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-specif ic Ca2+-binding protein: activates retinal guanylate cyclase 1 (GC1) d uring the recovery phase of phototransduction. In contrast to other Ca 2+-binding proteins from the calmodulin superfamily, the Ca2+-free for m of GCAP1 stimulates the effector enzyme, Ln this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis a nd mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity, The change from a Ca2+-bound to a Ca2+-fre e form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteol ysis by trypsin, Sequencing data revealed that in the Ca2+-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 Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inacti vation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivat ion of GCAP1. GCAP1((ED)-D-75,(ED)-D-111,(ED)-D-155) mutant did not bi nd Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and G CAP2, but not S-100 beta, a high [Ca2+](free) activator of GC1, compet ed with the triple mutant at high [Ca2+](free), inhibiting GC1 with si milar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs, Our data suggest that GCAP1 undergo es major conformational changes during Ca2+ binding and that EF3 and E F4 motifs are responsible for changes in the GCAP1 structure that conv erts this protein from the activator to the inhibitor of GC1.