A G86R mutation in the calcium-sensor protein GCAP1 alters regulation of retinal guanylyl cyclase and causes dominant cone-rod degeneration.

The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca concentrations decline, and decelerates the cyclase in the dark, when Ca concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 () gene in a family with a dominant retinopathy. The G86R substitution in a "hinge" region connecting EF-hand domains 2 and 3 in GCAP1 strongly interfered with its Ca-dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca concentrations characteristic of dark-adapted photoreceptors. Ca-dependent increase in Trp fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca, and the dose dependence of the G86R GCAP1-RetGC1 complex inhibition by retinal degeneration 3 (RD3) protein was shifted toward higher than normal concentrations. Our results indicate that the flexibility of the hinge region between EF-hands 2 and 3 is required for placing GCAP1-regulated Ca sensitivity of the cyclase within the physiological range of intracellular Ca at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly, leading to abnormally high affinity for the target enzyme and reduced Ca sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca influx in photoreceptors in the dark.