Cloning, Sequencing, and Expression of a 24-kDa Ca-binding Protein Activating Photoreceptor Guanylyl Cyclase

doi:10.1074/jbc.270.42.25200

Cloning, Sequencing, and Expression of a 24-kDa Ca-binding Protein Activating Photoreceptor Guanylyl Cyclase (*)

From the (1)Department of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195 and
(2) Genentech, Inc., Point San Bruno, South San Francisco, California 94080

^§ To whom correspondence should be addressed:
University of Washington, School of Medicine, P. O. Box 357370, Seattle, WA 98195.

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-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-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 recoverin family of Ca-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 homogenous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca inhibits this activation with an EC 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 Ca 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.

Footnotes

↵* This work was supported by grants from Human Frontier Science Program Organization (to A. M. D. and J. B. H.) and National Institutes of Health Grant EYO6641 (to J. B. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U32856[GenBank].
↵¹ The abbreviations used are:

OS

outer segment

GC

guanylyl cyclase

MOPS

4-morpholinepropanesulfonic acid

PCR

polymerase chain reaction

GCAP

guanylyl cyclase-activating protein

PAGE

polyacrylamide gel electrophoresis

HPLC

high pressure liquid chromatography.
↵²K. Palczewski, personal communication.
- Received July 18, 1995.
- Revision received August 3, 1995.