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Ca(2+)-dependent conformational changes in guanylyl cyclase-activating protein 2 (GCAP-2) revealed by site-specific phosphorylation and partial proteolysis
Journal article   Open access   Peer reviewed

Ca(2+)-dependent conformational changes in guanylyl cyclase-activating protein 2 (GCAP-2) revealed by site-specific phosphorylation and partial proteolysis

Igor V Peshenko, Elena V Olshevskaya and Alexander M Dizhoor
The Journal of biological chemistry, v 279(48), pp 50342-50349
26 Nov 2004
DOI: https://doi.org/10.1074/jbc.M408683200
PMID: 15448139
Featured in Collection :   UN Sustainable Development Goals @ Drexel
url
https://doi.org/10.1074/jbc.m408683200View
Published, Version of Record (VoR)Open Access (License Unspecified),  Open
url
https://doi.org/10.1074/jbc.M408683200View
Published, Version of Record (VoR) Open

Abstract

Amino Acid Sequence Animals Calcium - metabolism Calcium-Binding Proteins - metabolism Cattle Cyclic Nucleotide-Regulated Protein Kinases - metabolism Edetic Acid - metabolism Guanylate Cyclase-Activating Proteins Mice Molecular Sequence Data Phosphorylation Phosphotransferases - metabolism Protein Conformation Serine - metabolism
Guanylyl cyclase-activating proteins (GCAPs) are calcium sensor proteins of the EF-hand superfamily that inhibit retinal photoreceptor membrane guanylyl cyclase (retGC) in the dark when they bind Ca(2+) but activate retGC when Ca(2+) dissociates from GCAPs in response to light stimulus. We addressed the difference in exposure of GCAP-2 structure to protein kinase and a protease as indicators of conformational change caused by binding and release of Ca(2+). We have found that unlike its homolog, GCAP-1, the C terminus of GCAP-2 undergoes phosphorylation by cyclic nucleotide-dependent protein kinases (CNDPK) present in the retinal extract and rapid dephosphorylation by the protein phosphatase PP2C present in the retina. Inactivation of the CNDPK phosphorylation site in GCAP-2 by substitutions S201G or S201D, as well as phosphorylation or thiophosphorylation of Ser(201), had little effect on the ability of GCAP-2 to regulate retGC in reconstituted membranes in vitro. At the same time, Ca(2+) strongly inhibited phosphorylation of the wild-type GCAP-2 by retinal CNDPK but did not affect phosphorylation of a constitutively active Ca(2+)-insensitive GCAP-2 mutant. Partial digestion of purified GCAP-2 with Glu-C protease revealed at least two sites that become exposed or constrained in a Ca(2+)-sensitive manner. The Ca(2+)-dependent conformational changes in GCAP-2 affect the areas around Glu(62) residue in the entering helix of EF-hand 2, the areas proximal to the exiting helix of EF-hand 3, and Glu(136)-Glu (138) between EF-hand 3 and EF-hand 4. These changes also cause the release of the C-terminal Ser(201) from the constraint caused by the Ca(2+)-bound conformation.

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Web of Science research areas
Biochemistry & Molecular Biology
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