Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1

Abstract

Macular dystrophy leads to progressive loss of central vision and shows symptoms similar to age-related macular degeneration. Genetic screening of patients diagnosed with macular dystrophy disclosed a novel mutation in the GUCA1A gene, namely a c.526C > T substitution leading to the amino acid substitution p.L176F in the guanylate cyclase-activating protein 1 (GCAP1). The same variant was found in three families showing an autosomal dominant mode of inheritance. For a full functional characterization of the L176F mutant we expressed and purified the mutant protein and measured key parameters of its activating properties, its Ca2+/Mg2+-binding, and its Ca2+-induced conformational changes in comparison to the wildtype protein. The mutant was less sensitive to changes in free Ca2+, resulting in a constitutively active form under physiological Ca2+-concentration, showed significantly higher activation rates than the wildtype (90-fold versus 20-fold) and interacted with an higher apparent affinity with its target guanylate cyclase. However, direct Ca2+-binding of the mutant was nearly similar to the wildtype; binding of Mg2+ occurred with higher affinity. We performed molecular dynamics simulations for comparing the Ca2+-saturated inhibiting state of GCAP1 with the Mg2+-bound activating states. The L176F mutant exhibited significantly lower flexibility, when three Ca2+ or two Mg2+ were bound forming probably the structural basis for the modified GCAP1 function.

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

“…Leu 176 replacement by Phe, through the altered tug action, increases GCAP1 affinity for RetGC1 but also reduces the affinity of the EF-hand 4 for Ca 2ϩ and thus shifts Ca 2ϩ sensitivity of RetGC regulation outside the physiological range (43). The L176F substitution, originally used to change the calcium-myristoyl tug in GCAP1 in vitro (43), was soon after found in patients with a photoreceptor dystrophy (44), likely triggered by elevated cGMP production in the dark.…”

“…nant cone or cone-rod degenerations specifically suppress Ca 2ϩ sensitivity of RetGC1 isozyme in vivo (17,41,42), most often by directly altering GCAP1 EF-hand motifs and thus reducing metal binding in EF-hands 3 and 4 (reviewed in Ref. 21), but also indirectly, by affecting EF-hand 4 affinity for Ca 2ϩ via altering a "calcium-myristoyl tug" mechanism connecting the C-terminal EF-hand 4 with the N-terminal myristoyl group buried inside the N-proximal semiglobule of EF-hands 1 and 2 (1,43,44). Here we describe a new type of mutation in GCAP1, G86R, leading to a dominant retinopathy in humans.…”

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 .…”

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

“…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].…”

“…Detailed biochemical and biophysical studies reported a similar common molecular mechanism of dysregulation among the majority of GCAP1 variants [20,[27][28][29][30][31][32], namely, a reduced Ca 2+ -affinity due to mutations in EF3 and EF4. On the other hand, some disease-associated mutations resulted in an unaltered Ca 2+ -sensitivity [24,33], suggesting that GC dysregulation may arise from higher affinity for Mg 2+ or from alterations in the long-range allosteric communication between the EF-hands and the target interface [10].…”