1 7.61 Eukaryotic Cell 7.61 Eukaryotic Cell Biology: Principles and Biology: Principles and Practice Practice 2006 2006 Lecture 1 Lecture 1 Receptors Receptors Detect and process extracellular physiologic and pathophysiologic molecules Structure of Ligand determines structure of receptor and Mechanism of Action: Hydrophobic vs. Hydrophilic Hydrophobic: Nuclear Hormone Receptors Cytoplasmic/Nuclear Localization Complex with hsp90 and others to hold binding site open for the ligand Progesterone Receptor: Intracellular Pathway
Transcript
1
Growth Hormone & its Receptor (Growth Hormone & its Receptor (extracellular extracellular domains)domains)
7.61 Eukaryotic Cell7.61 Eukaryotic CellBiology: Principles andBiology: Principles and
PracticePractice20062006
Lecture 1Lecture 1ReceptorsReceptors
Detect and process extracellular physiologicand pathophysiologic molecules
Structure of Ligand determines structure ofreceptor and Mechanism of Action: Hydrophobic
Complex with hsp90 and others to hold bindingsite open for the ligand
Progesterone Receptor: Intracellular Pathway
2
Olefsky JM.J Biol Chem. 2001 Oct 5;276(40):36863-4
Nuclear Hormone Receptors:Four Mechanisms used by the Estrogen Receptor
Julie M. Hall, John F. Couse, and Kenneth S. Korach J. Biol. Chem., Vol. 276, Issue 40, 36869-36872, 2001
The retinoic acid receptor, in the absence of a hormone, is a repressor and it collects a series ofrepressor proteins like a magnet. Addition of the ligand reverses the polarity of the magnet,causing the repressors to fly off and the coactivators to bind, resulting in chromatin modification toactivate transcription. This is how the hormonal or physiological signals affect the recruitment of co-factors that modify the chromatin in target genes. Every cell has receptors and therefore canrespond, but each cell responds in its own unique way, and thus the same hormone can have adifferent effect in a neuron versus an epithelial skin cell versus a bone cell. Evans RM. 2003. PPARs andthe complex journey to obesity.Keio J Med. 2004 Jun;53(2):53-8.
48 HUMANNR GENES
3
Hydrophilic Ligand/Receptor SystemPlasma Membrane is Permeability Barrier
WHAT ARE THE CHARACTERISTICPROPERTIES OF A LIGAND/RECEPTOR INTERACTION?
How do you define a receptor, how do you find a receptor,how do you study receptors?
MEASURE BINDING
Receptor-preparation:Intact Cells:
Cultured cells, tissue slices, perfused organs, whole organism(37°C or 4°C)
Ligand-preparation:Detection
Labeling (direct or indirect)
Avoid artifacts of labeling(e.g. denaturation, crosslinking)
4
How to Label Ligands
Protein Ligands
Protein Labeling:
RadioisotopesFluorescent dyes
Epitope tagsOthers (biotin)
125I-Protein[3H]Protein
[35S]methionine
BINDING Assays
Variables:Concentration, Time, Temperature
Binding Characteristics:High Affinity
SaturableSpecific
201510500
No Additions
AcLDL
Specific
Control
A. Saturation Kinetics
I-AcLDL (µg protein/ml)125
0
100
200
300
400
Rece
ptor
Act
ivity
“Hot + Cold”
5
Look out for bad data and beware of people who only show delta!
Alternative to Chemical Modification of Ligand
Immunoreceptor vs Radioreceptor Assays
For immunorreceptor assay, nonspecific iscalculated by regression analysis
100%
time after binding
Reversible Bindingto Receptor
Off rates are criticalmust not loose signal during washing
Binding vs Cell Association:Inhibit uptake at 4°C
6
Quantitative Analysis:How many binding sites? How tight is the binding?
Rigorous analysis depends on system being in equilibrium
BINDING Assays
R + L RL
R = unbound receptorsL = unbound ligandRL = ligand/receptor complex
Rt = total # of receptors (binding sites) = R + RL
Classic Scatchard Analysis
Bound/Free
Bound (RL)
slope = -1/K
intercept = R
d
t
[RL]bound
free
Kdiss
[RL]
[L]
[R ]
Kd
tK
d
[RL]=
Rt = R + RL
free
bound=
=[R][L]
Simple Straight Line:Single Class of Binding
Sites
R = unbound receptorsL = unbound ligandRL = ligand/receptor complex
Rt = total # of receptors (binding sites) = R + RL
R + L RL
Complications!Error propagation
Bound
Bound/Free
0 2 4 6 8 100
2
4
6
7
Complications!Cannot always trust your eyes!
Direct Plot:
ScatchardPlot:
-ive coop. +ive coop.
B/F
BBound (RL)
Bound/Free
fewer high affinity
more abundent lower affinity
Non-linear Scatchard Plots:Multiple Classes of Binding Sites
8
.
0 10 20 0 10 20 300
2
4
B (nM) B (nM)
0
2
4B
ou
nd
/Fre
eB
ou
nd
/Fre
e
A. Correct Nonlinear Fit B. Correct Graphical Fit
C. Incorrect graphical fit D. Incorrect graphical fit
a
b
2
4
Boun
d/Fr
ee
00
Old FashionGraphicalAnalysis -
Not Good forQuantitativeDeterminations
2
4
Boun
d/Fr
ee
Correct Nonlinear Fit
0 10 20 30B (nM)
.
0 10 20 0 10 20 300
2
4
B (nM) B (nM)
0
2
4
Bo
un
d/F
ree
Bo
un
d/F
ree
A. Correct Nonlinear Fit B. Correct Graphical Fit
C. Incorrect graphical fit D. Incorrect graphical fit
Purified BINDING ACTIVITY, but how do you know that you'veactually purified the biologically relevant receptor?
Direct:2) Express cloned gene in cells or tissues and measurebio activity3) Inhibit activity in appropriate system (blockingantibody, siRNA, KO animal)
12
STRUCTURE OF THE ß-ADRENERGIC RECEPTOR
Out
In
Heptahelical (7-Transmembrane) domain receptor
7-TM or G-Protein Coupled Receptors (GPCR)
large family (>2000) - ~5% of worm genome codingsequences, perhaps 3% of mammals
α2-AR, ß1 and ß2-AR, dopamine receptors, muscarinicacetyl choline receptors (4 subclasses), sight(Rhodopsin), taste, smell, angiotensin receptor (masoncogene), serotonin receptor, a- and α- factor yeastreceptors, LH receptor, PAR (protease activated receptor)
many intronless genes!
all operate using similar signal transduction systems (G-proteins)
Major Drug Targets for Pharmaceutical Industry…
13
Family of structurally and functionally similar proteinsprovides special opportunity to explore structure/function
method: insert cDNA into expression vector, transfect into Lcells because they are ßAR -ive, look for binding and correctpharmacologic specificity, affinity, etc.-G-protein and cyclaserequired to work!
For example:Delete N- or C-terminus -> binding okDelete 5-6 loop (C3=i3) -> binding ok Conclusion -> not needed for binding
For example:Co-express SR(1-5, aa1-262) +SR(6-7, aa263-end) -> still works! Conclusion -> self assemble
Delete 6-7 loop (E4) or helices 6&7 -> No binding! Conclusion -> ???????????
What can you do?
Similar structures permittesting of chimeras ofGPCRs for structuraldeterminants of ligandspecificity and downstreamtarget (G-protein) specificity
α2-AR -> decrease inadenylate cyclase activityß2-AR -> increase in adenylatecyclase
different ligand specificities
Major determinant of specificbinding in 7th membranespanning domain
14
B3-AR from outside of cell bindingto norepinepherine
Combined Use of Chemistry (Pharmacology - alter structureof the ligand) and Molecular Biology (mutagenesis- modify
putative ligand binding sites) to define Ligand/ReceptorInteractions - ‘complementary mutations’
Side chain interactions within the transmembrane region of the !2-adrenergic receptor with agonist isoproterenol (A) and antagonistsalprenolol (B), as derived from mutagenesis and/or modelling studies.For clarity of pictorial representation the interaction of Phe-289 is notshown.
Agonist:Isoproterenol
Antagonist:Alprenolol
Scheme 3. Schematicdrawing of theinteraction of ATP withthe P2Y1 receptor asderived frommutagenesis andmodeling studies. Forreasons of clarity theinteraction withArg310(TM7) is notshown.
15
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE,Yamamoto M, Miyano M. Crystal structure of rhodopsin: A G protein-coupled receptor. Science. 2000 Aug 4;289(5480):739-45. Henry R. Bourne and Elaine C. Meng Rhodopsin Sees the Light Science 2000 289: 733-734
Figure 2. Ribbon drawing of rhodopsin (A) viewed parallel to the plane of the membrane and (B)seen from the extracellular (intradiscal) side of the membrane revealing a counterclockwisearrangement of the 7-TM helices.[8] The 11-cis-retinal molecule is shown in red. The figureshave been prepared using the programs Molscript[102] and Raster3d.[103]
11-cis-retinal
Figure 4. Model ofRS-102895 boundto the MCP-1receptor. RS-102895 is thespace-fillingmolecule in thecenter of the bundleof helices, indicatedby the ribbons.[33]Receptor residueGlu291 is shown inthe ball-and-stickpresentation; it isshown interactingwith the basicnitrogen atom of thespiropiperidinestructure. Pleasenote that in contrastto Figure 2 B theview from theintracellular side isdisplayed
Figure 3. Three-ligand binding sites ofthe 5-HT1A receptor in a rhodopsin-based 3D model according to Jacobyand co-workers.[24] Left: extracellularview; right: side view with extracellularside at the top. The three ligands areserotonin or 5-HT (yellow),propranolol (cyan), and 8-OH-DPAT(green). Residues identified bymutagenesis data are indicated. The 5-HT site is located between TM3 (TM III;with Asp116 as the key recognitionsite) and TM5 (TM V; providing Ser199and Thr200 to interact with the 5-OHgroup of serotonin). A second site, thepropranolol binding site, is locatedbetween TM3 and TM7 (contributing,for example, Asn386 to hydrogen bondthe oxygen atoms of the oxy-propanolamine fragment in beta -blockers). The third binding site, the 8-OH-DPAT binding site is also locatedbetween TM3 and TM7. Ligandsaddressing this site, like 8-OH-DPAT,are thought to be oriented parallel tothe helices (interactions by 8-OH toSer393 and Asn396 and by aminogroup to Asp116).
16
Desensitization: tendency of biological responses to wane overtime despite continuous presence of stimulus of constant intensity.How- inactivate, destroy, or sequestration
Homologous desensitization : ß-adrenergic receptor kinase(ßARK) phosphorylates receptor and lowers activity somewhat,after phosphorylation, ß-arrestin binds and really drops activity.NB at high ligand occupancy
Heterologous desensitization: general reduction on stimulation ofother receptors, protein kinase A (PKA) plays a role here, PKA isabout 6x slower than ßARK at phosphorylating the receptor also,ßARK mediated desensitization is t1/2<15 sec while PKA is 3.5min (>14 fold) see Roth et al, PNAS 88:6201-6204 (91)), arrestinindependent.
beta-Arrestin, a promiscuous mediator of receptorendocytosis. (A) Binding of ligand to its GPCR activatesheterotrimeric G proteins, causing dissociation of thealpha subunit from the beta/gamma dimer (not shown).The betagamma dimer, anchored to the inner surface ofthe plasma membrane by the prenylated gammasubunit, facilitates translocation of GRK to the plasmamembrane. This enables GRK to phosphorylate theserine/threonine motif in the GPCR carboxyl terminus,resulting in beta-arrestin translocation to the GPCR.beta-Arrestin binds to adaptor proteins in clathrin-coated pits, resulting in endocytosis of the GPCR, whichis then either recycled or degraded with concomitantactivation of G protein-independent signaling pathways.(B) On binding to its ligand, the Fz4 receptor inducestranslocation of Dvl2 to the plasma membrane.Phosphorylation of Dvl2 by protein kinase C (PKC) leadsto binding of beta-arrestin 2. Binding of Wnt5A to thecysteine-rich domain (CRD) in the extracellular aminoterminus of Fz4 leads to endocytosis of the Fz4complex. (C) Transforming growth factor-beta type IIreceptor (TbetaR-II) binds to, and its intracellular kinasedomain phosphorylates, the intracellular carboxylterminus of TbetaR-III. This leads to translocation ofbeta-arrestin 2 to the plasma membrane, followed byendocytosis of the TbetaR-II/TbetaR-III complex. Thisprocess does not require binding of TGF-beta to itsreceptor, hence the TbetaR-II extracellular binding site(triangle) is depicted as empty.
