FUNCTIONAL RECONSTITUTION OF PHOTORECEPTOR GUANYLATE-CYCLASE WITH NATIVE AND MUTANT FORMS OF GUANYLATE CYCLASE-ACTIVATING PROTEIN-1

Citation
A. Ottobruc et al., FUNCTIONAL RECONSTITUTION OF PHOTORECEPTOR GUANYLATE-CYCLASE WITH NATIVE AND MUTANT FORMS OF GUANYLATE CYCLASE-ACTIVATING PROTEIN-1, Biochemistry, 36(14), 1997, pp. 4295-4302
Citations number
36
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biology
Journal title
ISSN journal
0006-2960
Volume
36
Issue
14
Year of publication
1997
Pages
4295 - 4302
Database
ISI
SICI code
0006-2960(1997)36:14<4295:FROPGW>2.0.ZU;2-H
Abstract
In rod and cone photoreceptor cells, activation of particulate guanyla te 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 pho toreceptor 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, th e EF-1 motif, and the EF-3 motif, were likely involved in the interact ion with retGC1. Mutation of (2)Gly to Ala (GCAP1-G2A), which abolishe d myristoylation and a 25 amino acid truncation at the N-terminus (Del ta 25-GCAP1) reduced retGC1-stimulating activity dramatically, while d eletion of 10 amino acids (Delta 10-GCAP1) reduced the specific activi ty by only similar to 60% and modified the Ca2+ sensitivity. At 10(-6) M [Ca2+](free), in conditions that inactivated native GCAP1, retGC1 s howed significant activity in the presence of Delta 10-GCAP1. Native a nd all three mutant forms of GCAP1 had similar affinities for Ca2+ as demonstrated by gel filtration and the changes in tryptophan fluoresce nce. All mutants bound to ROS membranes in a Ca2+-independent manner, except Delta 25-GCAP1, which was mostly soluble. These findings sugges t that the N-terminal region is important in tethering of GCAP1 to the ROS membranes.