Identification of a domain in guanylyl cyclase-activating protein 1 that interacts with a complex of guanylyl cyclase and tubulin in photoreceptors.
The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.
Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Postfach 1913, D-52425 Jülich, Germany.
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D008969:Molecular Sequence Data; D019151:Peptide Library; D010449:Peptide Mapping; D020419:Photoreceptor Cells, Vertebrate / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D014404:Tubulin / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D019151:Peptide Library; D014404:Tubulin; C098768:guanylyl cyclase-activating protein, bovine; D006162:Guanylate Cyclase