Functional reconstitution of photoreceptor guanylate cyclase with native and mutant forms of guanylate cyclase-activating protein 1.
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.
Otto-Bruc
A
A
Department of Ophthalmology, School of Medicine, University of Washington, Seattle 98195, USA.
Buczylko
J
J
Surgucheva
I
I
Subbaraya
I
I
Rudnicka-Nawrot
M
M
Crabb
J W
JW
Arendt
A
A
Hargrave
P A
PA
Baehr
W
W
Palczewski
K
K
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D015870:Gene Expression; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012160:Retina / Q000201:enzymology; D017949:Retinal Cone Photoreceptor Cells / Q000201:enzymology*; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase; D002118:Calcium