CLONING, SEQUENCING, AND EXPRESSION OF A 24-KDA CA2-BINDING PROTEIN ACTIVATING PHOTORECEPTOR GUANYLYL CYCLASE()

Two vertebrate photoreceptor-specific membrane guanylyl cyclases, RetG C-1 and RetGC-2, are activated by a soluble 24-kDa retinal protein, p2 4, in a Ca2+-sensitive manner (Dizhoor, A. M., Lowe, D. G., Olshevskay a, E. V., Laura, R. P., and Hurley, J. B. (1994) Neuron 12, 1345-1352; Lowe, D. G., Dizhoor, A. M., Liu, K., Gu, O., Laura, R., Lu, L., and Hurley, J. B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5535-5539). T he primary structure of bovine p24 has been derived from peptide seque ncing and from its cDNA. p24 is a new EF-hand-type Ca2+ binding protei n, related but not identical to another guanylyl cyclase-activating pr otein, GCAP (Palczewski, K., Subbaraya, I., Gorczyca, W. A., Helekar, B. S., Ruiz, C. C., Ohguro, H. Huang, J., Zhao, X., Crabb, J. W., John son, R. S., Walsh, K. A., Gray-Keller, M. P., Detwiler, P. B., and Bae hr, W. (1994) Neuron 13, 395-404) and other members of the recoverin f amily of Ca2+-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically reco gnize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase by purified p24. p24 is found only in retina, and it copurifies with outer segment membranes. Immun ocytochemical analysis shows that it is present in rod photoreceptor c ells. An immobilized antibody column was used to purify p24 from a hea t-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogenous protein not contaminated with GCAP , and it activates photoreceptor guanylyl cyclase in vitro at submicro molar concentrations. Ca2+ inhibits this activation with an EC(50) nea r 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 2 93 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca2+ sensitivity to photoreceptor membrane guanylyl cyclase. We propose that p24 be referr ed to as GCAP-2 and that GCAP be referred to as GCAP-1.