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 CA²⁺ concentrations decline, and decelerates the cyclase in the dark, when CA²⁺ concentrations rise. Here, we report a novel mutation, G86R, in the GCAP1 (GUCA1A) 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 CA²⁺-dependent activator-to-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low CA²⁺ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the CA²⁺ concentrations characteristic of dark-adapted photoreceptors. CA²⁺-dependent increase in Trp⁹⁴ fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher CA²⁺ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to CA²⁺, 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 CA²⁺ sensitivity of the cyclase within the physiological range of intracellular CA²⁺ at the expense of reducing GCAP1 affinity for the target Enzyme. The disease-linked mutation of the hinge Gly⁸⁶, leading to abnormally high affinity for the target Enzyme and reduced CA²⁺ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and CA²⁺ influx in photoreceptors in the dark.

GUCA1A; RetGC; calcium sensor; calcium-binding protein; cyclic GMP (cGMP); eye; guanylate cyclase (guanylyl cyclase); photoreceptor; phototransduction; retinal degeneration; retinopathy.