Journal Article

PreJuSER-41869

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activation

Hwang, J.-Y.FZJ* ; Schlesinger, R.FZJ* ; Koch, K.-W.FZJ*

2004
Wiley-Blackwell Oxford [u.a.]

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Please use a persistent id in citations: doi:10.1111/j.1432-1033.2004.04320.x

Abstract: 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.

Keyword(s): Amino Acid Sequence (MeSH) ; Animals (MeSH) ; Calcium: metabolism (MeSH) ; Calcium-Binding Proteins: chemistry (MeSH) ; Calcium-Binding Proteins: genetics (MeSH) ; Calcium-Binding Proteins: isolation & purification (MeSH) ; Calcium-Binding Proteins: metabolism (MeSH) ; Chromatography, Gel (MeSH) ; Cysteine: metabolism (MeSH) ; Dimerization (MeSH) ; EF Hand Motifs: genetics (MeSH) ; Escherichia coli: genetics (MeSH) ; Guanylate Cyclase-Activating Proteins (MeSH) ; Light Signal Transduction (MeSH) ; Molecular Sequence Data (MeSH) ; Mutation (MeSH) ; Proline: metabolism (MeSH) ; Protein Conformation (MeSH) ; Reactive Oxygen Species: metabolism (MeSH) ; Recombinant Proteins: chemistry (MeSH) ; Recombinant Proteins: metabolism (MeSH) ; Sequence Homology, Amino Acid (MeSH) ; Spectrometry, Fluorescence (MeSH) ; Calcium-Binding Proteins ; Guanylate Cyclase-Activating Proteins ; Reactive Oxygen Species ; Recombinant Proteins ; Proline ; Cysteine ; Calcium ; J ; GCAP (auto) ; guanylate cyclase (auto) ; neuronal Ca2+ sensor (auto) ; phototransduction (auto)

Note: Record converted from VDB: 12.11.2012

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Contributing Institute(s):

1. Zelluläre Signalverarbeitung (IBI-1)
2. Biologische Strukturforschung (IBI-2)

Research Program(s):

1. Neurowissenschaften (L01)

Appears in the scientific report 2004

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Database coverage:
; JCR ; Science Citation Index Expanded ; Thomson Reuters Master Journal List ; Web of Science Core Collection

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