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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
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Hierarchical overviewSignaling molecule
G-Protein coupled receptor Receptor tyrosine kinase
Effector kinase cascades
Gene expression Phenotypic behaviorProtein activity
Second messengersNucleotide
cyclases
Phospholipases
Kinases
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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
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eg: Prostaglandin E2
• Locally generated
• Inflammatory mediator
• Labor
EPR
Gq Gi
PLC AC
Raf
MEKMAPKGene
transcriptionElk-1
PKCL-type Ca2+
Potentiation
Ca2+CaMIn/DecreasedContractility
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Receptor tyrosine kinases
• Single pass transmembrane protein
• Ligand induces dimerization– Kinase activity– Autophosphorylation
• Complex formationFGF FGF
Cytoplasmic
FGF receptor binding
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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
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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
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eg: FGFR phosphorylation
FGF
Y463
Y583
Y653Y730
Y766
Crk
PLCShc
GRB2
IP3+DAGCa2+, motility
DNA Synthesiscdc related kinase
MAPKgrowth
Sos
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Modulation & Termination
• Modulation– Receptor antagonists– Combinatorial control
• Termination– Protein Tyrosine Phosphatases (PTPs)– Internalization– Ubiquitinylation
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EGFR signaling network
Lemmon & Schlessinger 2010
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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
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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
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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
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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
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General Scheme
• Agonist binding triggers nucleotide exchange
• G subunits dissociate
• Ga binds effectors• GTP hydrolysis
restores inactive state
• Effector may be a GAP
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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
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GPCR Kinase (GRK)
• Receptor inactivation
–
– Gbg-binding
• Distributed signaling
Ligand Receptor
Ga
GRK B-arrestin
Clathrin-mediated internalization
Penela & al 2007
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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
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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
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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
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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
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-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