In rod phototransduction, cyclic GMP synthesis by membrane bound guanylate cyclase ROS-GC1 is under Ca 2+ -dependent negative feedback control mediated by guanylate cyclase-activating proteins, GCAP-1 and GCAP-2. The cellular concentration of GCAP-1 and GCAP-2 approximately sums to the cellular concentration of a functional ROS-GC1 dimer. Both GCAPs increase the catalytic efficiency (k cat /K m ) of ROS-GC1. However, the presence of a myristoyl group in GCAP-1 has a strong impact on the regulation of ROS-GC1, this is in contrast to GCAP-2. Catalytic efficiency of ROS-GC1 increases 25-fold when it is reconstituted with myristoylated GCAP-1, but only by a factor of 3.4 with nonmyristoylated GCAP-1. In contrast to GCAP1, myristoylation of GCAP-2 has only a minor effect on k cat /K m . The increase with both myristoylated and nonmyristoylated GCAP-2 is 10 to 13-fold. GCAPs also confer different Ca 2+ -sensitivities to ROS-GC1. Activation of the cyclase by GCAP-1 is half-maximal at 707 nM free [Ca 2+ ], while that by GCAP-2 is at 100 nM. The findings show that differences in catalytic efficiency and Ca 2+ -sensitivity of ROS-GC1 are conferred by GCAP-1 and GCAP-2. The results further indicate the concerted operation of two ÔGCAP modesÕ that would extend the dynamic range of cyclase regulation within the physiological range of free cytoplasmic Ca 2+ in photoreceptor cells.

Bovine photoreceptor guanylate cyclase (ROS-GC) consists of a single transmembrane polypeptide chain with extracellular and intracellular domains. In contrast to non-photoreceptor guanylate cyclases (GCs) which are activated by hormone peptides, ROS-GC is modulated in low Ca2+ by calmodulin-like Ca(2+)-binding proteins termed GCAPs (guanylate cyclase-activating proteins). In this communication we show that, like the native system, ROS-GC expressed in COS cells is activated 4-6-fold by recombinant GCAP1 at 10 nM Ca2+ and that the reconstituted system is inhibited at physiological levels of Ca2+ (1 microM). A mutant ROS-GC in which the extracellular domain was deleted was stimulated by GCAP1 indistinguishable from native ROS-GC indicating that this domain is not involved in Ca2+ modulation. Deletion of the intracellular kinase-like domain diminished the stimulation by GCAP1, indicating that this domain is at least in part involved in Ca2+ modulation. Replacement of the catalytic domain in a non-photoreceptor GC by the catalytic domain of ROS-GC yielded a chimeric GC that was sensitive to ANF/ATP and to a lesser extent to GCAP1. The results establish that GCAP1 acts at an intracellular domain, suggesting a mechanism of photoreceptor GC stimulation fundamentally distinct from hormone peptide stimulation of other cyclase receptors.