Illuminated rhodopsin is required for strong activation of retinal guanylate cyclase by guanylate cyclase-activating proteins.

We have recently shown that activation of retinal guanylate cyclase (retGC) by GC-activating proteins (GCAPs) is much stronger than that previously reported and that preincubation of photoreceptor outer segment homogenates with ATP or its analogue, adenylyl imidodiphosphate (AMP-PNP), is required for the strong activation [Yamazaki, A., Yu, H., Yamazaki, M., Honkawa, H., Matsuura, I., Usukura, J., and Yamazaki, R. K. (2003) J. Biol. Chem. 278, 33150-33160]. Here we show that illuminated rhodopsin is essential for development of the AMP-PNP incubation effect. This was demonstrated by illumination of dark homogenates and treatments of illuminated homogenates with 11-cis-retinal and hydroxylamine prior to the AMP-PNP incubation and by measurement of the GCAP2 concentration required for 50% activation. We also found that the AMP-PNP incubation effect was not altered by addition of guanosine 5'-O-(3-thiotriphosphate), indicating that transducin activation is not required. It is concluded that illuminated rhodopsin is involved in retGC activation in two ways: to initiate the ATP incubation effect for preparation of retGC activation as shown here and to reduce the Ca2+ concentrations through cGMP phosphodiesterase activation as already known. These two signal pathways may be activated in a parallel and perhaps proportional manner and finally converge for strong activation of retGC by Ca2+-free GCAPs.