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Abstract
Two vertebrate photoreceptor-specific membrane guanylyl cyclases, RetGC-1 and RetGC-2, are activated by a soluble 24-kDa retinal protein, p24, in a Ca(2+)-sensitive manner (Dizhoor, A.M., Lowe, D.G., Olshevskaya, 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). The primary structure of bovine p24 has been derived from peptide sequencing and from its cDNA. p24 is a new EF-hand-type Ca(2+)-binding protein, related but not identical to another guanylyl cyclase-activating protein, GCAP (Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H. Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray-Keller, M.P., Detwiler, P.B., and Baehr, W. (1994) Neuron 13, 395-404) and other members of the recovering family of Ca(2+)-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically recognize 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. Immunocytochemical analysis shows that it is present in rod photoreceptor cells. An immobilized antibody column was used to purify p24 from a heat-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogeneous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca2+ inhibits this activation with an EC50 near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293 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 referred to as GCAP-2 and that GCAP be referred to as GCAP-1.
Highlights
Light triggers hydrolysis of cyclic GMP and closure of cGMPgated cation channels in photoreceptor outer segment plasma membranes (OS)1 (reviewed in Stryer (1991), Lagnado and
Two membrane GCs are present in photoreceptor cells, RetGC-1 and RetGC-2 (Shyjan et al, 1992; Lowe et al, 1995)
We propose to refer to GCAP as GCAP-1 and to p24 as GCAP-2
Summary
Light triggers hydrolysis of cyclic GMP and closure of cGMPgated cation channels in photoreceptor outer segment plasma membranes (OS) (reviewed in Stryer (1991), Lagnado and. The decrease in free Ca2ϩ concentration allows a soluble activator protein to stimulate a membrane guanylyl cyclase (GC) (Lolley and Racz, 1982; Koch and Stryer, 1988). When cloned and expressed in HEK293 cells, both cyclases can be stimulated by a soluble protein purified from retina (Dizhoor et al, 1994; Lowe et al, 1995). This stimulation occurs only at free Ca2ϩ below 200 nM. A 21-kDa Ca2ϩ-binding protein referred to as GCAP (Gorczyca et al, 1994; Palczewski et al, 1994) stimulates GC activity in rod outer segment membranes. We propose to refer to GCAP as GCAP-1 and to p24 as GCAP-2 (guanylyl cyclase-activating protein-2)
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