Transcript Receptor mechanisms - Georgia Institute of Technology

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
Nucleotide
cyclases
Receptor tyrosine kinase
Second messengers
Kinases
Phospholipases
Gene expression
Effector kinase cascades
Protein activity
Phenotypic behavior
eg: Insulin
• Generated by pancreas
• Acts on muscle & other tissues
– Multiple mechanisms
– Multiplicative mechanisms
IR
IRS-1
Shc
Glut4
translocation
PKC
PI3-K
GRB2
Protein
synthesis
mTOR
Akt
Raf
Gene
transcription
Elk-1
MAPK
MEK
eg: Prostaglandin E2
• Locally generated
• Inflammatory mediator
• Labor
EPR
Gq
Gi
L-type Ca2+
Potentiation
PKC
PLC
AC
In/Decreased
Contractility
CaM
Ca2+
Raf
Gene
transcription
Elk-1
MAPK
MEK
Receptor tyrosine kinases
• Single pass transmembrane protein
FGF receptor binding
• Ligand induces dimerization
– Kinase activity
– Autophosphorylation
• Complex formation
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
FGF
DNA Synthesis
cdc related kinase
Crk Y463
Y583
Shc Y653
GRB2
Sos
MAPK
growth
Y730
Y766
PLC
IP3+DAG
Ca2+, motility
Modulation & Termination
• Modulation
– Receptor antagonists
– Combinatorial control
• Termination
– Protein Tyrosine Phosphatases (PTPs)
– Internalization
– Ubiquitinylation
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
Ga-GDP
Gbg
Bound Receptor
Ga-GTP
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
Gs
Gt
Gq
Gi
Gz
Gb
Agonist
Ubiquitous
Photons
ACh,
epinepherine
Ubiquitous
Dopamine,
adenosine
Effector
Adenylyl cyclase (AC)
cGMP phosphodiesterase
PLC
Ca2+ channels, AC (-)
AC(-), K+ channel (-)
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
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)
ROCK
Kinectin
mDia
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
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