Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1) (original) (raw)
Related papers
Biochemistry, 2010
Retinal membrane guanylyl cyclase (RetGC) and Ca 2þ /Mg 2þ sensor proteins (GCAPs) control the recovery of the photoresponse in vertebrate photoreceptors, through their molecular interactions that remain rather poorly understood and controversial. Here we have determined the main RetGC isozyme (RetGC1):GCAP1 binding stoichiometry at saturation in cyto, using fluorescently labeled RetGC1 and GCAP1 coexpressed in HEK293 cells. In a striking manner, the equimolar binding of RetGC1 with GCAP1 in transfected HEK293 cells typical for wild-type RetGC1 was eliminated by a substitution, D639Y, in the kinase homology domain of RetGC1 found in a patient with a severe form of retinal dystrophy, Leber congenital amaurosis (LCA). A similar effect was observed with another LCA-related mutation, R768W, in the same domain of RetGC1. In contrast to the completely suppressed binding and activation of RetGC1 by Mg 2þliganded GCAP1, neither of these two mutations eliminated the GCAP1-independent activity of RetGC stimulated by Mn 2þ . These results directly implicate the D639 (and possibly R768)-containing portion of the RetGC1 kinase homology domain in its primary recognition by the Mg 2þ -bound activator form of GCAP1.
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.
Structural Insights into Retinal Guanylate Cyclase Activator Proteins (GCAPs)
International Journal of Molecular Sciences, 2021
Retinal guanylate cyclases (RetGCs) promote the Ca2+-dependent synthesis of cGMP that coordinates the recovery phase of visual phototransduction in retinal rods and cones. The Ca2+-sensitive activation of RetGCs is controlled by a family of photoreceptor Ca2+ binding proteins known as guanylate cyclase activator proteins (GCAPs). The Mg2+-bound/Ca2+-free GCAPs bind to RetGCs and activate cGMP synthesis (cyclase activity) at low cytosolic Ca2+ levels in light-activated photoreceptors. By contrast, Ca2+-bound GCAPs bind to RetGCs and inactivate cyclase activity at high cytosolic Ca2+ levels found in dark-adapted photoreceptors. Mutations in both RetGCs and GCAPs that disrupt the Ca2+-dependent cyclase activity are genetically linked to various retinal diseases known as cone-rod dystrophies. In this review, I will provide an overview of the known atomic-level structures of various GCAP proteins to understand how protein dimerization and Ca2+-dependent conformational changes in GCAPs co...
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
Biochemistry, 1999
Regulation of cAMP and cGMP production is a fundamental step in a broad range of signal transduction systems, including phototransduction. To identify regions within photoreceptor guanylyl cyclase 1 (GC1) that interact with GC-activating proteins (GCAPs), we synthesized the intracellular fragment of GC1, residues 491-1110, as a set of 15 amino acid long, partially overlapping peptides on the surface of individual pins arranged in a microtiter plate format. This pin assay identified 8 peptides derived from different regions of the GC1 intracellular domain that bind GCAPs. Peptide variants containing these sequences were synthesized as free peptides and tested for their ability to inhibit GC1 stimulation by GCAPs. A free peptide, 968 GTFRMRHMPEVPVRIRIG, from the catalytic domain of GC1 was the strongest inhibitor of GCAP1/GCAP2-mediated activation. In native GC1, this polypeptide fragment is likely to form a loop between R-helix 3 and-strand 4. When this region in GC1 was replaced by the corresponding sequence of GCAP-insensitive GC type A, GCAPs did not stimulate the GC1 mutant. The corresponding loops in related adenylyl cyclase (AC) are involved in the activating and inhibiting interactions with G sR and G iR , respectively. Thus, despite interacting with different activating proteins, both AC and GC activity may be modulated through their respective regions within catalytic domains.
Molecular Characterization of a Third Member of the Guanylyl Cyclase-activating Protein Subfamily
Journal of Biological Chemistry, 1999
The mammalian retina contains at least two guanylyl cyclases (GC1 and GC2) and two guanylyl cyclase-activating proteins (GCAP1 and GCAP2). Here we present evidence of the presence of a new photoreceptor-specific GCAP, termed GCAP3, which is closely related to GCAP1. The sequence similarity of GCAP3 with GCAP1 and GCAP2 is 57 and 49%, respectively. Recombinant GCAP3 and GCAP2 stimulate GC1 and GC2 in low [Ca 2؉ ] free and inhibit GCs when [Ca 2؉ ] free is elevated, unlike GCAP1, which only stimulates GC1. GCAP3 is encoded by a distinct gene present in other mammalian species but could not be detected by genomic Southern blotting in rodents, amphibians, and lower vertebrates. The intron/exon arrangement of the GCAP3 gene is identical to that of the other GCAP genes. While the GCAP1 and GCAP2 genes are arranged in a tail-to-tail array on chromosome 6p in human, the GCAP3 gene is located on 3q13.1, suggesting an ancestral gene duplication/translocation event. The identification of multiple Ca 2؉ -binding proteins that interact with GC is suggestive of complex regulatory mechanisms for photoreceptor GC.
Experimental Eye Research, 1999
Guanylate cyclase activating proteins, GCAP-1 and GCAP-2, have a pivotal role in the activation of guanylate cyclase in phototransduction. Previous studies on the localization of GCAP-1 and GCAP-2 are contradictory. In this study, we tried to avoid possible artifacts accompanied by immunocytochemistry. Immunolabeling of a GCAP was carried out using antibodies pre-adsorbed with a different type of GCAP. In addition, immunolabeling was performed using three different animal species under different fixation and embedding. Electron microscopic immunocytochemistry was also performed to reveal subcellular localization of GCAPs as well as confirming data obtained by light microscopy. All data indicate that anti-GCAP-1 antibody binding sites were found predominantly in cone outer segments, in particular, in disk membrane regions. Sparse labeling was observed in rod outer segments, but the labeling was much lower than that seen in cone outer segments. Less labeling is also found in synaptic regions and inner segments of cones. No labeling was detected in connecting cilia and its cytoplasmic extensions. Such labeling patterns were similar among human, monkey and bovine retinas. The localization of GCAP-1 is consistent with the pattern of a recently reported human cone-specific degeneration. Anti-GCAP-2 antibody binding sites were detected in both inner and outer segments of rods and cones of all three animals although the labeling density was slightly different among species. Cryo-immuno-labeling of GCAP-2 in bovine retinas revealed that labeling sites were more concentrated in rods than those of cones, and that synaptic regions were also labeled. The different localization of GCAPs suggest that roles of GCAP-1 and GCAP-2 may be different.
Biochemistry, 2011
Retinal membrane guanylyl cyclase (RetGC) 1 in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca 2+ concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a non-competitive manner and it inhibits the GCAP-stimulated RetGC at low intracellular Ca 2+ levels. RD3 opposes the allosteric activation of the cyclase by GCAP, but does not significantly change Ca 2+ sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca 2+ and GCAPs.