Mapping Sites in Guanylyl Cyclase Activating Protein-1 Required for Regulation of Photoreceptor Membrane Guanylyl Cyclases

Guanylyl cyclase activating protein (GCAP)-1 regulates photoreceptor membrane guanylyl cyclase, RetGC, in a Ca 2+ -sensitive manner. It contains four Ca 2+ -binding motifs, EF-hands, three of which are capable of binding Ca 2+ . GCAP-1 activates RetGC in low Ca 2+ and inhibits it in high Ca 2+ . In this study we used deletion and substitution analysis to identify regions of GCAP-1 sequence that are specifically required for inhibition and activation. A COOH-terminal sequence within Met 157 to Arg 182 is required for activation but not for inhibition of RetGC. We localized one essential stretch to 5 residues from Arg 178 to Arg 182 . Another sequence essential for activation is within the N-terminal residues Trp 21 to Thr 27 . The region between EF-hands 1 and 3 of GCAP-1 also contains elements needed for activation of RetGC. Finally, we found that inhibition of RetGC requires the first 9 amino-terminal residues of GCAP-1, but none of the residues from Gln 33 to the COOH-terminal Gly 205 are specifically required for inhibition. The ability of GCAP-1 mutants to regulate RetGC was tested on total guanylyl cyclase activity present in rod outer segments. In addition, the key mutants were also shown to produce similar effects on recombinant bovine outer segment cyclases GC1 and GC2.