Functional Reconstitution of Photoreceptor Guanylate Cyclase with Native and Mutant Forms of Guanylate Cyclase-Activating Protein 1

Abstract

In rod and cone photoreceptor cells, activation of particulate guanylate cyclase (retGC1) is mediated by a Ca2+-binding protein termed GCAP1, that detects changes in [Ca2+]free. In this study, we show that N-acylated GCAP1 restored Ca2+ sensitivity of native and recombinant photoreceptor retGC1. ATP increased the affinity of retGC1 for GCAP1 and accelerated catalysis. Using peptides derived from the GCAP1 sequence, we found that at least three regions, encompassing the N-terminus, the EF-1 motif, and the EF-3 motif, were likely involved in the interaction with retGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation at the N-terminus (delta25-GCAP1) reduced retGC1-stimulating activity dramatically, while deletion of 10 amino acids (delta10-GCAP1) reduced the specific activity by only approximately 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+]free, in conditions that inactivated native GCAP1, retGC1 showed significant activity in the presence of delta10-GCAP1. Native and all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluorescence. All mutants bound to ROS membranes in a Ca2+-independent manner, except delta25-GCAP1, which was mostly soluble. These findings suggest that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.

Guanylyl Cyclase-activating Proteins (GCAPs) Are Ca2+/Mg2+ Sensors

“…Bovine GCAP-1 exhibits Ca 2ϩ -sensitive changes in the intrinsic tryptophan fluorescence as a result of cation binding to its EF-hands (23)(24)(25). The fluorescence of myristoylated bovine GCAP-1 is at its maximum in the absence of Ca 2ϩ and Mg 2ϩ , and the changes in fluorescence as a function of free Ca 2ϩ ([Ca] f ) are biphasic (Fig.…”

Calcium-sensitive Regions of GCAP1 as Observed by Chemical Modifications, Fluorescence, and EPR Spectroscopies

“…Washed ROS membranes were prepared as described previously (36), reconstituted with recombinant GCAPs, and assayed as described by Otto-Bruc et al (37). [Ca 2ϩ ] was calculated using the computer program Chelator 1.00 (38), and all assays were repeated at least twice.…”

Mapping Sites in Guanylyl Cyclase Activating Protein-1 Required for Regulation of Photoreceptor Membrane Guanylyl Cyclases

“…The first is between residues Gly 2 and Glu 28 . The second one is contiguous with the first; it runs from Glu 28 to Glu 57 (9), and the third one is the EF-hand 3 motif (17).…”