TY  - JOUR
AU  - Hwang, J.-Y.
AU  - Schlesinger, R.
AU  - Koch, K.-W.
TI  - Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activation
JO  - The FEBS journal
VL  - 271
SN  - 0014-2956
CY  - Oxford [u.a.]
PB  - Wiley-Blackwell
M1  - PreJuSER-41869
SP  - 3785 - 3793
PY  - 2004
N1  - Record converted from VDB: 12.11.2012
AB  - 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.
KW  - Amino Acid Sequence
KW  - Animals
KW  - Calcium: metabolism
KW  - Calcium-Binding Proteins: chemistry
KW  - Calcium-Binding Proteins: genetics
KW  - Calcium-Binding Proteins: isolation & purification
KW  - Calcium-Binding Proteins: metabolism
KW  - Chromatography, Gel
KW  - Cysteine: metabolism
KW  - Dimerization
KW  - EF Hand Motifs: genetics
KW  - Escherichia coli: genetics
KW  - Guanylate Cyclase-Activating Proteins
KW  - Light Signal Transduction
KW  - Molecular Sequence Data
KW  - Mutation
KW  - Proline: metabolism
KW  - Protein Conformation
KW  - Reactive Oxygen Species: metabolism
KW  - Recombinant Proteins: chemistry
KW  - Recombinant Proteins: metabolism
KW  - Sequence Homology, Amino Acid
KW  - Spectrometry, Fluorescence
KW  - Calcium-Binding Proteins (NLM Chemicals)
KW  - Guanylate Cyclase-Activating Proteins (NLM Chemicals)
KW  - Reactive Oxygen Species (NLM Chemicals)
KW  - Recombinant Proteins (NLM Chemicals)
KW  - Proline (NLM Chemicals)
KW  - Cysteine (NLM Chemicals)
KW  - Calcium (NLM Chemicals)
KW  - J (WoSType)
LB  - PUB:(DE-HGF)16
C6  - pmid:15355355
UR  - <Go to ISI:>//WOS:000223711400017
DO  - DOI:10.1111/j.1432-1033.2004.04320.x
UR  - https://juser.fz-juelich.de/record/41869
ER  -