Changes in Biological Activity and Folding of Guanylate Cyclase-Activating Protein 1 as a Function of Calcium †

Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca 2+ -binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca 2+ -binding proteins from the calmodulin superfamily, the Ca 2+ -free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca 2+ -dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca 2+ -sensitive modulation of GC1 activity. The change from a Ca 2+ -bound to a Ca 2+ -free form of GCAP1 increased susceptibility of Ca 2+ -free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca 2+ -bound form, only the N-terminus (myristoylated Gly 2 -Lys 9 ) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca 2+ -free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca 2+ -dependent inactivation of GCAP1. GCAP1(E 75 D,E 111 D,E 155 D) mutant did not bind Ca 2+ and stimulated GC1 in a (Ca 2+ )-independent manner. GCAP1 and GCAP2, but not S-100‚, a high (Ca 2+ )free activator of GC1, competed with the triple mutant at high (Ca 2+ )free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca 2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.