazing work their constant helpful advice and proofing. Special mention to Kacey Prentice and
Andrea Eversley for listening to me vent for hours on end. Your scientific insight and friendship
has been vital for my education, and my sanity.
I would like to thank my girlfriend Sabrina Parrotta for her unwavering support, her constant
encouragement, and her understanding when I can’t go to the movies because my cells need to be
split.
Last, I would like to thank my family, especially my parents Patricia and Daryl Froese for their
encouragement, support, and dedication to providing me with an education many would envy.
Most say they would do anything for their children, my parents have proved it.
iv
Table of Contents
Abstract……………………………………………………………………………………..........ii
Acknowledgments……………………………………………………………………………….iii
Table of Contents………………………………………………………………………………..iv
List of Figures………………………………………………………………………………......vii
Abbreviations………………………………………………………...…………...…………......ix
Chapter 1: General Introduction
1.1 Glucagon
1.1.1 Blood glucose homeostasis………………………………………………………………1
1.1.2 Glucagon expression and processing……………………………………………………2
1.1.3 Secretion…………………………………………………………………………………2
1.1.4 The effects of glucagon
1.1.4.1 Stimulatory effects…………………………………………………………………...5
1.1.4.2 Inhibitory effects……………………………………………………………………..6
1.2 Glucagon Receptor
1.2.1 Regulation of receptor expression………………………………………………………..6
1.2.2 Structure………………………………………………………………………………….7
1.2.3 Signalling
1.2.3.1 Activation and transduction…………………………………………………………..8
1.2.3.2 Glucagon receptor regulation………………………………………………………....9
1.3 Type 2 Diabetes and the Role of Glucagon
1.3.1 Pathogenic glucagon secretion………………………………………………………….11
1.3.2 Glucagon receptor antagonists………………………………………………………….12
1.4 Receptor Interactomes
1.4.1 Effects of accessory proteins on G-protein coupled receptors…………………………..13
1.4.2 Experimental approaches to receptor interactome identification
1.4.2.1 Membrane yeast two hybrid system…………………………………………………15
1.4.2.2 Fluorescence resonance energy transfer……………………………………………..16
v
1.4.2.3 Mass spectrometry…………………………………………………………………..16
1.5 Rationale and Hypothesis………………………………………………………………….…17
Chapter 2: Screening and Characterization of Glucagon Receptor Interactors
2.1 Introduction…………………………………………………………………………..………21
2.2 Materials and Methods
2.2.1 Animals and cell culture…………………………………………………………………22
2.2.2 Isolation of primary mouse hepatocytes…………………………………………………22
2.2.3 Plasmid preparation and transfection……………………………………………………23
2.2.4 Western blotting………………………………………………………………………....23
2.2.5 Glucose production assay………………………………………………………………..23
2.2.6 cAMP assay……………………………………………………………………………..23
2.2.7 Quantitative real-time PCR……………………………………………………………...24
2.2.8 Statistics and Bioinformatics…………………………………………………………….24
2.3 Results
2.3.1 Glucagon receptor interactors affect glucose production
in primary mouse hepatocytes………………………………………………………………….25
2.3.2 Changes to cAMP accumulation mediated by select glucagon receptor interactors
2.3.2.1 Primary mouse hepatocytes………………………………………………………….27
2.3.2.2 CHO and HepG2-GCGR cells………………………………………………………28
2.3.4 Expression of key gluconeogenic genes in primary mouse hepatocytes with glucagon
receptor interactor over expression……………………………………………………………..29
2.3.5 YWHAB does not alter cellular proliferation……………………………………………30
2.3.6 YWHAB overexpression decreases cell surface expression
of GCGR in HepG2-GCGR cells……………………………………………………………….31
2.3.7 siRNA knockdown of YWHAB enhances cAMP production
and decreases GCGR gene expression………………………………………………………….32
Chapter 3: Discussion
3.1 Summary
3.1.1 Effects of select glucagon receptor interactors on receptor function…………………….35
vi
3.1.2 Effects of interactor overexpression on expression of
key gluconeogenic genes……….................................................................................................36
3.1.3 Potential mechanism of YWHAB mediate reduction
in cAMP production……………………………………………………………………………37
Chapter 4: Future Direction and Conclusions
4.1 Future Directions…………………………………………………………………………..…39
4.2 Conclusions…………………………………………………………………………………..40
References……………………………………………………………………………………….41
vii
List of Figures
Figure 1. Diagram of proglucagon products that result from tissue specific processing
……………….………………………………………………………………………………….....2
Figure 2. Diagram of the glucagon secretory pathway in pancreatic α-cells……………….……...3
Figure 3. Crystal structure of the human glucagon receptor………………….…………….……...7
Figure 4. Schematic of the glucagon receptor signalling pathway in hepatocytes………….……...8
Figure 5. Diagram of the fate of internalized glucagon
receptor…………......…………….……………………………………………………………...10
Figure 6. GLP-1R interactome identified using the MYTH screening system……………..…….17
Figure 7. GLP-1R interactome identified using the affinity purification
tandem mass spectrometry approach……………….………………………………...……….….18
Figure 8. Co-immunoprecipitation validation of GCGR accessory protein interaction.……...….19
Figure 9. Glucose production in hepatocytes overexpressing interactors of interest……………..26
Figure 10. cAMP production in hepatocytes overexpressing interactors in
interest………………………………………………………………………………......….…….27
Figure 11. cAMP production in two cells lines overexpressing interactors of interest……..…….28
Figure 12. mRNA expression of gluconeogenic genes in interactor overexpressing
hepatocytes……………………………………………………………………………………….30
Figure 13. Cell proliferation in CHO cells overexpressing YWHAB……………...…...….…….31
Figure 14. Cell surface expression of the GCGR in YWHAB overexpressing
viii
HepG2-GCGR cells……………………………………………………………………………...32
Figure 15. mRNA expression of endogenous YWHAB and GCGR in primary hepatocytes…….33
Figure 16. cAMP production in hepatocytes following siRNA knockdown of YWHAB….…….33
Figure 17. mRNA expression of GCGR in primary hepatocytes following siRNA knockdown of
YWHAB…………………………………………………………………………………………34
Figure 18. Schematic of the proposed mechanism of YWHAB’s inhibition of
glucose production……………………………………………………………………...….…….38
ix
Abbreviations
CAMK Ca2+/calmodulin-dependant protein kinase
CAV1 Caveolin 1
cAMP cyclic adenosine monophosphate
CHO Chinese hamster ovary
CREB cAMP-response element binding protein
DMEM Dulbecco's Modified Eagle Medium
FRET Fluorescence resonance energy transfer
G-6-P Glucose-6-phosphate
G6Pase Glucose-6-phosphatase
GABA Gamma-amminobutyric acid
GALK1 Galactokinase 1
GASP G-protein-coupled receptor associated sorting protein
GCGR Glucagon receptor
GFP Green fluorescent protein
GLP-1/2 Glucagon-like peptide 1/2
GLP-1R Glucagon-like peptide-1 receptor
GPCR G-protein-coupled receptor
GRK G-protein-coupled receptor kinase
IBMX 3-isobutyl-1-methylxanthine
Katp ATP regulated potassium channel
LDLR Low density lipoprotein receptor
MPGF Major proglucagon fragment
MYTH Membrane yeast two-hybrid
PC1/3 Prohormone convertase 1/3
PC2 Prohormone convertase 2
PEPCK Phosphoenolpyruvate carboxykinase
PKA Protein kinase A
PLC Phospholipase C
RAMP Receptor activity-modifying protein
x
RGS Regulators of G-protein-coupled receptors
T2D Type 2 Diabetes
TMED2 Transmembrane Emp24 Domain Trafficking Protein 2
YWHAB Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein,
Beta
1
Chapter 1 General Introduction 1.1 Glucagon
1.1.1 Blood glucose homeostasis
The regulation of glucose metabolism and maintenance of normal blood glucose levels,
euglycemia, primarily involves the concerted action of hormones secreted from the pancreas,
insulin and glucagon, but also in part by hormones such as glucagon-like peptide-11. Euglycemia
is critical for healthy function, and disrupted blood glucose homeostasis leads to a number of
complications such as nephropathy and neuropathy2. In addition, neural control of the pancreas
(through branches of the vagus and splanchnic nerve) and liver (through the splanchnic nerve)
affect glucose metabolism3.
Ordinarily, an increase in blood glucose leads to an increase in glucose uptake into β-cells,
which is metabolised, creating ATP and ultimately leading to the influx of calcium causing
secretion of insulin4. Insulin, secreted from pancreatic β-cells lowers blood glucose through a
simultaneous stimulation of glucose uptake in skeletal muscle and adipose tissue, and a
suppression of glucose production in the liver5. On the other hand, the counterregulatory pancreatic
hormone glucagon increases blood glucose levels by stimulating gluconeogenesis and
glycogenolysis, and preventing the storage of glucose as glycogen6. In type 2 diabetes (T2D),
insulin resistance and β-cell dysfunction lead to a dysregulation of blood glucose homeostasis7.
Typically, normal glucose tolerant, non-diabetic individuals rely on glucagon to increase blood
glucose concentrations during periods of fasting, but this response is supressed following post-
prandial increases in blood glucose7. However, this suppression of blood glucose elevation is
absent in type 2 diabetics following a meal7. Complications related to T2D will then develop due
to the high levels of circulating glucose. The development of T2D is the result of a combination
of lifestyle factors (including lack of exercise and high fat diets) and genetics (possessing a strong
hereditary component7, 8).
2
1.1.2 Glucagon expression and processing
The preproglucagon gene encodes a large 180 amino acid preproprotein that serves as a
precursor to a number of hormones produced from proglucagon9. The functional products of this
proprotein differ between tissues (represented schematically in Figure 1.) due to the presence of
tissue specific prohormone
convertases (PCs)10. Glucagon,
produced in the α-cells of
pancreatic islets, is produced
upon posttranslational processing
of its proglucagon precursor by
prohormone convertase 2 (PC2)6.
This 29 amino acid peptide
hormone protects against
hypoglycemia during fasting,
raising plasma glucose levels
through its stimulation of gluconeogenesis and glycogenolysis11. In addition to glucagon, the major
proglucagon fragment (MPGF) is also derived from posttranslational processing of proglucagon
in α-cells11. In the intestines, proglucagon is processed by prohormone convertase 1/3 (PC1/PC3)
to produce glucagon-like peptide-1 and -2 (GLP-1, GLP-2)11. GLP-1 can also be released from the
MPGF10,12, however this full length N-terminally extended GLP-1 (1-37) and GLP-1 (1-36amide)
peptide have limited biological activity10,13. On the other hand, truncated GLP-1(7-37) directly
processed from proglucagon in intestinal L cells by PC1/3 (31 amino acids versus 37 for the full
length inactive GLP-1) is a potent potentiator of glucose-dependant insulin secretion10,13.
1.1.3 Secretion
Like insulin, glucagon secretion is primarily regulated through blood glucose levels that
lead to changes in intracellular ATP production14. Circulating glucose is brought into the α-cells
by facilitated transport through the GLUT 1 transporter. During periods of low blood glucose,
decreased ATP/ADP ratios keep potassium channels (KATP) open, leading to opening of T-type
Figure 1. Schematic diagram of some of the products that result
from tissue specific processing of the proglucagon precursor. This
tissue specific processing is the result of prohormone convertases
that differ between tissues.
3
calcium channels
when the
membrane is at a
potential of
approximately -60
mV15. Ca2+ then
enters the cell
causing
depolarization of
the membrane and
opening of voltage
gated Na+ channels.
Efflux of Na+
causes further
depolarization and opening of L-type calcium channels leading to sustained influx of Ca2+ into the
cell which ultimately lead to glucagon granule exocytosis. During periods of high blood glucose,
increased levels of glucose in the cell elevates the ATP/ADP ratio leading to closure of KATP
channel preventing the opening of T-type calcium channels and preventing the rest of the glucagon
exocytosis cascade from proceeding.
In addition to glucose sensing, glucagon secretion is also regulated in part by fatty
acids16,17,18 and amino acids19,20,21. Early studies involving the effects of fatty acids on glucagon
secretion concluded that fatty acids inhibited glucagon secretion, it has more recently been shown
that many fatty acids may actually stimulate glucagon secretion by pancreatic α-cells. Chain
length, spatial configuration and saturation all determine the action of specific fatty acids on
glucagon secretion16. While the action of many fatty acids at the cellular level remain unknown,
palmitate has been studied at the cellular level and was found to increase exocytosis of glucagon
from α-cells by increasing entry of calcium ions through L-type channels18. Furthermore, some
amino acids can also act as stimulators of glucagon secretion19,20,21. Glycine, for example, is able
to stimulate glucagon secretion by binding to the glycine receptor on the α-cell plasma membrane
leading to an influx of Ca2+ and thus secretion of glucagon20,22.
Figure 2. Schematic outline of the glucagon secretion pathway in pancreatic α-
cells in the presence and absence of glucose.
4
The proximity of α-cells to a number of other endocrine cells within pancreatic islets leads
to dynamic regulation of glucagon secretion via paracrine action. Insulin action on α-cells is one
of the most important paracrine signalling mechanisms which inhibits glucagon secretion14; in fact,
insulin has been shown to be essential for α-cell function23,24. In mouse α-cells, binding of insulin
to its receptor and subsequent activation of the insulin receptor-PIK3 pathway increases the
sensitivity of KATP channels to ATP25. In rats, insulin was also shown to inhibit glucagon secretion
by increasing KATP channel activity26. Taken together, this evidence suggests insulin regulates
glucose stimulated glucagon secretion in α-cells via changes in cell membrane potential.
Furthermore, insulin induces translocation of the gamma-aminobutyric acid (GABA) receptor A
in α-cells to the cell membrane14,27. Pancreatic β-cells contain glutamic acid decarboxylase (GAD),
the enzyme responsible for the synthesis of GABA, and therefore also possess high levels of
GABA27. With the increased level of GABA receptor A on the surface of α-cells, GABA can bind
to and activate the receptor, leading to membrane hyperpolarization and an inhibition of glucagon
secretion due to a decrease in intracellular Ca2+ 27. Last, somatostatin, secreted from δ-cells in the
islets of Langerhans, inhibits glucagon secretion from α-cells14,28. Activation of somatostatin
receptor 2 (SSTR2) in α-cells leads to efflux of K+ ions, hyperpolarizing the membrane and
inhibiting glucagon secretion. Additionally, activation of SSTR2 inhibits cAMP production in α-
cells, thereby reducing PKA-dependant secretion14,28,29,30.
In addition to the paracrine factors outlined above, the hormones GLP-1 and gastric
inhibitory polypeptide (GIP), are important endocrine regulators of glucagon secretion14. GLP-1,
produced from proglucagon in the intestinal L-cells, inhibits glucagon secretion from pancreatic
islets11,31. It is known that GLP-1 inhibits glucagon secretion in murine species and humans32,33,
however the exact mechanism through which this inhibition is achieved still remains
controversial11. While the GLP-1R is expressed in both pancreatic β-cells and δ-cells, most α-cells
do not express GLP-1R11,32,33. Although increased glucose stimulated insulin secretion due to
GLP-1 stimulation on β-cells could certainly explain some of the inhibitory effects of GLP-1 on
α-cell glucagon secretion, glucagon secretion is still inhibited in the absence of insulin, such as in
Type 1 diabetic patients11,34,35. Instead, GLP-1’s inhibition of glucagon secretion may be the result
of its stimulation of somatostatin which also inhibits glucagon secretion as outlined above11. While
GLP-1 supresses the post-prandial glucagon response by decreasing glucagon secretion, GIP,
another incretin hormone similarly released by the gut in response to a meal, enhances the post-
5
prandial glucagon response by stimulating glucagon secretion from α-cells36,37,38. Upon activation
of GIP receptors on the cell membrane of α-cells by GIP, increases in cAMP production leads to
increased PKA-dependant exocytosis and therefore an increase in glucagon exocytosis36,37,38.
Finally, glucagon has an autocrine function in pancreatic α-cells, enhancing its own
secretion14,39. Following exocytosis of glucagon from α-cells, glucagon can bind to the glucagon
receptor, stimulating the production of cAMP, and increasing PKA-dependant exocytosis of more
glucagon14,39.
1.1.4 The effects of glucagon
Glucagon’s role as a counter regulatory hormone to insulin involves the combined action
of stimulating glucose production and release of glucose from glycogen stores, and inhibiting
glucose breakdown and storage40.
1.1.4.1 Stimulatory effects
Glucagon enhances glycogenolysis in the liver to release stored glucose. Upon stimulation
of the glucagon receptor by glucagon, an increase in cAMP production leads to an activation of
protein kinase A (PKA), which phosphorylates and activates glycogen phosphorylase kinase,
which in turn phosphorylates and activates glycogen phosphorylase41,42. Once active, glycogen
phosphorylase releases single glucose-1-phosphate (G-1-P) molecules from glycogen, which are
converted first to G-6-P and then to glucose by the enzymes phosphoglucomutase and glucose-6-
phosphatase (G6Pase), respectively41,42. Once the phosphate group has been removed, glucose can
leave the liver through GLUT2 and enter the blood stream43.
In addition to enhancing the release of glucose stores, glucagon also increases
gluconeogenesis in the liver40. The entry and rate-limiting step of gluconeogenesis is regulated by
the enzyme phosphoenolpyruvate carboxykinase (PEPCK), which converts oxaloacetate into
phosphoenolpyruvate44. Glucagon stimulation leads to a downstream increase in PEPCK
expression and ultimately an increase in gluconeogenesis44,45. Activation of PKA leads to
phosphorylation of cAMP-response element binding protein (CREB). Phosphorylated CREB can
then bind to cAMP response element regions on the promotor of a gene, recruitment of CREB
6
binding protein (CBP) and an increase in transcription46,47. In the context of enhancing release of
stored glucose, phosphorylated CREB can bind to the promotor of the transcription co-factor PGC-
1, increasing its transcription and ultimately leading to an increase in PEPCK transcription46,47,48.
1.1.4.2 Inhibitory effects
While glucagon effectively raises blood glucose through the stimulation of glucose
producing pathways, the profound effects are also due in part to a simultaneous inhibition of
glucose storage. Glycogen synthase, an enzyme responsible for incorporating glucose molecules
into growing glycogen chains, is inhibited by glucagon49. Recall that when glucagon binds to its
receptor, PKA is activated. While PKA has a stimulatory effect on glycogen phosphorylase, PKA
inhibits glycogen synthase, preventing the storage of glucose in glycogen and increasing the
availability of free glucose50,51. In addition to inhibiting storage of glucose via inactivation of
glycogen synthase, glucagon stimulation also inhibits the breakdown of glucose by inhibiting
activity of phosphofructokinase-1 a key enzyme involved in the glycolytic pathway52,53.
1.2 Glucagon Receptor
1.2.1 Regulation of receptor expression
Glucagon exerts its effects upon binding to its cognate receptor, the glucagon receptor
(GCGR). GCGR is found abundantly in multiple tissues including liver, pancreas, brain and
heart6,54, and in lower levels in the adrenal glands, thyroid and skeletal muscle6,54. Expression of
the receptor is mediated by its own ligand: increased GCGR activity following glucagon binding
increases cAMP production and cAMP binds cAMP responsive elements on the GCGR gene
promotor to down-regulate GCGR transcription55,56,57. In fact, any stimulus that leads to an
increase in the production of the second messenger cAMP can lead to suppression of GCGR
transcription55,56,57. In addition to glucagon, artificial stimulation of the GCGR with 3-isobutyl-1-
methylxanthine (IBMX) (a phosphodiesterase inhibitor) and forskolin (an organic compound that
activates adenylyl cylcase), both raise cAMP production in hepatocytes and thus lower expression
of the GCGR57. Conversely, the hormone somatostatin which lowers the production of cAMP,
leads to an increase in the mRNA expression of GCGR57,58. Both mouse and human GCGR genes
contain two promotor regions that are regulated differentially. While GCGR transcription is
7
supressed by cAMP, transcription cofactor PGC-1α prevents the cAMP mediated suppression of
the receptor, providing fine tuning of GCGR expression55.
1.2.2 Structure
The GCGR is a member of the class B, or secretin, G-protein-coupled receptor (GPCR)
family. The GCGR is a seven transmembrane domain protein, 477 amino acids in length and
approximately 55 kDa in mass6,59. The N-terminal end of the GCGR lies outside of the cell at the
end of helix I, a region known as the “stalk” which maintains the extracellular domains orientation
and is also involved in
glucagon binding59.
Extracellular domains of
the GCGR are widely
spaced, forming a deep
ligand-binding pocket59.
Aspartic acid 63, tyrosine
65 and lysine 98 stabilize
the extra-cellular domain
of the GCGR59. The
intracellular C-terminal
end of the GCGR is rich in
serine residues which may
be phosphorylated to
begin the uncoupling
process, discussed further
in section 1.2.3.259,60.
Upon binding of glucagon
to the receptor binding
pocket, the stalk region
captures glucagon and
guides the N-terminus of
glucagon into the proper
Figure 3. Structure of the human GCGR determined through x-ray
crystallography with binding domain in purple, transmembrane spanning
region in blue and glucagon in green. (Siu et al., 2013)
8
orientation within the binding pocket, while tryptophan 36 provides a hydrophobic interaction site
for the C-terminus of glucagon59.
As seen in Figure 3. the extracellular stalk region of the GCGR (in purple) partially
occludes the binding pocket, orienting glucagon (in green) within the pocket and securing it within.
Additionally the stalk can be seen providing structural support to the rest of the extracellular
domain. Following binding of glucagon to the glucagon receptor, conformational changes in the
receptor occur leading to the second messenger cascade which will be discussed in section 1.2.3.
1.2.3 Signaling
1.2.3.1 Activation and transduction
The GCGR is a member of the class B G-protein-coupled receptor family, sharing this
classification with other important receptors involved in blood glucose regulation such the GLP-1
receptor and GIP receptor6,61. As a member of the class B G-protein-coupled receptor family, many
of the early steps of the GCGR signalling cascade mirror that of other class B GPCRs. Figure 4.
outlines schematically the glucagon signalling cascade. Glucagon is a potent hormone able to
rapidly increase blood glucose through simultaneous activation of Gαs and Gq signalling
Figure 4. Schematic overview of the primary glucagon-glucagon receptor signalling cascade. A
simultaneous stimulation of glucose production and inhibition of glucose storage and breakdown leads
profound increase in blood glucose.
9
pathways14,40,63,64,65. Upon binding of glucagon to the GCGR, conformational changes in the
GCGR allow the receptor to interact directly with the G-protein complex40,62. This interaction leads
to a subsequent change in the Gαs-subunit’s conformation allowing for the exchange of GDP for
GTP40,62. This exchange leads to dissociation of the α-subunit from the βγ dimer and activation of
adenylyl cyclase leading to the conversion of ATP to cyclic AMP (cAMP)40,62. Increases in
intracellular cAMP lead to activation of protein kinase A (PKA) which leading to a number of
effects as outlined in section 1.1.3. First, active PKA will phosphorylate phosphorylase kinase,
which in turn phosphorylates and activates glycogen phosphorylase increasing breakdown of
glycogen to release glucagon14,40. Second, PKA phosphorylates the cAMP response element
binding protein (CREB) ultimately leading to an increase in gluconeogenic gene expression14,40,46.
While signalling through the Gαs-cAMP cascade causes a profound increase in free glucose,
activation of a Gq-related signalling cascade leads to further changes in blood glucose.
In addition to Gαs, dissociation of Gq from the GCGR leads to an increase in inositol
trisphosphate production and a subsequent release of Ca2+ 14,40,63,64. Increased cytosolic Ca2+ leads
to activation of Ca2+/calmodulin-dependant protein kinase (CAMK). Activation of these kinases
leads to further reduction in glycolysis and glycogenesis via activation of the FOXO1 transcription
factor thereby increasing free glucose65. Furthermore activation of CAMK in addition to PKA
leads to an increase in CREB activity, further enhancing expression of gluconeogenic genes65,66.
These two independent GCGR signalling pathways, involving activation of both Gas and Gq
cascades, allow for a rapid and profound increase in blood glucose upon glucagon stimulation.
1.2.3.2 Glucagon receptor regulation
GCGR, like most GPCRs67,68,69, is subject to a number of regulatory processes that prevent
overstimulation of the receptor and an exaggerated response to glucagon70,71. Regulation of GCGR
signalling is accomplished through desensitization and uncoupling of the receptor67,68,69,70 followed
by internalization where it may be degraded or recycled back to the cell surface72,73. Following
binding of glucagon to the GCGR, serine residues, essential for desensitization of the receptor, are
phosphorylated71,73. Phosphorylation of serine residues is achieved through G-protein-coupled
receptor kinases (GRKs), causing recruitment of β-arrestin 1 and 2 which uncouple the receptor
from its associated G-proteins72,74. Additionally, other kinase, in particular PKC phosphorylate the
GCGR leading to enhanced recruitment of β-arrestins which can occur even without activation of
10
the GCGR by glucagon73,75,76. Thus, GCGRs are tightly regulated through both homologous
(ligand-mediated) and heterologous (ligand-independent) desensitization. Following uncoupling
of the receptor from the G-protein complex, β-arrestins also target the receptor for internalization
in clathrin-coated pits where they are internalized in endosomes73,77. Internalized GCGR is then
either recycled to the cell surface, or degraded72,73. Recycling of internalized GCGR to the cell
surface is critical for resensitizing cells to glucagon, since it would take significantly longer to
synthesize new GCGR72. Recycling of internalized GCGR is accomplished through multiple
recycling pathways involving both Rab4 and Rab11 positive endosomes72,78. Rab4 endosomes are
responsible for recycling of GCGR in early endosomes that remain close to the cells surface, while
Rab11 endosomes are involved in recycling of GCGR (and other GPCRs) from the trans-Golgi
network79,80. GCGR destined to be degraded involves sorting signals on the C-terminus tail of the
GCGR, like other GPCRs, which are ubiquitinated and targeted for degradation81. The exact
mechanism through which GCGR is sorted for either recycling or degradation is still poorly
understood72. The fates of stimulated glucagon receptor are represented schematically in Figure 5.
Figure 5. Overview of the two fates of internalized glucagon receptor following glucagon stimulation.
Recycling (left) allows for rapid resensitization of cells to glucagon without the need to synthesize new
receptor. Chronic stimulation of the receptor leads to degradation of the receptor (right).
11
Short term stimulation (30 minutes) of the GCGR with glucagon leads to recycling of the receptor
to the cells surface, while long term (5 hours) stimulation with glucagon targets the receptor for
degradation72. The observation that prolonged exposure of ligand causes a shift from recycling to
degradation has also been demonstrated for other GPCRs82. Recently, G-protein-coupled receptor
associated sorting proteins (GASPs) have been shown to be critical for the degradation of GPCRs,
where they recognize specific GASP binding motifs on the receptor and target it for degradation83.
Furthermore, GASPs have been shown to associate with Gαs, and depleted of Gαs was shown to
significantly reduce degradation of GPCRs, demonstrating a potential combined role for GASP
and Gαs in targeting GCGR for degradation as opposed to recycling84.
1.3 Type 2 Diabetes and the Role of Glucagon
1.3.1 Pathogenic glucagon secretion
The role of glucagon in the development of type 2 diabetes (T2D) has been known for
decades85,86,87. In type 2 diabetics, an overabundance of glucagon, known as hyperglucagonemia,
is the result of dysfunction in both β-cells and α-cells. Normally, an increase in insulin secretion
in response to high blood glucose results in (among other things) a decrease in glucagon secretion.
As discussed in section 1.1.3, insulin as a paracrine factor plays a key role in inhibiting glucagon
secretion in α-cells by increasing sensitivity of KATP channels and causing downstream
hyperpolarization of the cellular membrane preventing the influx of calcium23,24,25,26. In the early
stages of developing T2D, increased demand for insulin due to insulin resistance is met through a
compensatory increase in β-cell mass and insulin secretion, resulting in hyperinsulinemia during
early disease progression88. This increase in β-cell mass provides a stable compensatory
mechanism to combat chronically high blood glucose for a period of time88. In later T2D, in
addition to an increased resistance to insulin, insulin secretion from β-cells is also reduced88. With
constant challenge of insulin secretion, an increase in β-cell mass is no longer an adequate
compensatory mechanism88. With time, decompensation occurs along with β-cell exhaustion,
eventually leading to a decrease in β-cell mass and insulin secretion88,89. Thus in later T2D, insulin
secretion is diminished and paracrine regulation of glucagon secretion is greatly reduced which in
part explains the hyperglucagonemia present in type 2 diabetics. While decreased insulin secretion
12
in type 2 diabetics certainly contributes to hyperglucagonemia23,24,25,26,90, dysfunction in α-cells
has also been shown to be a significant contributor.
In addition to the direct role of β-cells in the development of T2D, α-cells have also been
shown to be responsible for the development of the disease. In fasted type 2 diabetics, elevated
blood glucagon levels have been observed in addition to impaired suppression of glucagon
secretion in response to glucose91. It has been previously reported that obese non-diabetics and
obese glucose-intolerant individuals already had an observed impairment in glucose-induced
glucagon suppression92. Since this impairment was noted despite the presence of hyperinsulinemia,
it was hypothesized that these individuals’ α-cells had become resistant to insulin’s paracrine
action in reducing glucagon secretion91,92,93. This resistance to insulin-induced glucagon
suppression in α-cells has been defined as paracrinopathy91,94. In addition to paracrinopathy,
dedifferentiation may play a role in the development of hyperglucagonemia91,95. Increased demand
for insulin in T2D and the resultant stress to β-cells has been shown to lead to dedifferentiation of
these β-cells to progenitor cells which begin to produce and secrete glucagon, further increasing
the inappropriate glucagon secretion91,95.
Thus, in T2D the absence of insulin is in itself not solely responsible for hyperglycemia.
Rather, hyperglucagonemia resulting from the lack of insulin suppression of glucagon secretion,
dysfunction in α-cells, and dedifferentiation of β-cells to glucagon secreting progenitors all
contribute to chronically elevated blood glucose. Thus, as opposed to the more “insulin-centric”
model of T2D in the past, development of the disease involves a complex dysregulation of both
insulin and glucagon. Initially, hyperinsulinemia as a compensatory mechanism leads to resistance
of insulin’s glucagon supressing effects in α-cells. In later T2D, there is a reduction in β-cell mass
and insulin secretion, dedifferentiation of β-cells to glucagon producing cells, and a lack of tonal
regulation of α-cell glucagon secretion.
1.3.2 Glucagon receptor antagonists
Potential avenues of T2D treatment typical fall within two developmental approaches:
increasing insulin secretion, biosynthesis and or sensitivity, or reducing glucagon section,
biosynthesis and or sensitivity. Defendants of the glucagon-focused approaches to the treatment
of T2D91,96 cite the fact that hyperglucagonemia is present in all poorly controlled diabetes, known
glucagon suppressors such as somatostatin eliminate inappropriate glucose production in total
13
insulin deficiency and, incredibly, glucagon-receptor knockout mice do not develop diabetes even
with complete β-cell destruction91,96,. Experiments involving glucagon receptor knockout mice
provide compelling support for the notion that glucagon may be at the center of diabetes, as
inducible diabetic mice with a whole-body glucagon receptor knockout have been shown to be
protected from diabetes97. Following destruction of β-cells with streptozotocin, GCGR+/+ mice
became hyperglycemic were sacrificed after 6 weeks. GCGR-/- mice on the other hand were
characterized by a complete absence of the manifestations of diabetes97. Even with destruction of
β-cells, these GCGR knockout mice had normal fasting glucose and oral glucose tolerance tests97.
The nature of the current project has focused on the glucagon receptor itself, therefore
discussion of the glucagon-centric approaches to the treatment of T2D will focus specifically on
regulation of the glucagon receptor itself. Some of the earliest GCGR antagonists (as far back as
1986) were peptides98,99,100. Despite their ability to bind to the GCGR and partially prevent the
activation of the cAMP-PKA pathway, these peptide antagonists were unable to lower plasma
glucose levels in diabetic rats99. Years later, Qureshi et al. identified a non-peptide antagonist of
the GCGR, Cpd 1101. Cpd 1 was found to prevent glucagon-mediated glycogenolysis, and blocked
the increase in blood glucose in mice following intraperitoneal injection of glucagon101. The small
molecule skyrin, a fungal bisanthroquinone, was found to bind to the GCGR and prevent glucagon-
induced activation of the cAMP-PKA pathway102.
While there have been numerous attempts to develop a clinically significant glucagon
receptor antagonist for the treatment of T2D, difficulties have arisen due to a lack of specificity of
the antagonist, failure to induce meaningful and long-term suppression of blood glucose levels in
diabetic animals, or lack of further investigation of the clinical impact. The development of an
effective GCGR antagonist for the treatment of T2D could be made easier with a deeper
understanding of GCGR function and how its activity is regulated in vivo.
1.4 Receptor Interactomes
1.4.1 Effects of accessory proteins on g-protein couple receptors
While the signalling pathway of many class B G-protein coupled receptors follow similar
cascades (i.e. activation of adenylyl cyclase and PKA), GPCRs have been shown to be involved
in protein-protein interactions with accessory proteins that can dramatically alter their function and
14
activity103. These protein-protein interactions can produce profound changes in not only ligand
binding or the downstream response to ligands, but also in receptor localization103,104. GPCRs may
be involved in single protein-protein interactions with an accessory protein,105 or complex
interacting networks (receptor interactomes) involving multiple accessory proteins that bind to the
receptor and to one another105.
Discussed in section, 1.2.3.2, β-arrestins and GRKs are one such example of GPCR
accessory proteins and their role in uncoupling GPCRs and endocytosis are critical to preventing
overstimulation of GPCRs106. Many other families of GPCR accessory proteins have been
identified which alter GPCRs in a variety of ways. Another family of GPCR accessory proteins,
known as receptor activity-modifying proteins (RAMPs), modify the cell surface expression and
pharmacology of certain GPCRs altering their trafficking and the strength of the response to
receptor ligand106,107. On the other hand, regulators of G-protein signalling (RGS) proteins have
been shown to negatively regulate G-proteins, turning off G-protein-coupled receptors106,108.
Another family, GPCR-associated sorting proteins (GASPs), are responsible for the removal of
GPCRs from the cell surface following ligand stimulation106,109,110.
Thus, while strategies to target GPCRs in the treatment of diabetes are promising
approaches, the presence of accessory proteins and interacting networks complicates the
development of these treatment options. Development of GCGR antagonists for the treatment of
T2D could be improved with a complete understanding of the GCGR interactome.
1.4.2 Experimental approaches to receptor interactome identification
A new experimental paradigm involving hypothesis generating studies has developed with
advances in high-throughput screening tools, leading to the development of many “omic” sub-
fields within biology. Omics refers to a full and complete set of all relevant biological molecules
in a field of study, such as genomics which explores the complete set of genes in an organism (the
genome), or metabolomics, which explorers the complete metabolic profile of a cell, tissue or
organism (the metabolome). Since GPCRs have been shown to be involved in a number of complex
interactions with accessory proteins, interactomics was developed which aims to reveal the
complete set of interacting proteins for a particular receptor (the interactome). The nature of
interactomics demands high-throughput screens of receptor interactors to make experiments cost
15
and time efficient. A number of a screening methods exist to this end, and a list of some of the
more common screening methods will be summarized below.
1.4.3.1 Membrane yeast two-hybrid system
The membrane yeast two-hybrid system (MYTH) takes advantage of the fact that in vivo,
the ubiquitin protein is fragmented into two moieties, a C-terminal fragment (Cub) and an N-
terminal fragment (Nub) that spontaneously re-associate with each other. However, by mutating
isoleucine 13 on the Nub fragment to glycine (NubG), this spontaneous re-association is prevented.
With these fragments, a bait protein (such as a GPCR), is fused to the Cub fragment, while a library
of prey proteins are fused to the NubG fragment. In addition, an artificial transcription factor is
fused to the Cub fragment. If a protein-protein interaction exists between the bait (receptor) and
prey (library) proteins, their physical interaction brings the two ubiquitin fragments into close
enough proximity to form pseudo-ubiquitin. This pseudo-ubiquitin protein can then be recognized
by deubiquitinating enzymes, releasing the transcription factor and leading to activation of a suite
of reporter genes that can be used to assess the interaction between the bait and prey. The MYTH
approach has been successfully employed by a number of groups to identify protein
interactomes111,112,113. Recently, our lab has successfully employed the MYTH system to screen
for interactors of the glucagon-like peptide-1 receptor (GLP-1R) revealing 38 novel GLP-1R
interacting candidates114.
While the MYTH system allows for high throughput screens of receptor interactomes, there
are significant limitations to this approach. 1) Interactions between bait and prey proteins must be
direct interactions in order to be detected. Indirect interactions in which another protein mediates
the interaction between bait and prey may not bring the ubiquitin fragments into close enough
proximity to fuse, preventing detection. 2) Screens are performed in a yeast vector using human
bait and prey proteins which may require other intracellular machinery and factors to properly
interact which may greatly differ in amino acid sequence and structure such as PKA and PLC 3)
Screening of receptor interactomes must be performed in the unliganded state. Since many
previously identified receptor interactors are responsible for desensitization or alteration of
receptor activity following agonist binding, the lack of receptor stimulation in the MYTH screen
means some potential receptor interactors may be missed.
16
1.4.3.2 Fluorescence resonance energy transfer
The florescence resonance energy transfer system (FRET) for detecting in vivo protein-
protein interactions is similar in principle to the MYTH system115. In FRET, two proteins of
interest are labelled with a donor molecule or an acceptor fluorophores, which can be a dye,
inorganic ion or fluorescent protein115,116. When excited, the donor molecule will transfer energy
to the acceptor molecule when in close proximity leading to an increase in the acceptor’s
fluorescent signal116. In MYTH, a protein-protein interaction allowed the ubiquitin fragments to
be in close enough proximity to re-associate. In FRET, binding of one protein to another brings
the conjugated donor and acceptor molecules in close enough proximity for the energy transfer
between the donor and acceptor to occur116. Unlike MYTH, liganded experiments are possible
with FRET screens allowing differences in protein interactions in the unliganded vs liganded state
to be determined.
While FRET has significant advantages over MYTH, it still possesses some of the
limitations inherent in the MYTH system. Because the FRET system requires conjugation of
fluorophores to two proteins of interest, a library of potential protein interactors with the protein
of interest must be constructed, which limits the potential high-throughput screening applications.
Like MYTH, constructing a library of potential interactors means each protein must be pre-
selected for inclusion in the screen, which may lead to missing potential protein-protein
interactions that were not predicted.
1.4.3.3 Mass spectrometry
Mass spectrometry has applications in both chemistry and biology for its ability to identify
unknown substances117. In biological applications of mass spectrometry, peptides are digested with
proteases and separated by various characteristics, such as polarity, using chromatography and
then ionized117. Since peptides were fragmented with proteases and separated, each fragment is
given a unique charge-to-mass ratio which can be plotted and used to identify the parent peptide
the fragment belongs to. In the study of receptor interactomes, the receptor of interest may be
stimulated with ligand, purified and fragmented with proteases. Following separation of the
fragments, ionization and detection, the data can be plotted as a mass spectrum, which plots the
17
intensity of fragments vs the charge-to-mass ratio. Since the receptor was purified from other
proteins prior to mass spectrometry, proteins identified other than the receptor itself may be
potential receptor interactors which can be further validated. Compared to MYTH and FRET, mass
spectrometry is an incredibly robust high throughput screening method for identifying receptor
interactors in both the liganded and unliganded state. Potential interactors are not required to be
labelled, drastically enhancing the number of novel interactors that may be detected and allowing
for hypothesis generating research. One of the first successful use of mass spectrometry to study
the interactome of a GPCR came from Daulet et al., who revealed the interactome of two melatonin
receptors and found 38 receptor interactors between the two118. Recently, our lab successfully
employed affinity-purification and mass spectrometry to study the interactome of the glucagon-
like peptide 1 receptor, and revealed 99 potential receptor interactors between two cell lines119.
1.5 Rationale and Hypothesis
The Wheeler labs first high-throughput
screen to identify receptor interactomes involved
the use of the membrane yeast two hybrid system
to screen for GLP-1 receptor interactors. Using a
human fetal liver cDNA prey library and the GLP-
1R as bait, Huang et al. were able to identify 38
potential GLP-1R interactors, seen in Figure 6, 36
of which were validated with co-
immunoprecipitation114. Functional
characterization of the interaction between these
novel interactors and the GLP-1R revealed a
number of significant alterations to cAMP
production114.
While the initial MYTH screen was
successful in identifying a number of novel GLP-
1R interactors (many of which were able to alter
Figure 6. Interactome of the GLP-1R as
identified by Huang et al. using the membrane
yeast two hybrid system (MYTH). In total, 38
potential GLP-1R interactors were identified
from the screen. (Huang et al., 2014)
18
receptor signalling), the limitations of MYTH, as outlined above, prompted the Wheeler lab to
explore more robust screening approaches for the identification of GPCR interactors. As a follow-
up study, Zhang et al. performed another screen of the GLP-1R, this time using affinity purification
and tandem mass spectrometry119. Following the GLP-1R mass spectrometry screen, Zhang et al.
identified 99 potential GLP-1R interactors within two cell lines119, as seen in Figure 7. One
interactor, PGRMC1, was shown to significantly enhance GLP-1R signalling and represents a
potential target for future therapeutics. The success of the mass spectrometry screen prompted the
Wheeler lab to explore other GPCR interactomes.
Figure 7. Interactome of the GLP-1R as identified by Zhang et al. using affinity purification and tandem
mass spectrometry. In total, 99 potential GLP-1R interactors were identified from the screen between two
cell lines, Min6 and CHO. (Zhang et al., 2014)
19
The role of glucagon in the development of T2D has made targeting the glucagon receptor
an attractive option for the treatment of the disease. A greater understanding regarding the
glucagon receptor’s interactome may improve the development of glucagon receptor antagonists.
Many members of the class B G-protein coupled receptor family have been shown to be involved
in many complex interactions with accessory proteins that dramatically alter their activity and
expression. Thus, it is reasonable to expect that there are a number of unknown glucagon receptor
interactors.
To this end, our lab has used affinity purification and tandem mass spectrometry to identify
a number of potential GCGR interactors. In total, 33 potential glucagon receptor interactors were
identified from the screen of human GCGR transfected in Chinese hamster ovary (CHO) cells in
both the unliganded and liganded state. After selecting 8 of these potential interactors (Figure 8.)
based on their previously reported functional roles in cell signalling and molecular transport, and
their known localization (on the cell surface), 5 of these potential interactors were validated with
co-immunoprecipitation: CAV1,
GALK1, LDLR, TMED2 and
YWHAB. These 5 validated
interactors were therefore selected for
functional and mechanistic studies in
this thesis.
Based on the previously
reported involvement of accessory
proteins in the regulation of other
class B GPCRs, it was hypothesized
that some or all of these interactors
will significantly alter the activity and
expression of the GCGR. The
interaction between the GCGR and
these accessory proteins will explain
in part the difficulties faced in the
generation of GCGR antagonists and
Figure 8. Validation of the interaction between GCGR and 8
potential interactors using co-immunoprecipitation.
YWHAE, TMED10 and YWHAQ were excluded due to
absence in the eluate or aspecific binding to the affinity gel,
respectively. (Han et al., 2015)
20
further reveal the methods in which fine tuning of the glucagon’s effects in the liver are achieved.
It was further hypothesized that altering the expression of these interactors may reveal their
therapeutic potential.
21
Chapter 2
High Throughput Screening and Characterization of Glucagon Receptor Interactors
The following chapter is based in part on work published in PloS One:
Han, J., Zhang, M., Froese, S., Dai, F. F., Robitaille, M., Bhattacharjee, A., Huang, X., Jia, W.,
Angers, S., Wheeler, M.B. & Wei, L. (2015). The Identification of Novel Protein-Protein
Interactions in Liver that Affect Glucagon Receptor Activity. PloS one, 10 e0129226 (2015).
Conceived and designed the experiments: MBW SA LW WJ. Performed the experiments: JH MZ
SF AB XH MR. Analyzed the data: JH MZ SF MR. Contributed reagents/materials/analysis
tools: SA. Wrote the paper: JH SF FD MB LW.
Glucose production, cAMP production, gene expression, cell proliferation, cell surface
expression and siRNA knockdown experiments were conducted by SF
2.1 Introduction
Glucagon is responsible for maintaining normal blood glucose during periods of fasting
through a simultaneous promotion of both glycogenolysis and gluconeogenesis. Synthesized in the
pancreatic α-cells in the islets of Langerhans, glucagon’s role in mediating blood glucose begins
upon binding to the glucagon receptor. The GCGR belongs to the class B G-protein-coupled
receptor superfamily, increasing glucose production primarily through the Gs alpha-cAMP-PKA
pathway as well as Gq and phospholipase C (PLC) pathway6. Following binding of glucagon to
the GCGR, the Gs alpha subunit is released from the G protein complex and activates adenylate
cyclase, leading to a downstream increase in cAMP levels, which in turn leads to increased
activation of PKA120. Furthermore, disassociation of Gq from the G protein complex leads to the
activation of PLC, causing increases in intracellular calcium121,122. Glucagon has been found to be
elevated in type 2 diabetics85, and disruption of glucagon’s activity improves hyperglycemia in
obese mice123. Because of these observations, GCGR antagonists represent a potential avenue of
diabetes treatment. A number of GCGR antagonists have been investigated, such as the fungal
bisanthroquinone skyrin102, or Cpd-1101. These antagonists fail due to their poor potency or a lack
of specificity. The development of GCGR antagonists would be greatly aided by a complete,
comprehensive understanding of each factor involved in regulating GCGR function.
22
G-protein coupled receptor interactors, or accessory proteins, have been the subject of a
great deal of study in recent years in an effort to reveal the diverse function and regulatory
mechanisms of the receptors. Many accessory proteins, such as the GABAB receptor accessory
proteins KCTDs, and GLP-1 receptor accessory protein beta-arrestin 1, dramatically alter receptor
function and are critical components of receptor activity124,125. Many GPCR interactomes have
been discovered in recent years yet the interactome of the GCGR remains unknown. Several
studies have employed affinity purification and mass spectrometry for high-throughput screening
of other GPCRs126,127,128. The Wheeler lab has recently revealed a number of novel GLP-1R
interactors in CHO and MIN6 cells expressing the GLP-1R using a similar affinity purification
mass and spectrometry method revealing 99 potential interactors, one of which significantly
augments GLP-1 stimulated insulin secretion119. In a more recent study, using this method the
Wheeler lab revealed 33 potential GCGR interactors, 8 of which were selected for further
functional and mechanistic study following validation of the interaction.
2.2 Materials and Methods
2.2.1 Animals and cell culture
Mice ages 8-12 weeks of age of C57BL/6 background were used for experiments. All
experiments have been approved by the Animal Care Committee (University of Toronto). Animals
were handled according to the Canadian Council of Animal Care guidelines. HepG2-GCGR cells,
the stable human glucagon receptor expressing human liver carcinoma cells, generated for this
study were cultured with high glucose Dulbecco’s Modified Eagle Medium (DMEM) with 10%
fetal bovine serum as well as 1% penicillin-streptomycin. Chinese hamster ovary (CHO) cells were
cultured under the same conditions and were passaged approximately every 4 days. Transient
transfections in primary hepatocytes as well as the two cell lines were performed using
Lipofectamine 2000 following the manufacturer’s instructions (Invitrogen, Carlsbad, California).
2.2.2 Isolation of primary mouse hepatocytes
Mice were fasted overnight and primary hepatocytes were isolated and cultured as
described previously129. Briefly, primary hepatocytes were isolated using collagenase IV (Sigma,
Canada) perfusion. Cells were seeded using DMEM supplemented with 1 g/L glucose, 10 M/L
sodium lactate, 0.01 μM/L dexamethasone, 5 mM/L HEPES, and 2 mM/L L-Glutamine.
23
2.2.3 Plasmid preparation and transfection
cDNA of the human GCGR (c-terminal Flag-tagged) and GCGR interactors (c-terminal
HA-tagged) were constructed in the pcDNA3.1 vector. Plasmids were purified using the Midi-
Prep kit according to the manufacturer’s protocol (Qiagen, Toronto, Canada).
2.2.4 Western blotting
Total cell lysate was collected using protein lysis buffer (10% glycerol, 50 mM Hepes, 150
mM NaCl, 2 mM EDTA, 0.25% n-dodecyl-b-d-maltoside, and complete protease inhibitor mixture
(Roche)). Anti-Flag antibody (1:1000 dilution; Sigma-Aldrich, United States), anti-GLUT2
antibody (1:1000 dilution; EMD Millipore, United States) and HRP-conjugated mouse secondary
antibody were used for detection of protein. The membranes were developed with the ECL
advance kit (GE Healthcare) and imaged using the Kodak ImageStation 4000 Pro (Care stream
Health Inc, Rochester, New York).
2.2.5 Glucose production assay
Primary mouse hepatocytes (2×105 cells per well in twelve-well plates) were first serum
starved overnight prior to stimulation. Following serum starving, primary hepatocytes were
preincubated with glucose-free DMEM without phenol red for 2 hours. Cells were then washed
with PBS and stimulated with adenylate cyclase activator, forskolin (10 μM/L), or glucagon (100
nM/L) in glucose-free DMEM without phenol red for 4 h. Culture media was collected for
measurement of glucose concentration using the Glucose (GO) assay kit (Sigma, Canada) and
readings were normalized to total protein content using the Bradford assay.
2.2.6 cAMP assay
Intracellular cAMP content was measured in primary hepatocytes as described
previously130. In brief, cells were washed with cold PBS and harvested using 80% ethanol. Lysates
were centrifuged and the supernatant was collected and lyophilized using a SpeedVac. The pellet
was resuspended in cAMP assay buffer (0.05 mM/L sodium acetate (pH 6.2) and 0.01% sodium
azide) and measured using an intracellular cAMP ELISA kit (Biomedical Technologies Inc, US).
For cAMP measurements in CHO and HepG2-GCGR cells, the Cisbio cAMP cell-based assay kit
was used according to the manufacturer’s instructions.
24
2.2.7 Quantitative real-time PCR
Total RNA from primary hepatocytes was extracted using an RNA-easy kit (Qiagen,
Canada) and cDNA was generated by SuperScript II enzyme (Invitrogen, Canada). mRNA
expression was analyzed by qPCR using Power SYBR Green PCR master mix according to the
manufacturer’s instructions (Applied Biosystems, Carlsbad, California) and ViiA 7 Real-Time
PCR System (Life-Technology, Canada). Data was normalized to β-actin expression. Primers for
PCR were designed using the Primer3 software program. Relative gene expression was estimated
by the standard curve method.
2.2.8 Statistics and Bioinformatics
The data are presented as the mean ± SE. Student’s t-test was used to measure the mean
difference for measurements of glucose production and cAMP in primary hepatocytes. One-way
ANOVA was used to measure the mean difference for cAMP production in CHO and HepG2-
GCGR cells and differences were considered statistically significant at p < 0.05.
25
2.3 Results
2.3.1 Glucagon receptor interactors affect glucose production in primary mouse
hepatocytes
The glucagon receptor is expressed in many tissues but it is found most abundantly in the
liver. In response to low blood glucose, glucagon is secreted from the pancreas and binds to the
glucagon receptor leading to glucose production in the liver through a simultaneous stimulation of
both glycogenolysis and gluconeogenesis. To determine the functional role that the identified
GCGR interactors have as GCGR accessory protein, glucose production was assessed in primary
mouse hepatocytes. Prior to over expression of the 5 GCGR interactors, the transfection efficiency
of lipofectamine 2000 in primary mouse hepatocytes was tested using overexpression of a green
fluorescent protein (GFP) plasmid. The transfection efficiency of GFP was confirmed to be
upwards of 70% (Figure. 9A). In pcDNA3.1 (empty vector) transfected primary hepatocytes,
glucose production in response to glucagon was shown to be dose-dependent (Figure. 9B),
indicating that isolated and transfected hepatocytes were healthy and the GCGR signalling
pathway was functional. Next, the five interactors validated by Co-IP and Western blot (Figure.
7) were transfected into primary hepatocytes and the EC50 concentration of 100 nM glucagon was
selected for treatment to examine glucose production. GCGR accessory proteins CAV1 and
GALK1 were both found to increase glucose production at the basal (without glucagon treatment)
concentration, however no changes to glucose production were found in the presence of glucagon
(118.87±9.4%, p<0.05, N = 6 and 120.03±13.0%, p<0.05, N = 6 respectively) (Figure. 9C). On
the other hand overexpression of two interactors (LDLR and TMED2) had no effect at the basal
level of glucagon, but were found to enhance glucagon-stimulated glucose production significantly
(128.97±12.6%, p<0.01, N = 6 and 131.15±10.3%, p<0.01, N = 6 respectively). Overexpression
of the fifth interactor, YWHAB, conversely reduced glucagon-induced glucose production
26
significantly (65.59±4.6%, p<0.01, N = 6) (Figure. 9D) while having no effect on glucose
production at the basal glucagon concentration. GCGR interactors CAV1 and GALK1 were found
to increase glucose production under basal conditions, but prevented glucose production in the
presence of glucagon which suggests they may block receptor function. While LDLR, TMED2
Figure 9. A. Expression of green fluorescent protein in primary hepatocytes, approximately 70%
transfection efficiency. B. Isolated hepatocytes transfected with pcDNA3.1 control produce glucose in
response to glucagon indicating healthy function of the cells. C. Glucose production in primary
hepatocytes with over expression of interactors that affect basal glucose production. D. Glucose
production in primary hepatocytes with over expression of interactors that affect glucagon stimulated
glucose production. Hepatocytes isolated by Han and Zhang, glucose production assays performed by
Zhang and Froese. * = p<0.05, ** = p<0.01. (Han et al., 2015)
27
and YWHAB were selected for further characterization, future studies should characterize the
effects of CAV1 and GALK on GCGR function.
2.3.2 Changes to cAMP accumulation mediated by select glucagon receptor
interactors
Prior to the activation of protein kinases responsible for increased glucose production,
GCGR activation in response to glucagon leads to activation of adenylyl cyclase, an increase in
cAMP production and activation of PKA. To determine whether the effects of LDLR, TMED2
and YWHAB were the result of regulation of the cAMP pathway, the effects that overexpression
of these interactors had on cAMP production in response to glucagon was assessed in isolated
primary mouse hepatocytes, and two cell lines: CHO and HepG2-GCGR.
2.3.2.1 Primary mouse hepatocytes
The effects of GCGR interactors on cAMP production in primary hepatocytes was assessed
using 1.0 nM glucagon, as determined from the EC50 in Figure. 10A. Overexpression of LDLR
and TMED2 significantly increased glucagon-induced cAMP accumulation to 127.74±2.1%
Figure 10. A. Glucagon-cAMP dose-response curve in primary hepatocytes demonstrates isolated
hepatocytes signalling pathway remains intact following isolation. B. cAMP production in primary
hepatocytes overexpressing GCGR accessory proteins of interest. LDLR and TMED2 significantly
increase cAMP production above control, while YWHAB significantly inhibits cAMP production. cAMP
assay performed by Froese. ** = p<0.01. (Han et al., 2015)
28
(p<0.01, N = 6) and to 80.4±2.13% (p<0.05, N = 6) respectively as anticipated based on the results
of the glucose production experiments (Figure. 10B). YWHAB significantly attenuated cAMP
production in response to glucagon by 57.24±7.9% (p<0.01, N = 6, Figure. 10B). Previous studies
have demonstrated that increased cAMP production is the key factor of GCGR mediated glucose
production6. Thus, the increase in cAMP production with LDLR and TMED2 overexpression, and
the decrease in cAMP production with YWHAB overexpression is a plausible explanation for the
changes to glucose production seen in Figure. 9.
2.3.2.2 CHO and HepG2-GCGR cells
Consistent with the cAMP production in primary hepatocytes, YWHAB overexpression
significantly decreased 1.0 nM glucagon induced cAMP accumulation in both CHO co-transfected
with GCGR and HepG2-GCGR cells (p<0.05, N = 3, Figure. 11A and 11B). In CHO cells,
overexpression of LDLR and TMED2 led to a significant increase in glucagon induced cAMP
production (Figure. 11A). Curiously, in HepG2-GCGR cells overexpressing LDLR and TMED2,
Figure 11. cAMP production in A CHO cells and B HepG2-GCGR cells overexpressing GCGR accessory
proteins of interest. Only YWHAB showed a consistent effect on cAMP production between cell lines,
that is, an inhibition of cAMP production. LDLR and TMED2 increase cAMP production in CHO cells
only. C. No change in forskolin stimulated cAMP production was found between YWHAB over
expressing CHO cells and control indicating YWHAB’s is not a general adenylyl cyclase inhibitor. cAMP
assay performed by Froese. * = p<0.05, ** = p<0.01. (Han et al., 2015)
29
no significant difference in cAMP production was found. To test whether the suppression of cAMP
production in cells overexpressing YWHAB was due to an overall suppression of adenylyl cyclase,
or the glucagon receptor itself, YWHAB overexpressing CHO cells were stimulated with
forskolin. Stimulation with forskolin did not affect cAMP production in CHO cells expressing the
GCGR (Figure. 11C), indicating YWHAB’s suppressive effects are mediated via action upstream
of adenylyl cyclase, likely the GCGR.
2.3.3 Expression of key gluconeogenic genes in primary mouse hepatocytes with
glucagon receptor interactor overexpression
Gluconeogenesis is in part responsible for the increased glucose production in the liver
following glucagon stimulation. Increased cAMP production leads to an increase in PKA activity,
enhancing gluconeogenesis131. Since LDLR, TMED2 and YWHAB were all shown to alter cAMP
production in primary mouse hepatocytes and two cells lines, changes in expression of key
gluconeogenic genes was assessed using qPCR. Two enzymes,
phosphoenolpyruvatecarboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) are critical
regulators of the rate limiting steps in gluconeogenesis and GCGR-mediated glucose
production132. One of the earliest steps in gluconeogenesis is the conversion of oxaloacetate to
phosphoenolpyruvate, catalyzed by PEPCK, and the final step of gluconeogenesis is the hydrolysis
of glucose-6-phosphate to glucose and inorganic phosphate, catalyzed by G6Pase. Following
overexpression of LDLR, TMED2, and YWHAB in primary hepatocytes, cells were treated with
or without glucagon, and prepared for qPCR. In the basal glucagon condition, LDLR, TMED2 and
YWHAB overexpression had no effect on gene expression of either PEPCK or G6Pase (Figure
12A and 12B). However, when stimulated with glucagon, expression of PEPCK and G6Pase in
LDLR- overexpressing hepatocytes was significantly increased to 76.52±2.3% and 108±1.7%
respectively (p<0.01, N = 6, Figure. 12A 12B). TMED2 was also shown to upregulate G6Pase (but
not PEPCK) gene expression, significantly to 114.15±3.5% (p<0.01, N = 6) following glucagon
stimulation (Figure 12B). Since LDLR and TMED2 over-expression led to increased expression
of gluconeogenic genes following stimulation with glucagon, it is possible that LDLR and TMED2
may be involved in enhancing gluconeogenesis in the liver. On the other hand, overexpression of
YWHAB lead to a significant reduction in PEPCK and G6Pase gene expression following
30
glucagon stimulation to
52.31±1.4% (p<0.05, N = 6)
and 41.61±0.8% (p<0.01, N =
6) respectively (Figure 11A
and 11B). Thus, as opposed to
LDLR and TMED2, YWHAB
may be involved in supressing
gluconeogenesis in the liver
following glucagon
stimulation.
In the context of a
potential treatment for TD2,
the ability of YWHAB to
supress glucagon-stimulated
cAMP and glucose production
as well as gluconeogenic gene
expression is a valuable
finding. For this reason,
YWHAB was selected as the
GCGR accessory protein of
primary interest, and
conducted further studies to
elucidate the mechanism
through which these effects are
accomplished.
2.3.4 YWHAB does not alter cellular proliferation
YWHAB has been found previously to be involved in proliferative pathways where it
interacts with RAF kinases and enhances their activity133,134. To determine if YWHAB’s effects
on cellular proliferation, the XTT Cell Proliferation Assay Kit was used. Following overexpression
Figure 12. mRNA expression of A PEPCK and B G6Pase in primary
hepatocytes transfected with GCGR interactors of interest. LDLR
significantly increased expression of both genes, while TMED2 only
increased expression of G6Pase. YWHAB overexpression led to a
significant decrease in expression of both gluconeogenic genes.
qPCR performed by Froese, Han, and Zhang. * = p<0.05, ** = p<0.01
(Han et al., 2015)
31
of YWHAB in CHO cells and
recovery for 24 hours, cells
XTT reagent was added and
absorbance was measured
every hour for 4 hours.
Overexpression of empty
vector, YWHAB, GCGR or
YWHAB and GCGR co-
transfection had no significant
effect on cellular proliferation
in CHO cells. Treatment of
cells overnight with 100 mM
hydrogen peroxide lead to a
significant decrease in cell
proliferation (not shown).
These results further support
the notion that YWHAB’s ability to lower gluconeogenesis is due specifically to its interaction
with the GCGR, and not on targets downstream of the receptor.
2.3.5 YWHAB overexpression decreases cell surface expression of GCGR in
HepG2-GCGR cells
Thus far, the mechanism through which YWHAB suppresses gluconeogenesis was not
found to be the result of inhibition of adenylyl cyclase (Figure. 11C), or changes to cellular
proliferation (Figure. 13). Since YWHAB physically interacts with the GCGR, as determined by
the initial AP-MS screen and Co-immunoprecipitation validation, it was hypothesised that
YWHAB may affect expression of the GCGR at the cell surface. To investigate this further, cell
surface isolation experiments were performed in HepG2-GCGR cells the GCGR was probed
following stimulation with glucagon for 15 minutes60, the time required for maximal endocytosis
of the glucagon receptor following stimulation by glucagon. Following stimulation with glucagon
and isolation of cell surface proteins, cell surface expression of the GCGR was found to be
Figure 13. Cellular proliferation in CHO cells under a variety of
transfection conditions determined with the XTT proliferation assay
after 4 hours. No change in proliferation was present between any
of the groups suggesting YWHAB’s effects may be independent of
proliferation. XTT assay performed by Froese. Unpublished.
32
decreased in HepG2-GCGR cells overexpressing YWHAB compared to PcDNA3.1 control
(Figure. 14A). Densitometry showed an approximate 33% reduction of cell surface GCGR
expression with YWHAB overexpression (Figure. 14B). These results indicate that YWHAB’s
physical interaction with the GCGR may be involved in mediating its endocytosis into the cell
following ligand stimulation. If YWHAB enhances endocytosis of the GCGR, this may explain
the YWHAB-mediated suppression of cAMP and glucose production.
2.3.6 siRNA knockdown of YWHAB enhances cAMP production and decreases
GCGR gene expression
Thus far, overexpression of YWHAB has been the approach to study the function and
mechanism of YWHAB’s interaction with GCGR. While YWHAB has been shown to inhibit
glucose production in primary hepatocytes, it is possible that YWHAB is part of a larger process
and its individual contribution to this suppression is minimal. To further determine the impact of
YWHAB on glucose production, YWHAB siRNA knockdown was performed in primary mouse
hepatocytes. qPCR revealed high level of expression of YWHAB in primary mouse hepatocytes,
nearly 3-fold great than β-actin, making it a suitable candidate for siRNA knockdown (Figure. 15).
Figure 14. A. Cell surface expression of GCGR and GLUT2 (control) in transfected HepG2-GCGR cells
following stimulation with glucagon. B. Densitometry used to quantify expression of the GCGR
revealed a decreased of nearly one-third in cell surface expression of the GCGR in YWHAB over
expressing cells after normalizing bands to the loading control. Cell surface isolation performed by
Froese and Zhang, Western blot performed by Froese. * = p<0.05. Unpublished.
A B
33
Following siRNA knockdown, primary hepatocytes
were either stimulated with glucagon and subjected to
measurements of cAMP production, or lysed and
prepared for qPCR. Stimulation of primary
hepatocytes with YWHAB knockdown showed a
significantly higher glucagon-induced cAMP response
compared to the scramble control (Figure. 16). The
fact that knockdown of YWHAB was able to reverse
the effects on cAMP production seen with
overexpression indicates that YWHAB alone may
contribute to the suppression of gluconeogenesis upon
glucagon stimulation of the GCGR. Endogenous
levels of YWHAB may be significant contributors to
suppression of GCGR signalling.
Interestingly,
qPCR in primary
mouse hepatocytes
with knockdown of
YWHAB showed a
significant reduction
in mRNA expression
of the GCGR
(Figure. 17). When
overexpressed,
YWHAB caused a
significant reduction
in expression of
GCGR on the cell
Figure 15. qPCR measurements of
endogenous expression of YWHAB and
GCGR in primary hepatocytes revealed an
abundance of both genes, indicating that
YWHAB and GCGR are viable targets for
siRNA knockdown. qPCR performed by
Froese. ** = p<0.01. Unpublished.
Figure 16. cAMP production in primary hepatocytes following siRNA
knockdown of YWHAB lead to an increase in glucagon-induced cAMP
production compared to the scramble control at a number of different
doses of glucagon. siRNA knockdown and cAMP assay performed by
Froese. * = p<0.05 Unpublished.
34
surface (Figure. 14). Ordinarily, an increase in endocytosis
of GCGR mediated by YWHAB would lead to an increase
in the recycling and degradation of the receptor73,74,79,80,81.
Degraded GCGR must be then replaced via transcription of
mRNA and translocation to the cell membrane. Decreased
YWHAB expression would lead to decreased endocytosis
of the GCGR, and thus less GCGR would need to be
replaced via transcription, which may explain in part the
observation of decreased GCGR mRNA expression. This
decrease in GCGR mRNA expression following
knockdown of YWHAB may further explain the
mechanism through which YWHAB inhibits
gluconeogenesis.
Figure 17. mRNA expression of the
GCGR in primary hepatocytes
following siRNA knockdown of
YWHAB. Knockdown of YWHAB caused
a significant decrease in the expression
of GCGR compared to control,
indicating endogenous YWHAB may
lead to increases in GCGR mRNA
expression. siRNA knockdown and
qPCR performed by Froese. ** =
p<0.05 Unpublished.
35
Chapter 3
Discussion
3.1 Summary
Protein-protein interactions have been shown to be critical regulators of GPCR function,
and vast interacting protein (interactome) networks have been identified for a number of receptors,
including the GLP-1R by our lab119. These accessory proteins can have significant effects on
receptor function, activity and cell surface expression, but little is known about the glucagon
receptor’s interactome. Affinity purification and tandem mass spectrometry performed previously
by the Wheeler lab revealed 33 potential GCGR interactors, 8 of which were selected based on
their previously reported functional roles in signal transduction, as well as their cellular locations.
Following selection of these 8 interactors, identification of their functional and mechanistic
characteristics was determined.
3.1.1 Effects of select glucagon receptor interactors on receptor function
From 8 select GCGR interactors, 5 were successfully validated with co-
immunoprecipitation and were selected for further functional and mechanistic characterisation. In
the context of T2D, chronic activation of the GCGR by elevated glucagon levels leads to an
exaggerated increase in gluconeogenesis. Glucose production was therefore selected as the initial
functional output to determine the effects of these 5 validated interactors. Two interactors, CAV1
and GALK1 were found to enhance glucose production under basal glucagon conditions, while
the remaining three, LDLR, TMED2 and YWHAB, were found to significantly alter glucose
production following glucagon stimulation in primary hepatocytes, with LDLR and TMED2
enhancing glucagon-stimulated glucose production and YWHAB supressing it. LDLR, TMED2
and YWHAB were selected for further analysis based on their function under elevated glucagon
conditions.
Since glucagon-stimulated glucose production is mediated by the cAMP-PKA pathway,
the effects of overexpression of these three interactors on cAMP production in primary mouse
hepatocytes was measured. Glucagon-cAMP dose response curves were determined for transfected
CHO and HepG2 cells to ensure proper pathway function, and to determine the optimal dose of
36
glucagon that would allow both increases and decreases in cAMP production to be measureable.
Mirroring the trends seen in glucose production, LDLR and TMED2 significantly enhanced
glucagon-stimulated cAMP production, while YWHAB overexpression significantly impaired
cAMP production. To validate these results, cAMP production was measured in interactor
overexpressing CHO and HepG2-GCGR cells. In CHO cells, LDLR and TMED2 significantly
increased cAMP production, while YWHAB supressed it, consistent with the results seen in
primary hepatocytes. Interestingly, in HepG2-GCGR cells, only YWHAB altered cAMP
production, lowering cAMP production as seen in the primary hepatocytes and CHO cells.
Forskolin stimulated cAMP production was consistent between control and YWHAB
overexpressing CHO cells indicating that YWHAB’s effects on cAMP production are likely not
the result of an overall inhibition of cAMP production, and is rather the result of its interaction
with the GCGR itself, or Gas.
3.1.2 Effects of interactor overexpression on expression of key
gluconeogenic genes
In addition to phosphorylation of key gluconeogenic enzymes by cAMP-PKA activation,
changes to gluconeogenic gene expression provide a further increase in glucose production and
suppression of glucose storage and breakdown following glucagon stimulation. Thus, the
expression of key gluconeogenic genes was measured in primary hepatocytes overexpressing
LDLR, TMED2 or YWHAB. Two genes, PEPCK and G6Pase, regulate key steps in
gluconeogenesis and were selected for qPCR analysis. qPCR revealed that LDLR overexpressing
primary hepatocytes significantly increased PEPCK and G6Pase expression following glucagon
stimulation. TMED2 had no effect on PEPCK expression, but was shown to enhance expression
of G6Pase. On the other hand, YWHAB overexpressing primary hepatocytes has significantly
lower levels of both PEPCK and G6Pase. These results suggest that in addition to their effects
exerted through direct interaction with the GCGR, LDLR, TMED2 and YWHAB may have
additional effects on glucose production by altering expressing of key gluconeogenic genes. CREB
regulates transcription of key gluconeogenic genes, and these alterations to cAMP production with
GCGR receptor overexpression may in part explain these results. Each of these three interactors
are highly interesting GCGR interactors which profoundly alter glucose production in primary
hepatocytes and two cells lines, and represent potential targets for future therapeutic research.
37
3.1.3 Potential mechanism of YWHAB mediated reduction in cAMP
production
YWHAB was shown to inhibit glucose production in primary hepatocytes, and inhibited
cAMP production in both primary hepatocytes and two cells lines. In the context of T2D, the
ability to supress glucose production is of particular importance. Elevated glucagon levels in type
2 diabetics leads to chronic activation of the GCGR, leading to exaggerated glucose production.
Since YWHAB lowers the activity of the GCGR, it was selected for further mechanistic studies
Since YWHAB has been shown to play a role in cell cycle regulation and proliferative
pathways, it was first determined whether the inhibition of glucose production previously
identified was the result of changes to cell proliferation. Cell proliferation assays revealed no
difference in proliferation of CHO cells overexpressing YWHAB compared to control, further
suggesting YWHAB’s ability to supress glucose production is the result of its direct interaction
with the GCGR.
Next, the cell surface expression of the glucagon receptor in cells overexpressing YWHAB,
both in the presence and absence of glucagon stimulation was investigated. Decreased cell surface
expression of the GCGR could in part explain the effects of YWHAB. Indeed, cell surface
expression studies revealed a significant decrease in cell surface expression of the GCGR
following overexpression of YWHAB. Thus, YWHAB may mediate the internalization of GCGR,
decreasing its cell surface expression and lowering cAMP-PKA pathway activation in hepatocytes.
Overexpression of YWHAB was shown to decrease glucose and cAMP production in
primary hepatocytes, but the significance of YWHAB alone was yet to be determined. To this end,
siRNA knockdown of YWHAB was used to determine the impact of YWHAB alone on cAMP
production. YWHAB knockdown lead to a partial rescue of the attenuated glucagon stimulated
cAMP production, indicating that YWHAB has a direct and significant role in regulating glucose
production in primary hepatocytes. High levels of endogenous YWHAB mRNA expression in
primary mouse hepatocytes as determined through qPCR further supports this notion.
Last, to further explore the mechanism of YWHAB’s suppression of glucose production,
mRNA expression of the GCGR was measured to determine if the observed decreased cell surface
expression of the GCGR was the result of changes to receptor gene transcription. Endogenous
38
mRNA expression of the GCGR in primary hepatocytes following knockdown of YWHAB was
significantly decreased compared to scramble control which may be explained by my hypothesis
that YWHAB increases internalization of the GCGR. Increased internalization and degradation of
the GCGR would lead to an increase in GCGR transcription. A decrease in YWHAB, as in my
knockout model, would prevent increased internalization and reduce GCGR degradation,
eliminating the need to synthesize new GCGR, decreasing the level of GCGR mRNA. The current
working model of YWHAB’s effects on the GCGR are summarized schematically in Figure. 18.
Figure 18. Schematic of the proposed mechanism through which YWHAB decreases glucose production
and GCGR signalling. YWHAB mediates the interaction between the GCGR and endocytotic machinery.
Increased YWHAB leads to an increase in the endocytosis of the GCGR, leading to an eventual increase
in degradation and an increase in gene expression to restore normal levels of the receptor.
39
Chapter 4 Future Directions and Conclusions
4.1 Future Directions
The proposed mechanism through which YWHAB lowers glucose production in primary
hepatocytes involves the mediation of receptor endocytosis following ligand stimulation,
decreasing cell surface expression of the GCGR and lowering activity of the GCGR-cAMP-PKA
pathway. Future studies involving fluorescently labelled receptor, such as the fluorogen activating
peptide system, in the presence and absence of YWHAB would allow for the visualization of
GCGR expression and would provide compelling evidence to support or refute this proposed
mechanism.
While PEPCK and G6Pase were selected as key gluconeogenic genes, glycogen synthase
also plays an important role in glucose regulation, as glycogen synthase is responsible for storing
glucose as glycogen. Future studies should investigate gene expression of glycogen synthase in
the presence of these GCGR interactors, as well as expression of glycogen synthase kinase, the
enzyme responsible for the activation of glycogen synthase.
While the focus of this thesis was the mechanism through which YWHAB inhibits glucose
production in primary hepatocytes, exploration of the mechanism of LDLR and TMED2 mediated
increases in glucose production would also be highly beneficial to the study of type 2 diabetes.
Determining the mechanism through which GCGR interactors enhance glucose production could
lead to the revelation of potential targets for suppression that would in turn decrease glucose
production. In addition, two of the other early identified GCGR interactors, CAV1 and GALK1
should be characterized and explored further.
Furthermore, future studies could identify the GCGR interactome in primary human
hepatocytes. In this study, the interactome of the human GCGR was performed in CHO cells,
which may lack proteins found in primary human hepatocytes. Performing an AP-MS screen in
primary hepatocytes may reveal addition GCGR accessory proteins that have potent effects on
receptor function.
40
The use of YWHAB knockout mice may prove invaluable in determining the true clinical
impact this GCGR accessory protein has. It is possible that knockout of YWHAB may be fatal, as
YWHAB belongs to a family of highly conserved proteins that may be critical for survival.
However if YWHAB knockout mice survive, fasting glucose and oral glucose tolerance tests may
reveal interesting changes to blood glucose regulation in diabetic mice. The true impact that
YWHAB may have on GCGR activity can only be assessed in animal models, and is a logical next
step for future investigation.
4.2 Conclusions
GPCR accessory proteins have been shown to have profound effects on receptor activity
and expression, with the GCGR being no exception. Three were shown to significantly alter
glucose production in primary hepatocytes following glucagon stimulation, with one, YWHAB,
significantly inhibiting glucose production. These effects appear to be the result of YWHAB’s
mediation of endocytosis of stimulated GCGR, reducing its cAMP-PKA activation. My project
has identified novel regulators of GCGR function, and has identified potential novel targets for
future T2D treatments.
41
References
1 Aronoff, S. L., Berkowitz, K., Shreiner, B. & Want, L. Glucose metabolism and regulation: beyond insulin
and glucagon. Diabetes Spectrum 17, 183-190 (2004).
2 Chang, Y. C., Chang, E. Y. C., & Chuang, L. M. Recent progress in the genetics of diabetic
microvascular complications. World Journal of Diabetes 6, 715-725 (2015).
3 Niijima, A. Neural mechanisms in the control of blood glucose concentration. The Journal of
nutrition 119, 833-840 (1989). 4 Leibiger, I. B., Leibiger, B. & Berggren, P. O. Insulin signaling in the pancreatic β-cell. Annual review of
nutrition, 28, 233-251 (2008).
5 Triplitt, C. L. Examining the mechanisms of glucose regulation. The American journal of managed care
18, S4-10 (2012).
6 Brubaker, P. L., & Drucker, D. J. Structure-function of the glucagon receptor family of G protein-coupled
receptors: the glucagon, GIP, GLP-1, and GLP-2 receptors. Receptors and Channels 8, 179-188 (2002).
7 Olokoba, A. B., Obateru, O. A. & Olokoba, L. B. Type 2 diabetes mellitus: a review of current
trends. Oman medical journal 27, 269-273 (2012). 8 Lovejoy, J. C. The influence of dietary fat on insulin resistance. Current diabetes reports 2, 435-440
(2002). 9 Mojsov, S., Heinrich, G., Wilson, I. B., Ravazzola, M., Orci, L. & Habener, J. F. Preproglucagon gene
expression in pancreas and intestine diversifies at the level of post-translational processing. Journal of Biological Chemistry 261, 11880-11889 (1986).
10 Sinclair, E. M. & Drucker, D. J. Proglucagon-derived peptides: mechanisms of action and therapeutic
potential. Physiology 20, 357-365 (2005). 11 Campbell, J. E. & Drucker, D. J. Islet α cells and glucagon - critical regulators of energy
homeostasis. Nature reviews endocrinology 11, 329-338 (2015). 12 Holst, J. J. The physiology of glucagon-like peptide 1. Physiological reviews 87, 1409-1439 (2007). 13 Mojsov, S., Weir, G. C. & Habener, J. F. Insulinotropin: glucagon-like peptide I (7-37) co-encoded in the
glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas. Journal of clinical investigation 79, 616 (1987). 14 Quesada, I., Tudurí, E., Ripoll, C. & Nadal, Á. Physiology of the pancreatic α-cell and glucagon
secretion: role in glucose homeostasis and diabetes. Journal of endocrinology 199, 5-19 (2008).
15 Bansal, P. & Wang, Q. Insulin as a physiological modulator of glucagon secretion. American Journal of
physiology-endocrinology and metabolism 295, E751-E761 (2010).
16 Hong, J., Abudula, R., Chen, J., Jeppesen, P. B., Dyrskog, S. E., Xiao, J., Colombo, M. & Hermansen,
K. The short-term effect of fatty acids on glucagon secretion is influenced by their chain length, spatial configuration, and degree of unsaturation: studies in vitro. Metabolism 54, 1329-1336 (2005).
42
17 Bollheimer, L. C., Landauer, H. C., Troll, S., Schweimer, J., Wrede, C. E., Schölmerich, J. & Buettner,
R. Stimulatory short-term effects of free fatty acids on glucagon secretion at low to normal glucose concentrations. Metabolism 53, 1443-1448 (2008). 18 Olofsson, C. S., Salehi, A., Göpel, S. O., Holm, C. & Rorsman, P. Palmitate stimulation of glucagon
secretion in mouse pancreatic α-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium. Diabetes 53, 2836-2843 (2004).
19 Leclercq-Meyer V, Marchand J, Woussen-Colle MC, Giroix MH & Malaisse WJ. Multiple effects of
leucine on glucagon, insulin, and somatostatin secretion from the perfused rat pancreas. Endocrinology 116, 1168-1174 (1985).
20 Kuhara, T., Ikeda, S., Ohneda, A. & Sasaki, Y. Effects of intravenous infusion of 17 amino acids on the
secretion of GH, glucagon, and insulin in sheep. American journal of physiology-endocrinology and metabolism 260, E21-E26 (1991).
21 Dumonteil, E., Magnan, C., Ritz-Laser, B., Ktorza, A., Meda, P. & Philippe, J. Glucose Regulates
Proinsulin and Prosomatostatin But Not Proglucagon Messenger Ribonucleic Acid Levels in Rat Pancreatic Islets 1. Endocrinology 141, 174-180 (2000).
22 Li, C., Liu, C., Nissim, I., Chen, J., Chen, P., Doliba, N., Zhang, T., Nissim, I., Daikhin, Y., Stokes, D.,
Yudkoff, M., Bennet, M.J., Stanley, C.A., Matschinsky, F.M. & Naji, A. Regulation of glucagon secretion in normal and diabetic human islets by γ-hydroxybutyrate and glycine. Journal of biological chemistry 288, 3938-3951 (2013). 23 Müller, W. A., Faloona, G. R. & Unger, R. H. The effect of experimental insulin deficiency on glucagon
secretion. Journal of clinical investigation 50, 1992-1999 (1997). 24 Diao, J., Asghar, Z., Chan, C. B. & Wheeler, M. B. Glucose-regulated glucagon secretion requires
insulin receptor expression in pancreatic α-cells. Journal of biological chemistry 280, 33487-33496 (2005).
25 Leung, Y. M., Ahmed, I., Sheu, L., Gao, X., Hara, M., Tsushima, R.G., Diamant, N.E. & Gaisano, H. Y.
Insulin regulates islet α-cell function by reducing KATP channel sensitivity to adenosine 5′-triphosphate inhibition. Endocrinology 147, 2155-2162 (2006).
26 Franklin, I., Gromada, J., Gjinovci, A., Theander, S. & Wollheim, C. B. β-cell secretory products activate
α-cell ATP-dependent potassium channels to inhibit glucagon release. Diabetes 54,1808-1815 (2005).
27 Xu, E., Kumar, M., Zhang, Y., Ju, W., Obata, T., Zhang, N., Liu, S., Wendt A., Deng, S., Ebina, Y.,
Wheeler, M.B., Braun, M. & Wang, Q. Intra-islet insulin suppresses glucagon release via GABA-GABA A receptor system. Cell metabolism 3, 47-58 (2006).
28 Gromada, J., Høy, M., Buschard, K., Salehi, A. & Rorsman, P. Somatostatin inhibits exocytosis in rat
pancreatic α‐cells by Gi2‐dependent activation of calcineurin and depriming of secretory granules. The Journal of physiology 535, 519-532 (2001). 29 Kailey, B., van de Bunt, M., Cheley, S., Johnson, P. R., MacDonald, P. E., Gloyn, A. L., Rorsman, P. &
Braun, M. SSTR2 is the functionally dominant somatostatin receptor in human pancreatic β-and α-cells. American journal of physiology-endocrinology and metabolism 303, E1107-E1116 (2012). 30 Fehmann, H. C., Strowski, M. & Goke, B. Functional characterization of somatostatin receptors
expressed on hamster glucagonoma cells. American journal of physiology-endocrinology and metabolism 268, E40-E47 (1995).
43
31 Weir, G. C., Mojsov, S., Hendrick, G. K., & Habener, J. F. Glucagonlike peptide I (7–37) actions on
endocrine pancreas. Diabetes 38, 338-342 (1989). 32 Pyke, C., Heller, R. S., Kirk, R. K., Ørskov, C., Reedtz-Runge, S., Kaastrup, P. Hvelplund, A., Bardram,
L., Calatayud, D. & Knudsen, L. B. GLP-1 receptor localization in monkey and human tissue: novel distribution revealed with extensively validated monoclonal antibody. Endocrinology 155, 1280-1290 (2014). 33 Richards, P., Parker, H. E., Adriaenssens, A. E., Hodgson, J. M., Cork, S. C., Trapp, S., Gribble, F.M. &
Reimann, F. Identification and characterization of GLP-1 receptor–expressing cells using a new transgenic mouse model. Diabetes 63, 1224-1233 (2014). 34 Dupre, J., Behme, M. T. & McDonald, T. J. Exendin-4 normalized postcibal glycemic excursions in type
1 diabetes. The journal of clinical endocrinology & metabolism 89, 3469-3473 (2004). 35 Farngren, J., Persson, M., Schweizer, A., Foley, J. E. & Ahrén, B. Vildagliptin reduces glucagon during
hyperglycemia and sustains glucagon counterregulation during hypoglycemia in type 1 diabetes. The journal of clinical endocrinology & metabolism 97, 3799-3806 (2012). 36 Moens, K., Heimberg, H., Flamez, D., Huypens, P., Quartier, E., Ling, Z. Pipeleers, D., Gremlich, S.,
Thorens, B. & Schuit, F. Expression and functional activity of glucagon, glucagon-like peptide I, and glucose-dependent insulinotropic peptide receptors in rat pancreatic islet cells. Diabetes 45, 257-261 (1996). 37 Ding, W. G., Renström, E., Rorsman, P., Buschard, K., & Gromada, J. Glucagon-like peptide I and
glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat α-cells by a protein kinase A–mediated mechanism. Diabetes 46, 792-800 (1997). 38 Seino, Y., Fukushima, M., & Yabe, D. GIP and GLP‐1, the two incretin hormones: similarities and
differences. Journal of diabetes investigation 1, 8-23 (2010). 39 Ma, X., Zhang, Y., Gromada, J., Sewing, S., Berggren, P. O., Buschard, K., Salehi, A., Vikman, J.,
Rorsman, P.& Eliasson, L. Glucagon stimulates exocytosis in mouse and rat pancreatic α-cells by binding to glucagon receptors. Molecular endocrinology 19, 198-212 (2005). 40 Jiang, G. & Zhang, B. B. Glucagon and regulation of glucose metabolism. American journal of
physiology-endocrinology and metabolism 284, E671-E678 (2003).
41 Johnson, L. N., Barford D., Own D.J., Noble M.E. & Garman, E.F. From phosphorylase to
phosphorylase kinase. Advances in second messenger and phosphoprotein research 31, 11-28 (1997). 42 Exton, J. H. Mechanisms of hormonal regulation of hepatic glucose metabolism. Diabetes and
metabolism research and reviews 3, 163-183 (2009).
43 Nordlie, R. C., Foster, J. D., & Lange, A. J. Regulation of glucose production by the liver. Annual review
of nutrition 19, 379-406 (1999). 44 Beale, E., Andreone, T., Koch, S., Granner, M. & Granner, D. Insulin and glucagon regulate cytosolic
phosphoenolpyruvate carboxykinase (GTP) mRNA in rat liver. Diabetes 33, 328-332 (1984). 45 Iynedjian, P. B., Auberger, P., Guigoz, Y. & Le Cam, A. Pretranslational regulation of tyrosine
aminotransferase and phosphoenolpyruvate carboxykinase (GTP) synthesis by glucagon and dexamethasone in adult rat hepatocytes. Biochemical journal 225, 77-84 (1985).
44
46 Herzig, S., Long, F., Jhala, U. S., Hedrick, S., Quinn, R., Bauer, A., Rudolph, D., Schutz, G., Yoon, C.,
Puigserver, P., Spiegelman, B. & Montminy, M. CREB regulates hepatic gluconeogenesis through the coactivator PGC-1. Nature 413, 179-183 (2001). 47 Vidal-Puig, A. & O'Rahilly, S. Metabolism: controlling the glucose factory. Nature 413, 125-126 (2001). 48 Yoon, J. C., Puigserver, P., Chen, G., Donovan, J., Wu, Z., Rhee, J., Adelmant, G., Stafford, J., Kahn,
C.R., Granner, D.K., Newgard, C.B. & Spiegelman, B. M. Control of hepatic gluconeogenesis through the transcriptional coactivator PGC-1. Nature 413, 131-138 (2001).
49 Akatsuka, A., Singh, T. J., Nakabayashi, H., Lin, M. C. & Huang, K. P. Glucagon-stimulated
phosphorylation of rat liver glycogen synthase in isolated hepatocytes. Journal of biological chemistry 260, 3239-3242 (1985).
50 Ciudad, C., Camici, M., Ahmad, Z., Wang, Y., DePaoli‐Roach, A. A. & Roach, P. J. Control of glycogen
synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon. European journal of biochemistry 142, 511-520 (1984). 51 Roach, P. J. Control of glycogen synthase by hierarchal protein phosphorylation. The FASEB
journal, 4(12), 2961-2968 (1990).
52 Okar, D. A. & Lange, A. J. Fructose‐2, 6‐bisphosphate and control of carbohydrate metabolism in
eukaryotes. Biofactors 10, 1-14 (1999). 53 Pilkis, S. J. & Claus, T. H. Hepatic gluconeogenesis/glycolysis: regulation and structure/function
relationships of substrate cycle enzymes. Annual review of nutrition 11, 465-515 (1991).
54 Hansen, L. H., Abrahamsen, N. & Nishimura, E. Glucagon receptor mRNA distribution in rat
tissues. Peptides 16, 1163-1166 (1995).
55 Mortensen, O. H., Dichmann, D. S., Abrahamsen, N., Grunnet, N. & Nishimura, E. Identification of a
novel human glucagon receptor promoter: Regulation by cAMP and PGC-1α. Gene 393, 127-136 (2007). 56 Abrahamsen, N., Lundgren, K. & Nishimura, E. Regulation of glucagon receptor mRNA in cultured
primary rat hepatocytes by glucose and cAMP. Journal of biological chemistry 270, 15853-15857 (1995). 57 Nishimura, E., Abrahamsen, N., Hansen, L. H., Lundgren, K. & Madsen, O. Regulation of glucagon
receptor expression. Acta physiologica scandinavica 157, 329-332 (2003).
58 Rutter, G. A. Regulating glucagon secretion: somatostatin in the spotlight. Diabetes 58, 299-301 (2009).
59 Siu, F. Y., He, M., de Graaf, C., Han, G. W., Yang, D., Zhang, Z., Zhou, C., Xu, Q., Wacker, D., Joseph,
J.S., Liu, W., Lau, J., Cherezov, V., Katritch, V., Wang, M.W. & Stevens, R. C. Structure of the human glucagon class B G-protein-coupled receptor. Nature 499, 444-449 (2013).
60 Merlen, C., Fabrega, S., Desbuquois, B., Unson, C. G. & Authier, F. Glucagon-mediated internalization
of serine-phosphorylated glucagon receptor and Gsα in rat liver. FEBS letters 580, 5697-5704 (2006).
61 Ravn, P., Madhurantakam, C., Kunze, S., Matthews, E., Priest, C., O'brien, S. Collinson, A., Papworth,
M., Fristch-Fredin, M., Jermutus, L., Benthem, L., Gruetter, M. & Jackson, R. H. Structural and pharmacological characterization of novel potent and selective monoclonal antibody antagonists of glucose-dependent insulinotropic polypeptide receptor. Journal of biological chemistry 288, 19760-19772 (2013).
45
62 Bissantz, C. Conformational Changes of G Protein‐Coupled Receptors During Their Activation by
Agonist Binding. Journal of receptors and signal transduction 23, 123-153 (2003). 63 Wakelam, M. J., Murphy, G. J., Hruby, V. J. & Houslay, M. D. Activation of two signal-transduction
systems in hepatocytes by glucagon. Nature 323, 68-71 (1986).
64 Christophe, J. Glucagon receptors: from genetic structure and expression to effector coupling and
biological responses. Biochimica et Biophysica Acta (BBA)-reviews on biomembranes 1241, 45-57 (1995). 65 Mieskes, G., Kuduz, J. & Söling, H. D. Are calcium‐dependent protein kinases involved in the regulation
of glycolytic/gluconeogenetic enzymes?. European journal of biochemistry 167, 383-389 (1987).
66 Hardingham, G. E., Arnold, F. J. & Bading, H. Nuclear calcium signaling controls CREB-mediated gene
expression triggered by synaptic activity. Nature neuroscience 4, 261-267 (2001).
67 Rockman, H. A. Uncoupling of G-protein coupled receptors in vivo: insights from transgenic mice.
In Analytical and quantitative cardiology, 67-72 (1997). 68 Gainetdinov, R. R., Premont, R. T., Bohn, L. M., Lefkowitz, R. J. & Caron, M. G. Desensitization of G
protein-coupled receptors and neuronal functions. Annual review of neuroscience 27, 107-144 (2004).
69 Kelly, E., Bailey, C. P. & Henderson, G. Agonist‐selective mechanisms of GPCR desensitization. British
journal of pharmacology 153, S379-S388 (2008).
70 Authier, F., Desbuquois, B. & De Galle, B. Ligand-mediated internalization of glucagon receptors in
intact rat liver. Endocrinology 131, 447 (1992).
71 Buggy, J. J., Heurich, R. O., MacDougall, M., Kelley, K. A., Livingston, J. N., Yoo-Warren, H. &
Rossomando, A. J. Role of the glucagon receptor COOH-terminal domain in glucagon-mediated signaling and receptor internalization. Diabetes 46, 1400-1405 (1997). 72 Krilov, L., Nguyen, A., Miyazaki, T., Unson, C. G. & Bouscarel, B. Glucagon receptor recycling: role of
carboxyl terminus, β-arrestins, and cytoskeleton. American journal of physiology-cell physiology 295, C1230-C1237 (2008). 73 Krilov, L., Nguyen, A., Miyazaki, T., Unson, C. G., Williams, R., Lee, N. H., Ceryak., S. & Bouscarel, B.
Dual mode of glucagon receptor internalization: role of PKCα, GRKs and β-arrestins. Experimental cell research 317, 2981-2994 (2011).
74 Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G. & Barak, L. S. Differential affinities of visual
arrestin, βarrestin1, and βarrestin2 for G protein-coupled receptors delineate two major classes of receptors. Journal of biological chemistry 275, 17201-17210 (2000).
75 Ikegami, T., Krilov, L., Meng, J., Patel, B., Chapin-Kennedy, K. & Bouscarel, B. Decreased glucagon
responsiveness by bile acids: A role for protein kinase Cα and glucagon receptor phosphorylation. Endocrinology 147, 5294-5302 (2006). 76 Murphy, G. J., Hruby, V. J., Trivedi, D., Wakelam, M. J. & Houslay, M. D. The rapid desensitization of
glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism. Journal of biochemistry. J 243, 39-46 (1987).
46
77 Luttrell, L. M. & Lefkowitz, R. J. The role of β-arrestins in the termination and transduction of G-protein-
coupled receptor signals. Journal of cell science 115, 455-465 (2002).
78 Esseltine, J. L. & Ferguson, S. S. Regulation of G protein-coupled receptor trafficking and signaling by
Rab GTPases. Small GTPases 4, 132-135 (2013). 79 Maxfield, F. R. McGraw, T. E. Endocytic recycling. Nature reviews molecular cell biology 5, 121-132
(2004). 80 Seachrist, J. L. Ferguson, S. S. Regulation of G protein-coupled receptor endocytosis and trafficking by
Rab GTPases. Life sciences 74, 225-235 (2003). 81 Marchese, A., Paing, M. M., Temple, B. R. & Trejo, J. G protein–coupled receptor sorting to endosomes
and lysosomes. Annual review of pharmacology and toxicology 48, 601 (2008).
82 Moore, R. H., Tuffaha, A., Millman, E. E., Dai, W., Hall, H. S., Dickey, B. F. Knoll, B. J. Agonist-induced
sorting of human beta2-adrenergic receptors to lysosomes during downregulation. Journal of cell science 112, 329-338 (1999).
83 Bornert, O., Møller, T. C., Boeuf, J., Candusso, M. P., Wagner, R., Martinez, K. L.Simonin, F.
Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors. PloS one 8, e56336 (2013).
84 Rosciglione, S., Theriault, C., Boily, M. O., Paquette, M. Lavoie, C. Gαs regulates the post-endocytic
sorting of G protein-coupled receptors. Nature communications 5 (2014). 85 Unger, R. H. Role of glucagon in the pathogenesis of diabetes: the status of the
controversy. Metabolism 27, 1691-1709 (1978).
86 Reaven, G. M., Chen, Y. D., Golay, A., Swislocki, A. L. M. Jaspan, J. B. Documentation of
Hyperglucagonemia Throughout the Day in Nonobese and Obese Patients with Noninsulin-Dependent Diabetes Mellitus.The Journal of Clinical Endocrinology & Metabolism 64, 106-110 (1987).
87 Shah, P., Vella, A., Basu, A., Basu, R., Schwenk, W. F. Rizza, R. A. Lack of Suppression of Glucagon
Contributes to Postprandial Hyperglycemia in Subjects with Type 2 Diabetes Mellitus 1. The journal of clinical endocrinology & metabolism 85, 4053-40599 (2000).
88 Weir, G. C. & Bonner-Weir, S. Five stages of evolving beta-cell dysfunction during progression to
diabetes. Diabetes 53, S16-S21 (2004). 89 Butler, A. E., Janson, J., Bonner-Weir, S., Ritzel, R., Rizza, R. A., & Butler, P. C. β-cell deficit and
increased β-cell apoptosis in humans with type 2 diabetes. Diabetes, 52, 102-110 (2003).
90 Kjems, L. L., Kirby, B. M., Welsh, E. M., Veldhuis, J. D., Straume, M., McIntyre, S. S., Yang, D.,
Lefebvre, P. & Butler, P. C. Decrease in β-cell mass leads to impaired pulsatile insulin secretion, reduced postprandial hepatic insulin clearance, and relative hyperglucagonemia in the minipig. Diabetes 50 (2001).
91 Godoy-Matos, A. F. The role of glucagon on type 2 diabetes at a glance. Diabetology & metabolic
syndrome 6, 91 (2014).
92 Borghi, V. C., Wajchenberg, B. L. Cesar, F. P. Plasma glucagon suppressibility after oral glucose in
obese subjects with normal and impaired glucose tolerance. Metabolism 33, 1068-1074 (1990).
47
93 Ferrannini, E., Muscelli, E., Natali, A., Gabriel, R., Mitrakou, A., Flyvbjerg, A., Golay, A.& Hojlund, K.
Association of fasting glucagon and proinsulin concentrations with insulin resistance. Diabetologia 50, 2342-2347 (2007).
94 Unger, R. H., & Orci, L. Paracrinology of islets and the paracrinopathy of diabetes. Proceedings of the
National academy of sciences 107, 16009-16012 (2010).
95 Dor, Y. Glaser, B. beta-cell dedifferentiation and type 2 diabetes. New England Journal of
Medicine 368, 572-573 (2013).
96 Unger, R. H. Cherrington, A. D. Glucagonocentric restructuring of diabetes: a pathophysiologic and
therapeutic makeover. The journal of clinical investigation 122, 4-12 (2012).
97 Lee, Y., Wang, M. Y., Du, X. Q., Charron, M. J. Unger, R. H. Glucagon receptor knockout prevents
insulin-deficient type 1 diabetes in mice. Diabetes 60, 391-397 (2011).
98 Gysin, B., Trivedi, D., Johnson, D. G. & Hruby, V. J. Design and synthesis of glucagon partial agonists
and antagonists. Biochemistry 25, 8278-8284 (1986). 99 Gysin, B., Johnson, D. G., Trivedi, D. & Hruby, V. J. Synthesis of two glucagon antagonists: receptor
binding, adenylate cyclase, and effects on blood plasma glucose levels. Journal of medicinal chemistry 30, 1409-1415 (1987). 100 Unson, C. G., Gurzenda, E. M. & Merrifield, R. B. Biological activities of des-His 1 [Glu 9] glucagon
amide, a glucagon antagonist. Peptides 10, 1171-1177 (1989).
101 Qureshi, S. A., Candelore, M. R., Xie, D., Yang, X., Tota, L. M., Ding, V. D. H., Li, Z., Bansal, A., Miller,
C., Cohen, S.M., Jiang, G., Brady, E., Saperstein, R., Duffy, J.L., Tata, J.R., Chapman K.T., Moller, D.E. & Zhang, B. B. A novel glucagon receptor antagonist inhibits glucagon-mediated biological effects. Diabetes 53, 3267-3273 (2004). 102 Parker, J. C., McPherson, R. K., Andrews, K. M., Levy, C. B., Dubins, J. S., Chin, J. E., Perry,
P.V., Hulin, B., Perry, D.D., Inagaki, T., Dekker, K.A., Tachikawa, K., Sugie, Y. & Treadway, J. L. Effects of skyrin, a receptor-selective glucagon antagonist, in rat and human hepatocytes. Diabetes 49, 2079-2086 (2000). 103 Presland, J. G-protein-coupled receptor accessory proteins: their potential role in future drug
discovery. Biochemical society transactions 32, 888 (2004).
104 Couvineau, A. & Laburthe, M. The family B1 GPCR: structural aspects and interaction with accessory
proteins. Current drug targets 13, 103-115 (2012).
105 Milligan, G., & White, J. H. Protein–protein interactions at G-protein-coupled receptors. Trends in
pharmacological sciences, 22, 513-518 (2001).
106 Magalhaes, A. C., Dunn, H. & Ferguson, S. S. Regulation of GPCR activity, trafficking and localization
by GPCR‐interacting proteins. British journal of pharmacology 165, 1717-1736 (2012).
107 Hay, D. L., Poyner, D. R. & Sexton, P. M. GPCR modulation by RAMPs. Pharmacology &
therapeutics 109, 173-197 (2006).
108 De Vries, L., Zheng, B., Fischer, T., Elenko, E. & Farquhar, M. G. The regulator of G protein signaling
family. Annual review of pharmacology and toxicology 40, 235-271 (2000).
48
109 Simonin, F., Karcher, P., Boeuf, J. J. M., Matifas, A. & Kieffer, B. L. Identification of a novel family of G
protein‐coupled receptor associated sorting proteins. Journal of neurochemistry 89, 766-775 (2004).
110 Kargl, J., Balenga, N. A., Platzer, W., Martini, L., Whistler, J. L. & Waldhoer, M. The GPCR‐associated
sorting protein 1 regulates ligand‐induced down‐regulation of GPR55. British journal of pharmacology 165, 2611-2619 (2012).
111 Thaminy, S., Auerbach, D., Arnoldo, A. & Stagljar, I. Identification of novel ErbB3-interacting factors
using the split-ubiquitin membrane yeast two-hybrid system. Genome research 13, 1744-1753 (2003).
112 Snider, J., Kittanakom, S., Damjanovic, D., Curak, J., Wong, V. & Stagljar, I. Detecting interactions with
membrane proteins using a membrane two-hybrid assay in yeast. Nature protocols, 5(7), 1281-1293 (2010). 113 Lam, M. H. Y., Snider, J., Rehal, M., Wong, V., Aboualizadeh, F., Drecun, L., Wong, O., Jubran, B., Li,
M., Ali, M., Jessulat, M., Deineko, V., Miller, R., Lee, M., Park, H.O., Davidson, A., Babu, M. & Stagljar, I. A Comprehensive Membrane Interactome Mapping of Sho1p Reveals Fps1p as a Novel Key Player in the Regulation of the HOG Pathway in S. cerevisiae. Journal of molecular biology 427, 2088-2103 (2015).
114 Huang, X., Dai, F. F., Gaisano, G., Giglou, K., Han, J., Zhang, M., Kittanakom, S., Wong, V., Wei, L.,
Showalter, A.D., Sloop, K.W., Stagljar, I. & Wheeler, M. B. The identification of novel proteins that interact with the GLP-1 receptor and restrain its activity. Molecular Endocrinology 27, 1550-1563 (2013). 115 Kenworthy, A. K. Imaging protein-protein interactions using fluorescence resonance energy transfer
microscopy. Methods 24, 289-296 (2001).
116 Ma, L., Yang, F. & Zheng, J. Application of fluorescence resonance energy transfer in protein
studies. Journal of molecular structure 1077, 87-100 (2014).
117 Kaltashov, I. A., Bobst, C. E., Nguyen, S. N. & Wang, S. Emerging mass spectrometry-based
approaches to probe protein–receptor interactions: Focus on overcoming physiological barriers. Advanced drug delivery reviews 65, 1020-1030 (2013).
118 Daulat, A. M., Maurice, P., Froment, C., Guillaume, J. L., Broussard, C., Monsarrat, B., Delagrange, P.
& Jockers, R. Purification and identification of G protein-coupled receptor protein complexes under native conditions. Molecular & cellular proteomics 6, 835-844 (2007). 119 Zhang, M., Robitaille, M., Showalter, A. D., Huang, X., Liu, Y., Bhattacharjee, A., Willard, F.S., Han, J.,
Froese, S., Wei, L., Gaisano, H.Y., Angers, S., Sloop, K.W., Dai, F.F. & Wheeler, M. B. Progesterone receptor membrane component 1 is a functional part of the glucagon-like peptide-1 (GLP-1) receptor complex in pancreatic β cells. Molecular & cellular proteomics 13, 3049-3062 (2014). 120 Li, X. C., Carretero, O. A., Shao, Y. & Zhuo, J. L. Glucagon Receptor–Mediated Extracellular Signal–
Regulated Kinase 1/2 Phosphorylation in Rat Mesangial Cells Role of Protein Kinase A and Phospholipase C. Hypertension 47, 580-585 (2006). 121 Xu, Y. & Xie, X. Glucagon receptor mediates calcium signaling by coupling to Gαq/11 and Gαi/o in
HEK293 cells. Journal of receptors and signal transduction 29, 318-325 (2009).
122 Cho, Y. M., Merchant, C. E. & Kieffer, T. J. Targeting the glucagon receptor family for diabetes and
obesity therapy. Pharmacology & therapeutics 135, 247-278 (2012). 123 Salehi, A., Vieira, E. Gylfe, E. Paradoxical stimulation of glucagon secretion by high glucose
concentrations. Diabetes 55, 2318-2323 (2006).
49
124 Schwenk, J., Metz, M., Zolles, G., Turecek, R., Fritzius, T., Bildl, W., Tarusawa, E., Kulik, A., Unger, A.,
Ivankova, K., Seddik, R., Tiao, J.Y., Rajalu, M., Trojanova, J., Rohde, V., Gassmann, M., Schulte, U., Fakler B. & Bettler, B. Native GABAB receptors are heteromultimers with a family of auxiliary subunits. Nature 465, 231-235 (2010). 125 Sonoda, N., Imamura, T., Yoshizaki, T., Babendure, J. L., Lu, J. C. Olefsky, J. M. β-arrestin-1 mediates
glucagon-like peptide-1 signaling to insulin secretion in cultured pancreatic β cells. Proceedings of the national academy of sciences 105, 6614-6619 (2008). 126 Ahmed, S. M., Daulat, A. M. Angers, S. Tandem affinity purification and identification of heterotrimeric
g protein-associated proteins. In Signal transduction protocols, 357-370 (2011). 127 Chen, G. I. Gingras, A. C. Affinity-purification mass spectrometry (AP-MS) of serine/threonine
phosphatases. Methods 42, 298-305 (2007). 128 Anastas, J. N., Biechele, T. L., Robitaille, M., Muster, J., Allison, K. H., Angers, S. & Moon, R. T. A
protein complex of SCRIB, NOS1AP and VANGL1 regulates cell polarity and migration, and is associated with breast cancer progression. Oncogene 31, 3696-3708 (2012).
129 Dentin, R., Liu, Y., Koo, S. H., Hedrick, S., Vargas, T., Heredia, J., Yates, J. & Montminy, M. Insulin
modulates gluconeogenesis by inhibition of the coactivator TORC2. Nature 449, 366-369 (2007). 130 Dai, F. F., Zhang, Y., Kang, Y., Wang, Q., Gaisano, H. Y., Braunewell, K. H., Chan, C.B. & Wheeler, M.
B. The neuronal Ca2+ sensor protein visinin-like protein-1 is expressed in pancreatic islets and regulates insulin secretion. Journal of biological chemistry 281, 21942-21953 (2006). 131 Ke, J., Zhang, C., Harikumar, K. G., Zylstra-Diegel, C. R., Wang, L., Mowry, L. E., Miller, L.J., Williams,
B.O. & Xu, H. E. Modulation of β-catenin signaling by glucagon receptor activation. PloS one 7, e33676 (2012).
132 Yabaluri, N. & Bashyam, M. D. Hormonal regulation of gluconeogenic gene transcription in the
liver. Journal of biosciences 35, 473-484 (2010).
133 Zhang, Y., Li, M., Wei, L., Zhu, L., Hu, S., Wu, S. Ma, S. & Gao, Y. Differential protein expression in
perfusates from metastasized rat livers. Proteome science 11, 1-8 (2013).
134 Freeman, A. K. & Morrison, D. K. 14-3-3 Proteins: diverse functions in cell proliferation and cancer
progression. In Seminars in cell & developmental biology 22, 681-687 (2011).