title: Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.
abstract: The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823).
authors.affiliation: From the Department of Research, Salus University, Elkins Park, Pennsylvania 19027.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Peshenko
authors.affiliation: From the Department of Research, Salus University, Elkins Park, Pennsylvania 19027.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Olshevskaya
authors.affiliation: From the Department of Research, Salus University, Elkins Park, Pennsylvania 19027 adizhoor@salus.edu.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Dizhoor
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D005136:Eye Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008715:Methionine / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015398:Signal Transduction
chemical_list: D005136:Eye Proteins; C496736:GUCA1A protein, human; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D008164:Luminescent Proteins; C477438:RD3 protein, human; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C413663:fluorescent protein 583; C110715:guanylate cyclase 1; D001120:Arginine; D008715:Methionine; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A
keywords: calcium-binding protein; cyclic GMP (cGMP); eye; guanylate cyclase (guanylyl cyclase); neurobiology; phototransduction