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J Biol Chem, Vol. 274, Issue 10, 6244-6249, March 5, 1999

Identification of a Domain in Guanylyl Cyclase-activating Protein 1 That Interacts with a Complex of Guanylyl Cyclase and Tubulin in Photoreceptors

Alexander Schrem, Christian Lange, Michael Beyermann^¶, and Karl-Wilhelm Koch

From the  Institut für Biologische Informationsverarbeitung, Forschungszentrum Jülich, Postfach 1913, D-52425 Jülich, Germany and the ^¶ Forschungsinstitut für Molekulare Pharmakologie, Alfred-Kowalke-Strasse 4, D-10315 Berlin, Germany

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.

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