Guanylyl Cyclase Activating Protein

doi:10.1074/jbc.270.37.22029

Guanylyl Cyclase Activating Protein

A CALCIUM-SENSITIVE REGULATOR OF PHOTOTRANSDUCTION (*)

From the (1) Departments of Ophthalmology and
(2) Pharmacology, University of Washington, Seattle, Washington 98195, the
(3) R. S. Dow Neurological Sciences Institute, Good Samaritan Hospital, Portland, Oregon 97209, and the
(4) Department of Ophthalmology, Baylor College of Medicine, Houston, Texas 77030

^¶Recipient of a Jules and Doris Stein Research to Prevent Blindness Professorship. To whom correspondence should be addressed:
Dept. of Ophthalmology, University of Washington, Box 356485, Seattle, WA 98195-6485.
Tel.: 206-543-9074; Fax: 206-543-4414.

↵^§ Present address: Moran Eye Center, University of Utah Health Sciences Center, Salt Lake City, UT 84132.

Abstract

Guanylyl cyclase activating protein (GCAP1) has been proposed to act as a calcium-dependent regulator of retinal photoreceptor guanylyl cyclase (GC) activity. Using immunocytochemical and biochemical methods, we show here that GCAP1 is present in rod and cone photoreceptor outer segments where phototransduction occurs. Recombinant and native GCAP1 activate recombinant human retGC (outer segment-specific GC) and endogenous GC(s) in rod outer segment (ROS) membranes at low calcium. In addition, we isolate and clone a retinal homolog, termed GCAP2, that shows 50% identity with GCAP1. Like GCAP1, GCAP2 activates photoreceptor GC in a calcium-dependent manner. Both GCAP1 and GCAP2 presumably act on GCs by a similar mechanism; however, GCAP1 specifically localizes to photoreceptor outer segments, while in these experiments GCAP2 was isolated from extracts of retina but not ROS. These results demonstrate that GCAP1 is an activator of ROS GC, while the finding of a second activator, GCAP2, suggests that a similar mechanism of GC regulation may be present in outer segments, other subcellular compartments of the photoreceptor, or other cell types.

Footnotes

↵* This work was supported in part by United States Public Health Service Grants EY08061 and EY01730 (to K. P.), EY07089 (to A. S. P.), and EY08123 (to W. B.) and an award from Research to Prevent Blindness, Inc. to the Departments of Ophthalmology at the University of Washington and at Baylor College of Medicine. 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) L43001[GenBank].
↵¹ The abbreviations used are;

ROS

rod outer segment

BTP

1,3-bis[tris(hydroxymethyl)methylamino]propane

HPLC

high performance liquid chromatography

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

pAb

polyclonal antibody

mAb

monoclonal antibody

GTPS

guanosine 5′-O-(thiotriphosphate).
↵²S. Semple-Rowland and W. Baehr, unpublished results.
↵³Polyclonal antibody pAb UW-14 and monoclonal antibody mAb G-2 were generated against truncated, bacterially expressed GCAP1 (Met-Pro); polyclonal antibodies pAb GS-35 and pAb GS-31 were raised against N-terminal myristoylated Gly-Glu and C-terminal Asp-Asp-Lys peptides encompassing portions of the GCAP1 sequence, respectively. pAbs were affinity-purified, while mAb G-2 was purified from ascites fluid by ammonium sulfate precipitation and ion exchange chromatography.
↵⁴Similarly, pAb UW-14 specifically bound GCAP1, but not GCAP2, further establishing the lack of cross-reactivity of this antibody.
- Received May 9, 1995.
- Revision received July 19, 1995.