%0 Journal Article
%A Hwang, J.-Y.
%A Schlesinger, R.
%A Koch, K.-W.
%T Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activation
%J The FEBS journal
%V 271
%@ 0014-2956
%C Oxford [u.a.]
%I Wiley-Blackwell
%M PreJuSER-41869
%P 3785 - 3793
%D 2004
%Z Record converted from VDB: 12.11.2012
%X 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.
%K Amino Acid Sequence
%K Animals
%K Calcium: metabolism
%K Calcium-Binding Proteins: chemistry
%K Calcium-Binding Proteins: genetics
%K Calcium-Binding Proteins: isolation & purification
%K Calcium-Binding Proteins: metabolism
%K Chromatography, Gel
%K Cysteine: metabolism
%K Dimerization
%K EF Hand Motifs: genetics
%K Escherichia coli: genetics
%K Guanylate Cyclase-Activating Proteins
%K Light Signal Transduction
%K Molecular Sequence Data
%K Mutation
%K Proline: metabolism
%K Protein Conformation
%K Reactive Oxygen Species: metabolism
%K Recombinant Proteins: chemistry
%K Recombinant Proteins: metabolism
%K Sequence Homology, Amino Acid
%K Spectrometry, Fluorescence
%K Calcium-Binding Proteins (NLM Chemicals)
%K Guanylate Cyclase-Activating Proteins (NLM Chemicals)
%K Reactive Oxygen Species (NLM Chemicals)
%K Recombinant Proteins (NLM Chemicals)
%K Proline (NLM Chemicals)
%K Cysteine (NLM Chemicals)
%K Calcium (NLM Chemicals)
%K J (WoSType)
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:15355355
%U <Go to ISI:>//WOS:000223711400017
%R 10.1111/j.1432-1033.2004.04320.x
%U https://juser.fz-juelich.de/record/41869