Two receptor classes

• Receptor tyrosine kinases (RTKs)– Ligand induced dimerization– Autophosphorylation– Substrate phosphorylation– Adapter proteins

• G-Protein coupled receptors (GPCRs)– Ligand induced activation– Guanine exchange factor (GEF)– Second messenger cascade

Hierarchical overviewSignaling molecule

G-Protein coupled receptor Receptor tyrosine kinase

Effector kinase cascades

Gene expression Phenotypic behaviorProtein activity

Second messengersNucleotide

cyclases

Phospholipases

Kinases

eg: Insulin

• Generated by pancreas

• Acts on muscle & other tissues– Multiple mechanisms– Multiplicative mechanisms

IR

IRS-1 Shc

PI3-K GRB2

Raf

MEKMAPKGene

transcriptionElk-1

AS160Glut4

translocation

AktmTORProtein

synthesis

Rab 8/14

eg: Prostaglandin E2

• Locally generated

• Inflammatory mediator

• Labor

EPR

Gq Gi

PLC AC

Raf

MEKMAPKGene

transcriptionElk-1

PKCL-type Ca2+

Potentiation

Ca2+CaMIn/DecreasedContractility

Receptor tyrosine kinases

• Single pass transmembrane protein

• Ligand induces dimerization– Kinase activity– Autophosphorylation

• Complex formationFGF FGF

Cytoplasmic

FGF receptor binding

Phosphotyrosine binding

• Phospho-Tyrosine Binding (PTB)

• Src homology (SH2, SH3) domain– Common amino acid motif

– Phosphotyrosine binding pocket

– Phosphorylation dependent association

Interaction with both pY and nearby residues

xnnletyewy nksisrdkae kllldtgkeg afmvrdsrtp gtytvsvftk aiisenpcik

hyhiketnds pkryyvaeky vfdsiplliq yhqynggglv trlrypvcg

Sh2 domain from Itk PDB:2etz

Phosphotyrosine binding

• Recruit biologically active molecules– Phospholipases, PI3-K– GTPase modulators (Sos, DOCK180)– Adapter proteins (Grb-2, Shc, Nck, Crk)

• Increase effective availability of substrate– Membrane phospholipids– Other pY-bound proteins

• Increase biological activity– Phosphorylation dependent activation– pY-binding dependent activation

eg: FGFR phosphorylation

FGF

Y463

Y583

Y653Y730

Y766

Crk

PLCShc

GRB2

IP3+DAGCa2+, motility

DNA Synthesiscdc related kinase

MAPKgrowth

Sos

Modulation & Termination

• Modulation– Receptor antagonists– Combinatorial control

• Termination– Protein Tyrosine Phosphatases (PTPs)– Internalization– Ubiquitinylation

EGFR signaling network

Lemmon & Schlessinger 2010

G-Protein Coupled Receptor

• GPCR are 7 pass transmembrane proteins– Rhodopsin/-adrenergic– Secretin/vasointestinal peptide– Metabotropic glutamate

• G Guanine exchange factor (GEF)– Heterotrimeric G-Protein– G - G binding

• Can function monomeric

• Also dimerize

GPCR

• Receptor ligation catalyzes GDP-GTP exchange on G

• GTP bound G dissociates from G• G modulates secondary signaling

• G may also modulate secondary signalingUnligated Receptor

G-GDP

G

Bound Receptor

G-GTP

G

G- mediated signaling

• Acylated, membrane bound G and target

• G allosterically regulated by GTP

• Target allosterically regulated by G• Membrane association decreases diffusion distance

Gs Adenylate cyclase

GTP

Substrate ATP

G Protein Classes

G Protein Agonist Effector

Gs Ubiquitous Adenylyl cyclase (AC)

Gt Photons cGMP phosphodiesterase

Gq ACh, epinepherine

PLC

Gi Ubiquitous Ca2+ channels, AC (-)

Gz Dopamine, adenosine

AC(-), K+ channel (-)

G K+ Channel, PLA2, AC, PLC

Metabotropic neurotransmitter receptors are all GPCRs

General Scheme

• Agonist binding triggers nucleotide exchange

• G subunits dissociate

• Ga binds effectors• GTP hydrolysis

restores inactive state

• Effector may be a GAP

Receptor regulation, negative feedback

• Activity depends on association of intracellular loops

• Rapid desensitization – G-protein coupled Receptor Kinases (GRK)– PKA, PKC

• Internalization– Arrestin– Clathrin/caveolae

• Long-term desensitization– Downregulation– G-Protein deactivation

GPCR Kinase (GRK)

• Receptor inactivation

–

– Gbg-binding

• Distributed signaling

Ligand Receptor

Ga

GRK B-arrestin

Clathrin-mediated internalization

Penela & al 2007

G-Protein regulation

• In vivo signaling much faster than reconstituted systems

• Regulator of G-protein Signaling (RGS)– Ga GAP– Esp Gi, Gq– PLCRhoGEF are RGS

• Kinetics– Ga: minutes– Ga-RGS: tenths of second

eg: Synaptic remodeling

• Rearrangement of neural networks

• Synaptic reinforcement– Long term potentiation

• Remodeling of dendritic spines– Calcium dependent cell motility

Stimulation of cultured neuron with NMDA results in rapid development of a new dendritic spineGoldin, et al., 2001

Glutamate - Ga12/13

• Metabotropic glutamate receptor 1 is Ga12 coupled GPCR

• Ga12 is a Rho-GEF

• RhoA small GTPases– RhoA, Rac1 and Cdc42– Subcellular transport– Cytoskeletal remodeling

• Actin filament growth(mDia)

• Stress fiber anchorage (ROCK)ROCK

mDiaKinectin

eg: Competitive control of AC (heart)Sympathetic NS(fight or flight)

Parasympathetic NS(rest or relax)

Wettschureck & Offermanns, 2005Huang & al., 2011

Increaseddepolarization cAMP increases

Ca2+ influx

Reduced Ca2+ effluxIncreased Ca2+ influx

Increased force

Isoproterenol-induced contractile desensitization ~20 min

-adrenergic heart failure

• Persistent -stimulation induces heart failure– Reduced 1 receptor– Exaggerated BARK/GRK2

• GRK2 knockout reduces mortality

Cardiac, inducible GRK2 ko before or after MI improves long-term survivalRaake & al 2008