Mutations in theGUCA1Agene involved in hereditary cone dystrophies impair calcium-mediated regulation of guanylate cyclase

Abstract

ABSTRACT:The GUCA1A gene encodes the guanylate cyclase activating protein 1 (GCAP1) of mammalian rod and cone photoreceptor cells, which is involved in the Ca 2+ -dependent negative feedback regulation of membrane bound guanylate cyclases in the retina. Mutations in the GUCA1A gene have been associated with different forms of cone dystrophies leading to impaired cone vision and retinal degeneration. Here we report the identification of three novel and one previously detected GUCA1A mutations: c.265G>A (p.Glu89Lys), c.300T>A (p.Asp100Glu), c.476G>T (p.Gly159Val) and c.451C>T (p.Leu151Phe). The clinical data of the patients carrying these mutations were compared with the functional consequences of the mutant GCAP1 forms. For this purpose we purified the heterologously expressed GCAP1 forms and investigated whether the mutations affected the Ca 2+ -triggered conformational changes and the apparent interaction affinity with the membrane bound guanylate cyclase. Furthermore, we analyzed Ca 2+ -dependent regulatory modes of wildtype and mutant GCAP1 forms. Although all novel mutants were able to act as a Ca 2+ -sensor protein, they differed in their Ca 2+ -dependent activation profiles leading to a persistent stimulation of guanylate cyclase activities at physiological intracellular Ca 2+ concentration.

Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1)

“…1B). This was consistent with earlier observations that the region downstream from Arg-182 tolerates replacement with the C terminus from recoverin (32). However, we also found that, contrary to earlier expectations based on the substitution of larger fragments in GCAP primary structure (32,47), none of the individually tested surface residues in the exiting helix of EF-hand 4 or in ␣-helices 10 and 11 was essential for the cyclase activation.…”

“…The binding patch in GCAP1 extends from the EF-2 loop to the exiting helix ␣5 of EF-hand 2 and the entering helix ␣6 of EF-3. The hinge region between the two semi-globules in the GCAPs has been implicated in cyclase regulation (32,47). We found that three non-charged residues exposed on the surface of exiting helix ␣5 of EF-2 (Met-74, Val-77, and Ala-78) participate in the cyclase binding.…”

“…The activity of the cyclase was assayed using [␣- 32 P]GTP as a substrate, and the [ 32 P]cGMP product was quantified using TLC as described previously (11,50). Briefly, the assay mixture (25 l) incubated at 30°C contained 30 mM MOPS-KOH (pH 7.2), 60 mM KCl, 4 mM NaCl, 1 mM DTT, 2 mM Ca 2ϩ /EGTA buffer, 1 mM free Mg 2ϩ , 0.3 mM ATP, 4 mM cGMP, 1 mM GTP, and 1 Ci of [␣-…”

“…Despite the importance of RetGC regulation for the normal retinal physiology and disease and the ample physiological and biochemical data on the regulation of cGMP synthesis in the photoreceptor outer segment, the molecular mechanism of RetGC activation by GCAP remains obscure. To date, there have been several attempts to identify the possible sites of target recognition in GCAPs using chimeras with other neuronal calcium sensor (NCS) proteins (32,47,49), implicating several regions in GCAP primary structure as likely parts of the cyclase-binding interface. However, the precise identity of the binding interface(s) with the cyclase could not be derived directly from the earlier low-resolution studies.…”

Evaluating the Role of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Domains in Binding Guanylyl Cyclase-activating Proteins (GCAPs)

“…The mOrangeRetGC1VenusN chi- Fig. 2; the a max values for stimulation by GCAPs in the absence or presence of 1 and 2 M M26R GCAP1 only slightly change for stimulation by GCAP1 (27,26, and 25 nmol of cGMP/min/mg, respectively) or GCAP2 (22,21, and 19 nmol of cGMP/min/mg, respectively), but the K 1/2GCAP increased in the presence of M26R GCAP1 for wild type GCAP1 (1.5, 4, and 6.7 M, respectively) and GCAP2 (1.7, 4.7, and 7.4 M, respectively). The Hill coefficient increased for GCAP1 (from 1.17 in the absence to 1.53 in the presence of 2 M M26R GCAP1) and for GCAP2 (from 1.14 in the absence to 1.54 in the presence of 2 M M26R GCAP1).…”