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.

Rod and cone cells of the mammalian retina harbor two types of a membrane bound guanylate cyclase (GC), rod outer segment guanylate cyclase type 1 (ROS-GC1) and ROS-GC2. Both enzymes are regulated by small Ca 2+ -binding proteins named GC-activating proteins that operate as Ca 2+ sensors and enable cyclases to respond to changes of intracellular Ca 2+ after illumination. We determined the expression level of ROS-GC2 in bovine ROS preparations and compared it with the level of ROS-GC1 in ROSs. The molar ratio of a ROS-GC2 dimer to rhodopsin was 1 : 13 200. The amount of ROS-GC1 was 25-fold higher than the amount of ROS-GC2. Heterologously expressed ROS-GC2 was differentially activated by GC-activating protein 1 and 2 at low free Ca 2+ concentrations. Mutants of GC-activating protein 2 modulated ROS-GC2 in a manner different from their action on ROS-GC1 indicating that the Ca 2+ sensitivity of the Ca 2+ sensor is controlled by the mode of target-sensor interaction.

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