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.
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.
Arshavsky VYu
Laboratory of Molecular Biology, University of Wisconsin, Madison 53706.
Gray-Keller
M P
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Bownds
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MD
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*
D006160:Guanosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D020558:GTP Phosphohydrolases; D015293:Transducin; D006152:Cyclic GMP; D002118:Calcium