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

Abstract

94 fluorescence, indicative of the GCAP1 transition to its RetGC inhibiting state, was suppressed and shifted to a higher Ca 2؉ range. Conformational changes in G86R GCAP1 detectable by isothermal titration calorimetry (ITC) also became less sensitive to Ca 2؉ , 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 2؉ sensitivity of the cyclase within the physiological range of intracellular Ca 2؉ at the expense of reducing GCAP1 affinity for the target enzyme. The disease-linked mutation of the hinge Gly 86 , leading to abnormally high affinity for the target enzyme and reduced Ca 2؉ sensitivity of GCAP1, is predicted to abnormally elevate cGMP production and Ca 2؉ influx in photoreceptors in the dark.

Constitutive Activation of Guanylate Cyclase by the G86R GCAP1 Variant Is Due to “Locking” Cation-π Interactions that Impair the Activator-to-Inhibitor Structural Transition

“…Biochemical experiments performed with the GC-GCAP1 reconstituted system in the presence of the G86R mutation showed that the complex forms with higher affinity than the WT and it is unable to decelerate the GC at high Ca 2+ -levels corresponding to the dark-adapted state [26]. This mechanism was found to be in line with the clinical cone-rod dystrophy phenotype of the proband and with the autosomal dominant inheritance pattern proven by the segregation study [26].…”

“…Mutations in GUCA1A, encoding for GCAP1 result in aberrant GC regulation and in retinal degeneration, a set of progressive diseases involving cones, the macula, and in some cases rods, ultimately leading to blindness [11][12][13]. To date, 20 GCAP1 variants have been associated with retinal dystrophy [14][15][16][17][18][19][20][21][22][23][24][25], with one of the most recently discovered being the G86R mutation, located in the previously mentioned hinge region [26]. Biochemical experiments performed with the GC-GCAP1 reconstituted system in the presence of the G86R mutation showed that the complex forms with higher affinity than the WT and it is unable to decelerate the GC at high Ca 2+ -levels corresponding to the dark-adapted state [26].…”

“…To date, 20 GCAP1 variants have been associated with retinal dystrophy [14][15][16][17][18][19][20][21][22][23][24][25], with one of the most recently discovered being the G86R mutation, located in the previously mentioned hinge region [26]. Biochemical experiments performed with the GC-GCAP1 reconstituted system in the presence of the G86R mutation showed that the complex forms with higher affinity than the WT and it is unable to decelerate the GC at high Ca 2+ -levels corresponding to the dark-adapted state [26]. This mechanism was found to be in line with the clinical cone-rod dystrophy phenotype of the proband and with the autosomal dominant inheritance pattern proven by the segregation study [26].…”

“…In this respect, the molecular characterization of the G86R GCAP1 mutant showed features in between the two groups, as the constitutive activation of the GC was due to a concerted effect of: (i) increased affinity for Mg 2+ ; (ii) increased affinity for the target; and (iii) lower Ca 2+ -sensitivity. The molecular mechanism underlying these differences was found to reside in the limited flexibility of the hinge region affecting the "myristoyl tug" [26]. Moreover, a close inspection of the three-dimensional structural model of human GCAP1 suggested that the geometry of the hinge region would bring the positively charged guanidino group of R86 sufficiently close to the indole of W94, thus, potentially leading to a stabilizing cation-π interaction ( Figure 1A).…”

GCAP neuronal calcium sensor proteins mediate photoreceptor cell death in the rd3 mouse model of LCA12 congenital blindness by involving endoplasmic reticulum stress

“…Synthesis of cyclic GMP (cGMP) is an essential process in photoreceptor cells of the retina, as cGMP is the signaltransducing molecule in the light response 1,2 . Mutations in a number of genes that impair or alter cGMP synthesis in rods and cones have been associated to different forms of blindness [3][4][5][6][7][8][9][10][11][12] . Loss-of-function mutations in the RD3 gene (name from the natural strain of "retinal degeneration 3" mice, rd3 locus mutated) cause Leber's congenital amaurosis 12 (LCA12) 13,14 .…”