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 overview Signaling molecule G-Protein coupled receptor Receptor tyrosine kinase Nucleotide cyclases Phospholipases Kinases Second messengers Effector kinase cascades Gene expression Protein activity Phenotypic behavior

eg: Insulin Generated by pancreas Acts on muscle & other tissues Multiple mechanisms Multiplicative mechanisms IR IRS-1 Shc Glut4 translocation Rab 8/14 AS160 PI3-K GRB2 Protein synthesis mTOR Akt Raf MEK Gene transcription Elk-1 MAPK

eg: Prostaglandin E2 Locally generated Inflammatory mediator Labor EPR Gq Gi L-type Ca2+ Potentiation PKC PLC AC In/Decreased Contractility CaM Ca2+ Raf MEK Gene transcription Elk-1 MAPK

Receptor tyrosine kinases Single pass transmembrane protein Ligand induces dimerization Kinase activity Autophosphorylation Complex formation FGF receptor binding FGF FGF Cytoplasmic

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 Sh2 domain from Itk PDB:2etz xnnletyewy nksisrdkae kllldtgkeg afmvrdsrtp gtytvsvftk aiisenpcik hyhiketnds pkryyvaeky vfdsiplliq yhqynggglv trlrypvcg

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 DNA Synthesis cdc related kinase Crk Y463 Y583 Shc Y653 PLC Y730 GRB2 Y766 IP3+DAG Ca2+, motility Sos MAPK growth

Modulation & Termination 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/b-adrenergic Secretin/vasointestinal peptide Metabotropic glutamate Ga Guanine exchange factor (GEF) Heterotrimeric G-Protein Ga - Gbg binding Can function monomeric Also dimerize

GPCR Receptor ligation catalyzes GDP-GTP exchange on Ga GTP bound Ga dissociates from Gbg Ga modulates secondary signaling Gbg may also modulate secondary signaling Unligated Receptor Bound Receptor Ga-GDP Ga-GTP Gbg Gbg

G-a mediated signaling Acylated, membrane bound Ga and target Ga allosterically regulated by GTP Target allosterically regulated by Ga Membrane association decreases diffusion distance Gas 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 Ca2+ channels, AC (-) Gz Dopamine, adenosine AC(-), K+ channel (-) Gb 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

Clathrin-mediated internalization GPCR Kinase (GRK) Receptor inactivation Gbg-binding Distributed signaling Ga Ligand Receptor 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 PLCb, RhoGEF 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 spine Goldin, 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) Kinectin ROCK mDia

eg: Competitive control of AC (heart) Sympathetic NS (fight or flight) Parasympathetic NS (rest or relax) Increased depolarization cAMP increases Ca2+ influx Increased force Reduced Ca2+ efflux Increased Ca2+ influx Isoproterenol-induced contractile desensitization ~20 min Wettschureck & Offermanns, 2005 Huang & al., 2011

b-adrenergic heart failure Persistent b-stimulation induces heart failure Reduced b1 receptor Exaggerated BARK/GRK2 GRK2 knockout reduces mortality Cardiac, inducible GRK2 ko before or after MI improves long-term survival Raake & al 2008