title: cGMP suppresses GTPase activity of a portion of transducin equimolar to phosphodiesterase in frog rod outer segments. Light-induced cGMP decreases as a putative feedback mechanism of the photoresponse.
abstract: In rod photoreceptor cells, the light response is triggered by an enzymatic cascade that causes cGMP levels to fall: excited rhodopsin (Rho*)----rod G-protein (transducin, Gt)----cGMP-phosphodiesterase (PDE). This results in the closure of plasma membrane channels that are gated by cGMP. PDE activation by Gt occurs when GDP bound to the alpha-subunit of Gt (Gt alpha) is exchanged with free GTP. The interaction of Gt alpha-GTP with the gamma-subunits of PDE releases their inhibitory action and causes cGMP hydrolysis. Inactivation is thought to be caused by subsequent hydrolysis of Gt alpha-GTP by an intrinsic Gt-GTPase activity. Here we report that there are two portions of Gt in frog rod outer segments (ROS) expressing different rates of GTP hydrolysis: 19.5 +/- 3 mmol of Gt/mol of Rho, equivalent to that amount which participates in PDE activation, hydrolyzing GTP at a rate of approximately 0.6 turnover/s ("fast") and the remaining Gt (80.5 +/- 3 mmol/mol Rho) hydrolyzing GTP at a rate of 0.058 +/- 0.009 turnover/s. Fast GTPase activity is abolished in the presence of cGMP. This effect occurs over the physiological range of cGMP concentration changes in ROS, half-saturating at approximately 2 microM and saturating at 5 microM cGMP. cGMP-dependent suppression of GTPase is specific for cGMP; cAMP in millimolar concentration does not affect GTPase, while the poorly hydrolyzable cGMP analogue, 8-bromo-cGMP, mimics the effect. GTPase regulation by cGMP is not affected by Ca2+ over the concentration range 5-500 nM, which spans the physiological changes in cytoplasmic Ca2+ in rod cells. We suggest that the fast cGMP-sensitive GTPase activity is a property of the Gt that activates PDE. In this model, cGMP serves not only as a messenger of excitation but also modulates GTPase activity, thereby mediating negative feedback regulation of the pathway via PDE turnoff: a light-dependent decrease in cGMP accelerates the hydrolysis of GTP bound to Gt, resulting in the rapid inactivation of PDE.
authors.affiliation: Laboratory of Molecular Biology, University of Wisconsin, Madison 53706.
authors.lastname: Arshavsky VYu
authors.forename: M P
authors.initials: MP
authors.lastname: Gray-Keller
authors.forename: M D
authors.initials: MD
authors.lastname: Bownds
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D006152:Cyclic GMP / Q000502:physiology* / Q000528:radiation effects; D005246:Feedback; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008027:Light; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010786:Photoreceptor Cells / Q000378:metabolism; D011892:Rana catesbeiana; D011898:Ranidae; D012374:Rod Cell Outer Segment / Q000201:enzymology* / Q000528:radiation effects; D015290:Second Messenger Systems; D015293:Transducin / Q000378:metabolism*
chemical_list: D006160:Guanosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D020558:GTP Phosphohydrolases; D015293:Transducin; D006152:Cyclic GMP; D002118:Calcium