Chapter 20

Cell - Cell Signaling:Hormones and Receptors

Three general types ofextracellular signaling

• endocrine signaling

• paracrine signaling

• autocrine signaling

Fig 20-1a

Endocrine Signaling - signaling molecules (hormones) act ontarget cells distant from their site of synthesis

Are affected if they express theappropriate hormone receptor

Paracrine Signaling - signaling molecules released by a cellonly affect target cells in close proximity.

Fig 20-1b

Conduction of signals from one nerve cell to another acrossa synapse is a special example of paracrine signaling.

Fig. 21-4a

Fig 20-1c

Autocrine Signaling - cells respond to substances thatthey themselves release.

Fig 20-1d

Proteins attached to the plasma membrane of one cell can interactdirectly with receptors on an adjacent cell.

Receptors Exhibit Two Types of Specificity

(1) Ligand Specificity-which ligand(s) are bound. This issimilar to the specificity of an enzyme for its substrate.(2) Effector Specificity-the response to ligand binding

•Same ligand binding to a receptor can have differenteffects in different cell types� same ligand specificty, different effector specificity

Different ligands binding to different receptors on thesame cell can have the same effect� different ligand specificity, same effector specificity

Hormones May Be Classified by Receptor Location

•Intracellular Receptors – lipophilic molecules (hydrophobic)•Steroids•Thyroxine•Retinoids

•Cell Surface Receptors -...may be hydrophilic

•Peptide Hormones - insulin, glucagon, growth factors•Small Charged Molecules: e.g. epinephrine, histamine

or lipophilic•Prostaglandins

Fig 20-2a

Intracellular Receptors located in the cytosol-signaling molecules must be membrane permeable (lipophilic)-they are carried in the blood bound to carrier proteins

CH3

CH3C O

CH3

O

Progesterone, see page 851

Other examples include Thyroxine and Retinoic Acid

Fig 20-2b

Cell Surface Receptors-receptor must have mechanism totransmit signal across membrane-involves “Second Messengers”

Fig 20-4

Examples of second messengers

Property Steroids Thyroxine Peptides and Catecholamines Proteins

Feedback regulation Yes Yes Yes Yesof synthesis

Storage of preformed Very little Several weeks One day Several days, inHormone adrenal medulla

Mechanism of Diffusion through Proteolysis of Exocytosis of Exocytosis ofSecretion plasma membrae thyroglobulin storage vesicles storage vesicles

Binding to plasma Yes Yes Rarely NoProteins

Lifetimein blood Hours Days Minutes SecondsPlasma

Time course of action Hours to days Days Minutes to hours Seconds or less

Receptors Cytosolic or nuclear Nuclear Plasma membrane Plasma membrane

Mechanism of action Receptor-hormone complex controls Hormone binding Hormone binding transcription and stability of mRNA’s triggers synthesis causes change in of cytosolic second membrane potential messengers of triggers synthesis protein kinase of cytosolic second activity messengers

For transport in blood

Source: Adapted from E.L. Smith et al., 1983, Principles of Biochemistry: Mammalian Biochemistry, 6th ed., McGraw-Hill, p 358

TABLE 20-1 Characteristic Properties of Principal Types of Mammalian Hormones

Cell-surface receptors can becategorized into four major classes

• G protein-linked receptors• Ion-channel receptors• Receptors lacking intrinsic catalytic activity

– tyrosine kinase linked receptor

• Receptors with intrinsic enzymatic activity– receptor serine/threonine kinases– receptor tyrosine kinases (RTKs)

Fig 20-3a

G protein-linked receptors: ligand binding activates aG protein that in turn activates or inhibits an enzyme thatgenerates a specific second messenger.

Fig 20-3b

Ion-channel receptors: ligand binding changes the conformationof the receptor so that specific ions flow through it.

Ligand-Gated Channel

Fig 20-3c

Tyrosine kinase-linked receptors (Receptors lacking intrinisiccatalytic activity): ligand binding causes receptor monomers to dimerize;the dimeric receptor then activates a cytosolic protein tyrosine kinase.

Fig 20-3d

Receptors with intrinsic catalytic activity-binding of ligand activates Catalysis; Receptor is also an enzyme

How are signals involving protein phosphorylation turned off?

Secondmessenger

Receptor Tyrosine Kinase

R H RH+

Text Page 858-860

“Basics” of binding interactions

KD =[R][H][RH]

Total Concentrationof Receptors:

Fraction of receptorswith bound hormone:

Text Page 858-860

This equation is analogous to the Michaelis-Menton Equation thatdescribes enzyme activity. KD is similar to a Michaelis Constant, KM

In general, the KDvalue of a cell-surfacehormone receptorapproximates the bloodlevel of its ligand.

KD =[R][H][RH][RT ] = [R]+ [RH]

[RH][RT ]

=1

1+ KD [H]

Cells with surface receptor

Hormone radioactively labeled H*[0.1] [1] [10] [100] [1000]

H* H* H* H*

Isolate cells and remove excess unbound hormone

Measure radioactivity (amount of H* bound)

Fig 20-7

Amount of H* added

Total Concentration of Receptors

1/2 of total Receptors

K D

Amount of H* Bound to cell

Receptor

KD[R] [H][RH]

=

RH R + HKD

affinity - how tightly does the receptor bind hormone (KD)specificity - ability to distinquish a specific interaction from anonspecific interaction.

The KD values for two different receptor /hormone systems are indicated in the table below.

20 nM5,000 nMHistamine Receptor

200 nM1 nMEpinephrine Receptor

HistamineEpinephrine

Which exhibits the…greatest binding Affinity?

greatest binding Specificity?

Greatest Affinity 1 nM

Greatest Specificity

5,000 nM/20 nM > 200 nM/1 nM

20 nM

Agonist

Antagonists

Required for bindingRequired for

Activity

Hormone

Isoproterenol 0.4 x 10-6M

Alprenolol 0.0034 x 10-6M

Propranolol 0.0046 x 10-6M

Practolol 21 x 10-6M

Table 20-2

KD for Binding to the ReceptorOn Frog Erythrocytes

Highest Affinity

Epinephrine 5 x 10-6M Structure Compound

Structure of Typical Agonists and Antagonists of the B-Adrenergic Receptor

Fig 20-8

Maximum physiological response can be achievedbefore the receptor is saturated with Ligand.

Fig. 20-11: Comparison of the abilities of 3 catecholamines to activateadenylyl cyclase and to bind to cell-surface β-adrenergic receptors

IP EP EPIPNEP NEP

Fig 20-3a

G protein - heterotrimeric guanine nucleotide-binding proteins thatusually are linked to a seven-membrane spanning receptor on the cellsurface.

Fig20-10

General structure of a G protein-linked Receptor:

G-Proteins are Heterotrimers consisting of α, β, and γ subunits:♦ββ and γ subunits act as one unit and bind to α-subunits ♦αα subunits act on the effector molecule and are of two types

•Gsα stimulates the effector when GTP is bound•Giα inhibits the effector when GTP is bound

Gsα or Giαγ β

GDP GTP

β-Adrenergic Receptors•Liver & Adipose Cells==> liberation of Glucose & FA’s•Heart Cells ==> Increased rate of contraction•Intestinal Smooth Muscle ==> Relax

α-Adrenergic Receptors•Blood vessels of intestinal tract, skin, kidneys

==> Arteries constrict limiting blood flow

The net effect is:•Increased blood flow to skeletal muscle and•More fuel for the muscles, Glucose and FA’s (Fatty Acids)

Fig. 20-13

Fig. 20-14

α Agonists Inhibits (binds Gi)

β Agonists Activates (binds Gs)

β Agonists Activates (binds Gs)

α Agonists Activates (binds Gs)

β Agonists Inhibits (binds Gi)

Helix number1 2 3 4 5 6 7

β2−Adrenergic receptor

Chimeric receptor 1

Chimeric receptor 2

Chimeric receptor 3

Region bindingto G protein(comparechimeras 1 and 3)

Region bindingto agonists (comparechimeras 1 and 2)Conclusion

α2−Adrenergic receptor

β1 & β2 Adrenergic Receptors are Coupled to Gs ==> Activates Adenylyl Cyclase

α2 Adrenergic Receptor are Coupled to Gi

==> Inhibits Adenylyl Cyclase

Fig 20-16

GDP

αβγ

Binding of hormoneProduces conformationalchange in receptor

Receptor binds to Gsα protein

Binding to receptor induces aconformational change in GsαGDP bound to Gsα is replacedby GTP and the subunitdissociates from Gβγ

Gsα

Gsα

Gsα

Fig 20-16

GDP

αβγ

GTP

α

β1 & β2 Adrenergic Receptorsare Coupled to Gs

==> Activates Adenylyl Cyclase

Binding to receptor induces aconformational change in GsαGDP bound to Gsα isreplaced by GTP and thesubunit dissociates from Gβγ

Gsα binds to adenylyl cyclase, activatingsynthesis of cAMP; hormone dissociates

Hydrolysis of GTP to GDP causes Gsα todissociate from adenylyl cyclase and bind to Gβγ

Gsα

Gsα

Gsα

Gsα

Fig 20-18

β1/β2 α2

Fig. 15-24: Molecular Biology of the Cell 3rd EdAlberts et al. Garland Pub.

Fig. 15-12: Voet, Voet, & PrattRegulation of Glycogen Phosphorylase bycAMP-Dependent Protein Kinase

Glycogenphosphorylase

Fig. 20-34b: Degradation of glycogen.

Fig 20-17: Effect of cholera toxin on cycling ofGsα between the active and inactive forms

Fig. 20-38a: Several second messengers arederived from phosphatidylinositol (PI)

α1 Adrenergic Receptor are Coupled to Gq

==> Stimulates Phospholipase C (PLC) producing

Diacylglycerol (DAG) and Inositol Trisphosphate IP3

InositolPhosphatidylinositol (PI) PI 4-phosphate (PIP) PI 4,5-bisphosphate (PIP2)

Cytosol

Inositol 1,4,5-trisphosphate (IP3)

Figure 20-39: Elevation of cytosolic Ca++ via the inositol-lipidsignaling pathway.

Stimulated by Gq

Fig 20-37: Intracellular transduction of an extracellular signal via acascade of sequential reactions produces a large amplification of the signal.

Fig 20-6

Changes in… •Enzyme activities•Gene expresssion•Cytoskeletal structures

AutophosphorylationFig 20-21

Receptor Tyrosine Kinases: ligand binding causes receptormonomers to dimerize; the dimeric receptor autophosphorylates.

Like Gα, Ras alternates between anactive state with bound GTPand an inactive state with boundGDP. Both are molecular switches.Unlike Gα, cycling of Ras requirestwo proteins: and

Fig 20-22

Ras is a monomeric guanine nucleotide bindingprotein that interacts with RTK’s.

GEF GAP

Guanine Exchange Factor

GTPase Activating Protein

GEF - guanine nucleotide-exchange factor - binds to Ras-GDPcomplex, causing dissociation of the bound GDP. GTP bindsspontaneously to “empty” Ras.

Fig 20-22

is usuallydelivered to Ras byan activated receptor.

Lifetime of Ras-GTP ~ 1 min.

GEF

Fig 20-22

- activates GTPase activityof Ras; functions as an inhibitor by promoting formationof the inactive Ras-GDP complex

GAP- GTPase activating protein

Fig 20-23

Binding of hormonecauses dimerization andautophosphorylation oftyrosine residues

Receptormonomer

Inactive Ras

Dimeric receptor

EGF

GDP

Fig 20-23

Sos is a GEF

Binding of GRB2 andSos couples receptorto inactive Ras

Dimeric receptor

Sos promotes dissociationof GDP from Ras; GTPbinds and Sos dissociatesfrom active Ras

Signaling

ActiveRas

Sos SH3

Sos SH3

GRB2

GRB2

GDP

GTP

Receptor Tyrosine Kinases (RTK’s) and Ras

Ligands - soluble or membrane bound peptide/protein hormones•NGF - Nerve Growth Factor•PDGF - Platelet Derived Growth Factor•FGF - Fibroblast Growth Factor•EGF - Epidermal Growth Factor•Insulin

Ligand Binding stimulates Tyrosine Protein Kinase ActivityEffects include...

•Regulation of Cell Proliferation and Differentiation•Promotion of Cell Survival•Modulation of Cellular Metabolism

Mutant RTK’s stimulate cell growth in absence of hormone=> Cancer

GTPase superfamily

Group of Guanine Nucleotide-BindingProteins that cycle between...– Inactive State with bound GDP– Active state with bound GTP

Examples:•Gα proteins•Ras proteins•Rab proteins -regulation vesicle fusion•Rho proteins - regulate actin cytoskeleton