Molecular Characterization of a Third Member of the Guanylyl Cyclase-activating Protein Subfamily (original) (raw)
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The Journal of biological chemistry, 1994
Guanylate cyclase-activating protein (GCAP) is a novel Ca(2+)-binding protein that stimulates synthesis of cGMP in photoreceptors. Molecular cloning of human and mouse GCAP cDNA revealed that the known mammalian GCAPs are more than 90% similar, consist of 201-205 amino acids, and contain three identically conserved EF hand Ca2+ binding sites. The sequence homology with recoverin, a related photoreceptor Ca(2+)-binding protein, is less than 35%. In situ hybridization in primate retinas shows that the GCAP gene is expressed exclusively in photoreceptor inner segments. To investigate the GCAP gene structure, we probed 10 eucaryotic genomic DNAs with a bovine GCAP cDNA under stringent conditions. The results demonstrate that the GCAP gene has been well conserved during evolution of vertebrate species and that each gene is most likely present as a single copy. By genomic cloning, polymerase chain reaction, mapping, and direct sequencing, we show that the human GCAP gene spans approximate...
Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1)
Journal of Biological Chemistry, 2014
Background: GCAP1 regulates cGMP synthesis in photoreceptors in response to light. Results: Mutagenesis of the entire GCAP1 surface reveals its guanylyl cyclase interface. Conclusion: The interface forms a compact patch that enables both primary binding to and allosteric activation of the target enzyme. Significance: Guanylyl cyclase activation by GCAP1 is indispensable for vision and survival of photoreceptors. Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metalbinding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca 2؉ binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the "binding patch" prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94. Retinal membrane guanylyl cyclase (RetGC) 2 and RetGCactivating proteins (GCAPs) play a critical role in the physiol
Retinal Guanylyl Cyclase-Activating Protein 1 and 2
Encyclopedia of Signaling Molecules, 2016
Historical Background Retinal guanylyl cyclases (RetGCs) in retinal rod and cone photoreceptors are regulated by a family of EF-hand Ca 2+ sensor proteins called guanylyl cyclase-activating proteins (GCAP1-8) that belong to the neuronal calcium sensor (NCS) family. Mammalian GCAPs (GCAP1 and GCAP2) activate RetGCs at low Ca 2+ levels in light-activated photoreceptor cells and inhibit RetGC activity at higher Ca 2+ levels in darkadapted photoreceptors. The Ca 2+-sensitive RetGC activity controlled by GCAPs is an important mechanism of visual recovery and light adaptation of phototransduction. Mutations in either RetGCs or GCAPs that disable this Ca 2+-sensitive cyclase activity are genetically linked to retinal disease. Here I review atomic-level structures of GCAP1 in both Ca 2+-free/Mg 2+-bound (activator) and Ca 2+-saturated (inhibitory) states, as well as the structure of Ca 2+-saturated GCAP2. The structure of GCAP2 reveals an exposed N-terminus that may be important for Ca 2+-dependent membrane anchoring of the myristoyl group. By contrast, the structures of Ca 2+-free and Ca 2+-bound forms of GCAP1 each contain a covalently attached myristoyl group that is sequestered in a hydrophobic protein cavity formed by helices at both the N-and C-terminus. Hence, myristoylated GCAP1 is not targeted to bilayer membranes. The Ca 2+-free activator form of GCAP1 contains Mg 2+ bound at the second EF-hand (EF2) that is essential for activating RetGC. The Ca 2+ saturated form of GCAP1 contains Ca 2+ bound at EF2, EF3, and EF4. Ca 2+-dependent conformational changes are most apparent in EF2 and in the Ca 2+ switch helix (residues 169-174) and will be discussed in terms of a proposed mechanism for Ca 2+-dependent activation of retinal guanylyl cyclases.
Localization of guanylate cyclase-activating protein 2 in mammalian retinas
Proceedings of the National Academy of Sciences, 1997
Guanylate cyclase-activating proteins (GCAP1 and GCAP2) are thought to mediate the intracellular stimulation of guanylate cyclase (GC) by Ca 2+ , a key event in recovery of the dark state of rod photoreceptors after exposure to light. GCAP1 has been localized to rod and cone outer segments, the sites of phototransduction, and to photoreceptor synaptic terminals and some cone somata. We used in situ hybridization and immunocytochemistry to localize GCAP2 in human, monkey, and bovine retinas. In human and monkey retinas, the most intense immunolabeling with anti-GCAP2 antibodies was in the cone inner segments, somata, and synaptic terminals and, to a lesser degree, in rod inner segments and inner retinal neurons. In bovine retina, the most intense immunolabeling was in the rod inner segments, with weaker labeling of cone myoids, somata, and synapses. By using a GCAP2-specific antibody in enzymatic assays, we confirmed that GCAP1 but not GCAP2 is the major component that stimulates GC ...
Journal of Biological Chemistry, 2019
The guanylyl cyclase-activating protein, GCAP1, activates photoreceptor membrane guanylyl cyclase (RetGC) in the light, when free Ca 2؉ concentrations decline, and decelerates the cyclase in the dark, when Ca 2؉ 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 Ca 2؉-dependent activatorto-inhibitor conformational transition. The G86R-GCAP1 variant activated RetGC at low Ca 2؉ concentrations with higher affinity than did the WT GCAP1, but failed to decelerate the cyclase at the Ca 2؉ concentrations characteristic of darkadapted photoreceptors. Ca 2؉-dependent increase in Trp 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. Guanylyl cyclase-activating proteins (GCAPs), 4 N-myristoylated calcium/magnesium-binding proteins of the EF-hand superfamily, are comprised of two pairs of EF-hand domains connected via a "hinge" region (reviewed in Refs. 1 and 2). Among several isoforms of GCAPs expressed in the vertebrate retinas (3-6) two, GCAP1 and GCAP2, regulate visual signaling in all species by properly shaping the sensitivity and kinetics of rod and cone responses (7-10). Vertebrate rods and cones respond to light stimuli by closing cGMP-gated channels in their outer segments via phototransduction cascade-mediated hydrolysis of cGMP (reviewed in Refs. 11 and 12). Following the excitation, cGMP production by retinal membrane guanylyl cyclase (RetGC) (13-15) first becomes accelerated, to speed up the recovery and light adaptation of photoreceptors, and then decelerated again as photoreceptors recover from the excitation back to their dark-adapted state (7, 16). Negative Ca 2ϩ feedback regulates the activity of RetGC via its Ca 2ϩ sensor proteins, GCAPs, such that in the light, when cGMP channels are closed and the influx of Ca 2ϩ through the channels stops, GCAPs release Ca 2ϩ and convert into a Mg 2ϩ-liganded state that stimulates RetGC. Once the photoreceptors return to their dark-adapted state, when cGMP channels reopen and the influx of Ca 2ϩ resumes, GCAPs undergo the reverse, activatorto-inhibitor, transition, by replacing Mg 2ϩ in their EF-hands with Ca 2ϩ , and decelerate RetGC (reviewed in Refs. 2 and 12). Failure of RetGC to accelerate or decelerate cGMP production within the normal range of the intracellular free Ca 2ϩ alters light sensitivity and kinetics of rod and cone response to light (7-9, 16-18) and has been linked to various forms of retinal blindness in humans, such as Leber congenital amaurosis, dominant cone or cone-rod degenerations (reviewed in Ref. 19-22), and a recessive night blindness (23). Multiple mutations linked to these blinding disorders have been found in the genes coding for RetGC1 isozyme (GUCY2D) (19-27) and GCAP1 (GUCA1A) (28-40). GUCA1A mutations linked to the domi
European Journal of Biochemistry, 1998
Two guanylate-cyclase-activating proteins (GCAP) encoded by a tail-to-tail gene array have been characterized in the mammalian retina. Using frog retina as a model, we obtained evidence for the presence of a photoreceptor Ca 2ϩ -binding protein closely related to GCAP. This protein (206 amino acids) does not stimulate guanylate cyclase (GC) in low [Ca 2ϩ ], but inhibits GC in high [Ca 2ϩ ], and is therefore termed guanylate-cyclase-inhibitory protein (GCIP). Sequence analysis indicates that GCIP and GCAP1 and GCAP2 have diverged substantially, but conserved domains present in all vertebrate GCAP are present in GCIP. Moreover, partial characterization of the GCIP gene showed that the positions of two introns in the GCIP gene are identical to positions of corresponding introns of the mammalian GCAP gene array. As to the major differences between GCIP and GCAP, the fourth EF hand Ca 2ϩ -binding motif of GCIP is disabled for Ca 2ϩ binding, and GCIP does not stimulate GC. Monoclonal and polyclonal antibodies raised against recombinant GCIP identified high levels of GCIP in the inner segments, somata and synaptic terminals of frog cone photoreceptors. The results suggest that GCIP is a Ca 2ϩ -binding protein of the GCAP/recoverin subfamily. Its localization in frog cones closely resembles that of GC in mammalian cones. GCIP inhibits GC at high free [Ca 2ϩ ], competing with GCAP1 and GCAP2 for GC regulatory sites.