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

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... 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 IC 50 '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. Calcium ions, Ca 2+ , play a crucial role in cellular signaling. Because they are nondegradable, several systems have evolved to regulate the cellular concentration of free Ca 2+ ([Ca 2+ ] free ), including intracellular compartmentalization/ sequestration, pumping to the extracellular space, and buffering by Ca 2+ -binding proteins. Some of these Ca 2+ -binding proteins are also poised to take advantage of transient changes in [Ca 2+ ] free to affect properties of regulatory enzymes and ion channels. In cells that lower their internal [Ca 2+ ] free upon excitation, such as rod and cone photoreceptor cells, distinct types of proteins have evolved that act as activators of effector enzymes when they are in the Ca 2+free state. Guanylate cyclase-activating proteins, GCAP1 1 and GCAP2, were found to fulfill such functions in the regulation of photoreceptor guanylate cyclase (GC1) (1-4). GCAP1 and GCAP2 are acidic, ∼23-kDa, homologous proteins that contain three functional high-affinity, EF-hand Ca 2+ -chelating motifs (reviewed in ref 5). At low [Ca 2+ ] free , GCAPs increase the activity of GC1 (6) at least 10-fold (1) by an unknown mechanism. GCAP1 forms a stable complex with GC1, independent of [Ca 2+ ] free . The GC1/GCAP1 complex may switch between two conformations, active and inactive, with the binding or dissociation of Ca 2+ (2, 7). GCAP2 may translocate from the cytosol to the membranebound cyclase when it is free of Ca 2+ and stimulates GC1 activity (8). The N-terminal fatty acid-acylated regions of both GCAPs show weak sequence conservation, and the function of this region remains speculative. It is possible that the N-terminus is flexible and exposed, providing hydrophobic tethering to the membranes for the most efficient stimulation of GC1, as proposed for GCAP1 by , but this modification is functionally unrelated in the GC1 stimulation by GCAP2, as proposed by Olshevskaya et al. (8).