title: The second C2-domain of copine-2, copine-6 and copine-7 is responsible for their calcium-dependent membrane association.
abstract: The copine family of proteins contains nine members with a similar domain structure, namely two N-terminal C2-domains (C2A and C2B) and a C-terminal A-domain. The former are thought to be responsible for binding to the inner face of the plasma membrane following increases in intracellular calcium levels, whereas the A-domain has been suggested to be a protein-binding structure. In this study, we examined the effects of mutagenesis of selected residues in the linker area between the C2-domains and the A-domain, and mutagenesis of the aspartates of the C2-domains, which are predicted to bind calcium and promote membrane association of the copines. We found that Lys282-Lys284 of the linker area are important for the folding of the intact protein. We showed that substitution with asparagine, single or multiple, of the aspartates in the C2A-domain had no effect on the calcium-mediated membrane association of copine-2, copine-6, or copine-7. Similar mutagenesis of a single residue in the C2B-domain of copine-6 (but not copine-2 and copine-7) was sufficient to eliminate its calcium-mediated membrane binding, and simultaneous substitution of all four of the asparagines in the C2B-domain resulted in constitutive membrane association of copine-2, copine-6 and copine-7 with the plasma membrane. These data show that the C2B-domains of copine-2, copine-6 and copine-7 are the domains responsible for the protein calcium-dependent membrane association.
authors.affiliation: Medical Research Council Brain Network Dynamics Unit at the University of Oxford, UK.
authors.forename: Pavel
authors.initials: P
authors.lastname: Perestenko
authors.affiliation: Medical Research Council Brain Network Dynamics Unit at the University of Oxford, UK.
authors.forename: Masanori
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Medical Research Council Brain Network Dynamics Unit at the University of Oxford, UK.
authors.forename: Tobias
authors.initials: T
authors.lastname: Beusnard-Bee
authors.affiliation: Medical Research Council Brain Network Dynamics Unit at the University of Oxford, UK.
authors.forename: Prakash
authors.initials: P
authors.lastname: Guna
authors.affiliation: Medical Research Council Brain Network Dynamics Unit at the University of Oxford, UK.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: McIlhinney
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D002352:Carrier Proteins; C000607100:Cpne7 protein, rat; D008565:Membrane Proteins; C110203:copine; C000607099:copine 6 protein, mouse; C000607097:copine-2 protein, mouse; D002217:Carbachol; D002118:Calcium
keywords: C2-domain; copine; intracellular calcium; membrane binding; protein folding