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Functional reconstitution of photoreceptor guanylate cyclase with native and mutant forms of guanylate cyclase-activating protein 1.
- Source :
-
Biochemistry [Biochemistry] 1997 Apr 08; Vol. 36 (14), pp. 4295-302. - Publication Year :
- 1997
-
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.
- Subjects :
- Adenosine Triphosphate pharmacology
Amino Acid Sequence
Animals
Calcium pharmacology
Calcium-Binding Proteins genetics
Calcium-Binding Proteins metabolism
Calcium-Binding Proteins pharmacology
Cattle
Cell Membrane metabolism
Electrophoresis, Polyacrylamide Gel
Enzyme Activation drug effects
Gene Expression
Guanylate Cyclase-Activating Proteins
Molecular Sequence Data
Mutagenesis, Site-Directed
Peptide Fragments chemistry
Peptide Fragments pharmacology
Protein Binding
Recombinant Proteins chemistry
Recombinant Proteins metabolism
Retina enzymology
Rod Cell Outer Segment enzymology
Spectrometry, Fluorescence
Calcium metabolism
Calcium-Binding Proteins chemistry
Guanylate Cyclase metabolism
Retinal Cone Photoreceptor Cells enzymology
Retinal Rod Photoreceptor Cells enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 36
- Issue :
- 14
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 9100025
- Full Text :
- https://doi.org/10.1021/bi963000d