Outline

• GPCRs
• RTKs

GPCRs

• Amazingly this class of receptors all have 7 TM domain topology
• They are a very large family
• Bind diverse ligands: hormones, neurotransmitters, oderants, "light"
• Regulate diverse effectors (all through trimeric G switch proteins)
• Classic example is beta adrenergic receptor regulation of adenylate cyclase
• Ligand is epinephrin or norepi (catechol based hormone)
• Acts on multiple tissues for flight response in stress
• Binds liver & fat tissues to mobilize glucose & fatty acids etc.
• Heart to increase contraction-> blood supply to tissues
• Smooth muscle of intestine-> relax
• Arteries-> constrict to prevent flow to peripheral organs
• Acts in seconds, short lived
• Many agonists & antagonists known
• Beta blockers: antagonists that bind specifically beta1 receptor
• Slow heart contractions
• Beta-adrenergic receptor is 7 TM domain protein
• Trimeric G:
• Gs binds & hydrolyzes GTP
• beta/gamma act in a regulatory fashion inhib when bound
• both alpha and gamma are acylated to promote membrane binding
• Adenylate Cyclase
• Bilaterally symetric 12 TMD protein
• Each half has a catalytic site in a cytoplasmic domain
• Thought to act together in ATP->cAMP + PPi
• Key is series of conformational changes that lead to rapid cAMP increase by Ad Cyc
  And that GTPase activity acts as timer to control time of activation
• Ligand binds receptor in core of TM domains involving H7 (see 20-13)
• TMDs rotate which causes rotation of cytoplasmic C3 loop
• Cyto loop now can interact with Gs-GDP bound to beta/gamma
• This interaction opens binding pocket allowing release of GDP & binding of GTP
• GTP binding causes conf change in switch domains at interface w/ beta/gamma releasing beta/gamma
• This same conformational change generates an Ad cyclase binding site
• The site involves switch II and the a3-b5 loop
• This interaction near one catalytic site may cause rotation in the other
• The rotated enzyme stabilizes the transition state in cAMP production
• GTP is hydrolyzed, destroying Ad cyclase site & restoring beta/gamma site
• cAMP is destroyed by phosphodiesterase (cAMP->5’AMP)
• The net result is:
• Every occupied receptor complex activates about 100 G proteins
• Each G activates a single Ad Cyclase
• Each Ad Cyclase generates hundreds of cAMP
• GPCRs also negatively regulate Ad Cyclase
• Occupied receptor activates Gi
• Gi-GTP binds Ad cyclase in slightly different region masking Gs binding site
• Bacterial toxins modify G proteins
• Cholera toxin irreversibly ribosylates Gs
• this binds GTP & activates Ad Cy, but no GTP hydrolysis=> locked active
• Pertussis toxin ribosylates Gi, this prevents GDP release=> locked inactive
• These effects lead to massive trans epithelial H2O loss-> death by dehydration

RTKs

• Ligands
• Peptides: eg NGF, PDGF, FGF, EGF, insulin
• Leads to changes in cell physiol or gene expression
• These are involved in cell proliferation, differentiation, cell survival, metabolic regulation
• Mutations give cancers, developmental defects etc.
• Receptors (EGFR etc.)
• Extracellular ligand binding domain, single TMD, cytoplasmic catalytic domain
• In most, ligand binding induces dimerization-> activates the kinase domain->autophosphoryl
• Some are already dimerized so conformational changes induced by binding activate kinase
• Autophos occurs in two stages
• Phosp of tyr at lip of kinase domain-> increases ATP or substrate binding
• Phosph of other sites-> generates binding sites for SH2, and PTB adaptors
• Other substrates are also phosph. by activated RTK
• Receptor activation (EGFR) leads to Ras activation via adaptor & GEF
• Adaptor (GRB2) contains SH2 domain that binds P-tyr in receptor in context of adjacent residues
• Src SH2 binds Ptyr-glu-glu-ile (fig 20-26); different SH2s bind diff surround seqs.
• GRB2 using SH3 domains simultaneously binds pro-rich domain in ras GEF (SOS)
• This brings SOS from cytosol to membrane
• Note binding pockets (Fig 20-26)
• Also note that specificity comes from adjacent non-Pro seq. =>certain residues define structural motif for binding, spec. from surround seq.
• Binding displaces an inhibitory domain in SOS, allowing it to interact w/ ras
• SOS (RasGEF) binding to Ras opens binding pocket allowing diffusion of GDP out
• And diffusion of GTP (10X more conc than GDP) in
• SOS interacts with 2 switch domains in effect prying open ras GTP binding site
• Activated Ras (RasGTP) binds Raf kinase N-terminus recruiting it to membrane and
• the binding drives a conformational change in Raf that prevents its binding to 14-3-3 dimer
• each subunit in 14-3-3 dimer binds a phospho serine in Raf (259,621)
• thus 14-3-3 disociation exposes these to a phosphatase
• dephosph of 259 activates Raf kinase
• Activated Raf phosphorylates & thus activates MEK
• MEK is a dual specificity kinase (serine & tyrosine)
• Activated MEK phosphorylates and thus activates MAP kinase
• MAPK is inactive because the phosph. lip excludes ATP access to the binding pocket
• MEK binding destabilizes lip exposing a buried tyrosine
• phosph of the exposed tyr and a nearby thr cause the lip to shift its conformation
• this allows ATP binding, MAPK dimerization (NLS exposed) & substrate inteactions
• Ksr forms signaling complex by binding raf, 14-3-3, MEK and MAP
• Ksr also has kinase activity that may be used to help turn off signal
• RasGap also localized to site by SH2 domain interaction w/ RTKs
• This allows turn off of the ras signal.