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

  • Igor V. Peshenko
    Affiliations
    Department of Basic Sciences and the Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania 19027
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  • Elena V. Olshevskaya
    Affiliations
    Department of Basic Sciences and the Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania 19027
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  • Sunghyuk Lim
    Affiliations
    Department of Chemistry, University of California, Davis, California 95616
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  • James B. Ames
    Affiliations
    Department of Chemistry, University of California, Davis, California 95616
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  • Alexander M. Dizhoor
    Correspondence
    Martin and Florence Hafter Chair Professor of Pharmacology. To whom correspondence should be addressed: Dept. of Basic Sciences and Pennsylvania College of Optometry, Salus University, 8360 Old York Rd., Elkins Park, PA 19027. Tel.: 215-780-1468; Fax: 215-780-1464
    Affiliations
    Department of Basic Sciences and the Pennsylvania College of Optometry, Salus University, Elkins Park, Pennsylvania 19027
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants EY11522 (to A. M. D.) and EY012347 (to J. B. A.) from NEI. This work was also supported by a CURE Formula Grant (to A. M. D.) from the Pennsylvania Department of Health.
Open AccessPublished:February 24, 2014DOI:https://doi.org/10.1074/jbc.M113.540716
      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-metal-binding 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 Ca2+ 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.

      Introduction

      Retinal membrane guanylyl cyclase (RetGC)
      The abbreviations used are: RetGC
      retinal membrane guanylyl cyclase
      GCAP
      guanylyl cyclase-activating protein
      NEM
      N-ethylmaleimide
      NCS
      neuronal calcium sensor
      PCC
      Pearson correlation coefficient.
      and RetGC-activating proteins (GCAPs) play a critical role in the physiology of vertebrate photoreceptors by producing the second messenger of phototransduction, cGMP, and regulating its synthesis in a light-sensitive manner (
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      ). Photoactivated pigment (e.g. rhodopsin), via Gt protein-dependent stimulation of PDE6 phosphodiesterase, triggers the decay of cGMP and shuts off cGMP-gated channels in the outer segment, thus hyperpolarizing the photoreceptors in response to light (reviewed in Ref.
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      ) are ubiquitously present throughout the subfilum and are the only two isoforms encoded by the genome in some mammalian species such as mice and rats. Structures of Ca2+-liganded GCAP1 (Fig. 1A), GCAP2, and GCAP3 have been solved mostly by NMR spectroscopy and x-ray crystallography (
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      ). GCAPs have different Ca2+ sensitivities and target specificities. GCAP1 is present in mammalian rods and cones and primarily regulates RetGC1 (
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      ), whereas GCAP2 regulates both RetGC1 and RetGC2 in rods (
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      ).
      Figure thumbnail gr1
      FIGURE 1Effect of amino acid substitutions in GCAP1 on RetGC1 activation. A, three-dimensional model of Ca2+-liganded GCAP1 (
      • Stephen R.
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      ) annotated as follows: Myr, N-myristoyl moiety; EF-1–EF-4, EF-hand domains; α1–α11, α-helices numbered beginning from the amino terminus; hinge, the loop connecting two semi-globules (I and II) between the α5 and α6 helices. Ca2+ ions bound in three metal-binding EF-hand loops (EF-2 through EF-4) are shown as yellow spheres. B, RetGC1 activation by 5 μm GCAP1 mutants normalized to the wild type activation in control samples (mean ± S.D., n = 3). The following mutations were tested: K8E, S9R; E11,12K; S15R,T16A; E17K,C18D; C18T,C106T,C125T; H19R; Q20R; Y22D; K23D; K24D; M26R; T27K; T27E; E28R; C29T; P30Y; S31Y; G32N; Q33R; T35R; L36E; Y37R; E38R; Q41R;; K46D; N47R; P50G; W51N,S53R; E57R; Q58R; E61R; F65N; K67D; Y70A; F73E; M74K; V77E; A78E; S81A; L82S; K85E; K87D; V88R; E89R; Q90R; R93E; W94A; K97S; V101Y; G103R; C106D; R109D; D110R; R117D; R120D; D127R; A132R; E133R; E134R; D137R; F140A; S141Y; K142D; V145R; G147R; E150Y; S152E; L153R; E154C; E155G; M157R; E158R; K162E; Q164R; L166R; L167R; R172E; D175K; R178D; R181E, Q184R; and deletion, ΔGln-184—Gly-205. Additional substitutions, I122E, N1123A, P124E, C125Q, S126Q, D127G,S128K, T129L, M130L, T138R, S141L, and V145E, and the Val-160–Gly-205 region replacement with the corresponding region from GCAP2 were tested as a single chimera construct (
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      ). The assay contained 10 mm MgCl2 and 2 mm EGTA. The threshold level of 50% activation (dashed line) was selected for segregating the mutants for suspected damage of the RetGC1-binding interface. The mutations that caused this decrease are shown in open diamonds, and the substitutions are labeled next to the data points. C, positions of the mutations causing major decrease in RetGC1 activating capacity in the GCAP1 primary structure. All mutated side chains are marked in bold; those in which replacement rendered RetGC1 activation ≤50% of the wild type level are marked in red and underlined, and those in which mutations reduced activation below 80% are marked with red asterisks. The 12-residue loops of EF-1 through EF-4 are shaded.
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      • Peshenko I.V.
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      • Dizhoor A.M.
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      ). 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 (
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      ,
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      ,
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      Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activation.
      ), 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. In the present study, we have described a refined mapping of the residues in GCAP1 using global mutagenesis of the surface-exposed residues combined with functional tests that allowed us to distinguish between the primary binding to the cyclase versus its activation. We found that the residues required for GCAP1 binding to RetGC1 formed a distinct “binding patch” on one side of the molecule that also contains at least two residues, Met-26 and Trp-94, that are not essential for the primary binding but affect secondary interactions required for RetGC1 activation.

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