Specific GEFs and GAPs for heterotrimeric G proteins
Active heterotrimer dissociates into a G subunit and a G dimer
G protein-coupled receptors as GEFs for heterotrimeric G proteins GPCRs are the largest family of cell-surface receptors for extracellular signals (superfamily of >1000 members) GPCRs respond to a wide range of inputs: hormones, neurotransmitters, odorants, tastants, photons of light, etc. ~60% of all clinical therapeutics act by affecting some aspect of GPCR signaling (e.g., agonist, antagonist, inhibition of natural ligand metabolism)
The G subunit : Lipid modifications for membrane binding Exotoxin from Bordetella pertussis (whooping cough) catalyzes ADP-ribosylation of i-class G : Result? De-coupling from receptor Exotoxin from Vibrio cholerae (cholera diarrhea) catalyzes ADP-ribosylation of s-class G Result? Constitutive activity since crippled as a GTPase
G contains a Ras-like domain and an all-helical domain Three “switch regions” have conformations that depend on which nucleotide is bound
G contains a Ras-like domain and an all-helical domain
G t. GDP. AlF 4 Ras/p120 GAP. GDP. AlF 3 S1 S2 S3 S1 S2 p120 GAP Ras Helical domain Ras-like domain “Arginine finger” is critical for GTP hydrolysis. R174 R789 transducin Aluminum tetrafluoride ion mimics third phosphate of GTP and thereby “activates” GDP-bound G subunits.
Mg 2+ Q200 R174 AlF 4 S2 S1 H2OH2O Q61 R789 S1 S2 AlF 3 Mg 2+ H2OH2O GDP G t. GDP. AlF 4 Ras/p120 GAP. GDP. AlF 3 For Ras-like GTPases, arginine finger is supplied in trans by GAPs. For heterotrimeric G proteins, the catalytic arginine is part of the alpha subunit. “Arginine finger” is critical for GTP hydrolysis Site of cholera activation; R>C mutation activates. Q>L mutation also activates by crippling GTPase function.
Primary sequence characteristics of G dimers
G forms a propeller; G is an extended helical peptide. CAAX Sondek et al. Nature (1996) 379: 369
The switches of G are primary interaction sites with G (no interactions between G and G !) Lambright et al. Nature (1996) 379: 311.
The switches of G are primary interaction sites with G (the N-terminal helix of G is also used) Lambright et al. Nature (1996) 379: 311.
Portions (purple) of all three subunits contact receptor Area of ADP-ribosylation via pertussis toxin ( i-class) Swap receptor specificity by switching C-termini? Inhibit coupling via C-terminal minigenes? Gilchrist et al. (2001) JBC 276:25672
m retinal sugars rhodopsin GGGG GGGG S2 S1 S3 S-Prenyl (GG or F) N-Myristoyl, S-palmitoyl GDP Interface defined by structural features and biochemical evidence Regions of protein in purple are implicated in receptor interactions membrane
G switches (I, II, III) are sensitive to bound nucleotide
G switch regions (I, II) directly interact with GTP
The four families of G subunits Adenylyl cyclase (+) (-)(-) Phospholipase C-beta (+) Rho-GTP (+) Pertussis-toxin sensitive G subunits
Examples of G-protein effector systems G-protein subunitEffector“Second messenger” G s, G olf adenylyl cyclase [cAMP] G i1/i2/i3 adenylyl cyclase [cAMP] G q/11 phospholipase-C [IP 3 ] & [DAG] G 12/13 RGS-box RhoGEFs [RhoA-GTP] phospholipase-C [IP 3 ] & [DAG] G (transducin) cyclic GMP phosphodiesterase [cGMP] G dimer or adenylyl cyclase or [cAMP] PLC- PLC- [IP 3 ] & [DAG] or ion channel flux K+ and Ca2+
Blake et al. (2001) JBC 276:49267 Plenty o’ G subunits
Gs = “stimulatory” G-protein linked to adenylyl cyclase activation (2nd-messenger generation) s GTP AC Adenylyl cyclase 2-adrenergic receptor on vasculature of skeletal muscles Adrenaline or Isoproterenol (“1st messenger”) + ATPcAMP Second messenger Net result: Five-fold increase in [cAMP] i in seconds
S3 S2 S1 GsGs IIC2 VC1 forskolin GTP S Forskolin favors dimerization of cyclase domains G s interacts with cyclase primarily through switches 1 and 2 Complex of G s with cytoplasmic portions of Adenylyl cyclase Tesmer (1997) Science 278:1907
Turning off the signal Multiple levels & multiple time-frames Reuptake/destruction of agonist (~millisec) Hydrolysis of GTP bound to G subunit (~sec) Reuptake/destruction of second messenger (~sec) Uncouple receptor from signal machinery (~sec/min) Remove receptor from cell-surface (~min/hr)
Adapted from Hollinger & Hepler (2002). The R7 family
RGS proteins stabilize the transition state for GTP hydrolysis