Activation of RGS9-1GTPase acceleration by its membrane anchor, R9AP.
The GTPase-accelerating protein (GAP) complex RGS9-1.G beta(5) plays an important role in the kinetics of light responses by accelerating the GTP hydrolysis of G alpha(t) in vertebrate photoreceptors. Much, but not all, of this complex is tethered to disk membranes by the transmembrane protein R9AP. To determine the effect of the R9AP membrane complex on GAP activity, we purified recombinant R9AP and reconstituted it into lipid vesicles along with the photon receptor rhodopsin. Full-length RGS9-1.G beta(5) bound to R9AP-containing vesicles with high affinity (K(d) < 10 nm), but constructs lacking the DEP (dishevelled/EGL-10/pleckstrin) domain bound with much lower affinity, and binding of those lacking the entire N-terminal domain (i.e. the dishevelled/EGL-10/pleckstrin domain plus intervening domain) was not detectable. Formation of the membrane-bound complex with R9AP increased RGS9-1 GAP activity by a factor of 4. Vesicle titrations revealed that on the time scale of phototransduction, the entire reaction sequence from GTP uptake to GAP-catalyzed hydrolysis is a membrane-delimited process, and exchange of G alpha(t) between membrane surfaces is much slower than hydrolysis. Because in rod cells different pools exist of RGS9-1.G beta(5) that are either associated with R9AP or not, regulation of the association between R9AP and RGS9-1.G beta(5) represents a potential mechanism for the regulation of recovery kinetics.
Hu
Guang
G
Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Zhang
Zhixian
Z
Wensel
Theodore G
TG
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D050356:Lipid Metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D017785:Photons; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D015293:Transducin / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D008565:Membrane Proteins; D020710:RGS Proteins; C464657:RGS9BP protein, human; D011993:Recombinant Fusion Proteins; C116925:regulator of g-protein signaling 9; D020558:GTP Phosphohydrolases; D015293:Transducin