Involvement of Illuminated Rhodopsin in Adenine Nucleotide–Enhancement of GCAP–Stimulated retGC Activity

Abstract

Abstract: : Purpose: In the current model, retinal guanylyl cyclase (retGC) is activated by guanylyl cyclase–activating proteins (GCAPs) in a Ca2+–sensitive manner (one–step activation). We have recently suggested that a conformational change of retGC by ATP–binding to its kinase–homology domain greatly enhances retGC activity stimulated by GCAPs (two–step activation) (Yamazaki et al, JBC, 278, 33150, 2003). Here we investigate involvement of illuminated rhodopsin as a signal for the ATP binding and the resulting enhancement of retGC. Methods: Homogenates of bovine retinal photoreceptor outer segments (OS), pretreated in various ways, were incubated on ice with or without adenylyl imidodiphosphate (AMP–PNP). After removing free AMP–PNP, retGC activity was measured with GCAP2. Results: Illumination in the presence of AMP–PNP clearly enhanced GCAP2–stimulated retGC activity, with the light treatment yielding an AMP–PNP effect that was 50% greater than that in membranes incubated with AMP–PNP in the dark. No enhancement of GCAP–stimulated retGC activities was seen with illuminated incubations lacking AMP–PNP. To test the involvement of illuminated rhodopsin, we used NH2OH as an agent to quench active rhodopsin. Treatment of illuminated OS homogenates with NH2OH prior to the AMP–PNP incubation reduced the AMP–PNP effect by 70%. Without AMP–PNP, the treatment only marginally affected retGC activity stimulated with S100B or GCAP2. GTPgS did not further enhance the AMP–PNP effect, suggesting that transducin is not involved in the retGC activation. The observation that the AMP–PNP preincubation effect is evident after various treatments implies that the putative adenine nucleotide–induced conformational state is stable if these nucleotids are hydrolysis–resistant. Conclusions: These results suggest that illumination of rhodopsin is a trigger for ATP binding to retGC and the resulting conformational change allows the large–scale GCAP stimulation. Thus, retGC and cGMP phosphodiesterase may be activated in a parallel and perhaps proportional manner by rhodopsin although the expressions of their activities are separated in time since retGC is ultimately activated by GCAPs when [Ca2+] is reduced.

Keywords: signal transduction • protein structure/function • photoreceptors