Functional Reconstitution of Photoreceptor Guanylate Cyclase with Native and Mutant Forms of Guanylate Cyclase-Activating Protein 1
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- 作者：Annie Otto-Bruc ; Janina Buczy
//pubs.acs.org/images/entities/lstrok.gif" border="0">ko ; Irina Surgucheva ; Iswari Subbaraya ; Maria Rudnicka-Nawrot ; John W. Crabb ; Anatol Arendt ; Paul A. H
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：April 8, 1997
- 年：1997
- 卷：36
- 期：14
- 页码：4295 - 4302
- 全文大小：210K
- 年卷期：v.36,no.14(April 8, 1997)
- ISSN：1520-4995
文摘
In rod and cone photoreceptor cells, activation of particulateguanylate cyclase (retGC1) ismediated by a Ca2+-binding protein termed GCAP1, thatdetects changes in [Ca2+]free. In thisstudy, weshow that N-acylated GCAP1 restored Ca2+ sensitivity ofnative and recombinant photoreceptor retGC1.ATP increased the affinity of retGC1 for GCAP1 and acceleratedcatalysis. Using peptides derived fromthe GCAP1 sequence, we found that at least three regions, encompassingthe N-terminus, the EF-1 motif,and the EF-3 motif, were likely involved in the interaction withretGC1. Mutation of 2Gly to Ala (GCAP1-G2A), which abolished myristoylation and a 25 amino acid truncation atthe N-terminus (
25-GCAP1)reduced retGC1-stimulating activity dramatically, while deletion of 10amino acids (10-GCAP1) reducedthe specific activity by only ~60% and modified theCa2+ sensitivity. At 10-6 M[Ca2+]free, in conditionsthat inactivated native GCAP1, retGC1 showed significant activity inthe presence of 10-GCAP1. Nativeand all three mutant forms of GCAP1 had similar affinities forCa2+ as demonstrated by gel filtration andthe changes in tryptophan fluorescence. All mutants bound to ROSmembranes in a Ca2+-independentmanner, except 25-GCAP1, which was mostly soluble. Thesefindings suggest that the N-terminal regionis important in tethering of GCAP1 to the ROS membranes.
25-GCAP1)reduced retGC1-stimulating activity dramatically, while deletion of 10amino acids (10-GCAP1) reducedthe specific activity by only ~60% and modified theCa2+ sensitivity. At 10-6 M[Ca2+]free, in conditionsthat inactivated native GCAP1, retGC1 showed significant activity inthe presence of 10-GCAP1. Nativeand all three mutant forms of GCAP1 had similar affinities forCa2+ as demonstrated by gel filtration andthe changes in tryptophan fluorescence. All mutants bound to ROSmembranes in a Ca2+-independentmanner, except 25-GCAP1, which was mostly soluble. Thesefindings suggest that the N-terminal regionis important in tethering of GCAP1 to the ROS membranes.