Cloning, sequencing, and expression of a 24-kDa Ca super(2+)-binding protein activating photoreceptor guanylyl cylase

• Published in: The Journal of biological chemistry 1995-01, Vol.270 (42), p.25200-25206
• Main Authors: Dizhoor, A M, Olshevskaya, E V, Henzel, W J, Wong, S C, Stults, J T, Ankoudinova, I, Hurley, J B
• Format: Article
• Language: English
• ISSN: 0021-9258

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 super(2+)-sentive manner. The primary structure of bovine p24 has been derived peptide sequencing and from its cDNA. p24 is a new EF-hand-type Ca super(2+)-binding protein, related but not identical to another guanylyl cyclase-activating protein, GCAP and other members of the recoverin family of Ca super(2+)-binding proteins. Anti-bodies against a truncated fusion protein and against p24-specific synthetic peptide specifically recognize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase 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-polyaclamide gel electrophoresis as a homogenous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca super(2+) inhibits this activation with an EC sub(50) near 200 nM and a Hill coefficient 1.7. Recombinant p24 expressed in 293 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca super(2+) 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.