Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activation.
Guanylate cyclase-activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination. GCAPs contain four EF-hand Ca2+-binding motifs, but the first EF-hand is nonfunctional. It was concluded that for GCAP-2, the loss of Ca2+-binding ability of EF-hand 1 resulted in a region that is crucial for targeting guanylate cyclase [Ermilov, A.N., Olshevskaya, E.V. & Dizhoor, A.M. (2001) J. Biol. Chem.276, 48143-48148]. In this study we tested the consequences of mutations in EF-hand 1 of GCAP-1 with respect to Ca2+ binding, Ca2+-induced conformational changes and target activation. When the nonfunctional first EF-hand in GCAP-1 is replaced by a functional EF-hand the chimeric mutant CaM-GCAP-1 bound four Ca2+ and showed similar Ca2+-dependent changes in tryptophan fluorescence as the wild-type. CaM-GCAP-1 neither activated nor interacted with guanylate cyclase. Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild-type. Critical amino acids in EF-hand 1 of GCAP-1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+-induced conformational changes. The latter mutation also promoted dimerization of the protein. Our results show that EF-hand 1 in wild-type GCAP-1 is critical for providing the correct conformation for target activation.
Hwang
Ji-Young
JY
Institut für Biologische Informationsverarbeitung 1, Jülich, Germany.
Schlesinger
Ramona
R
Koch
Karl-Wilhelm
KW
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism; D019281:Dimerization; D020832:EF Hand Motifs / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D051604:Guanylate Cyclase-Activating Proteins; D055537:Light Signal Transduction; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000378:metabolism; D011487:Protein Conformation; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D011392:Proline; D003545:Cysteine; D002118:Calcium