Interactions of Visinin-like proteins with phospho-inositides

Abstract

The family of neuronal calcium sensor (NCS) proteins comprises five subfamilies, namely recoverins, frequenins, Visinin-like Proteins (VILIPs), GCAPs and KChlPs, which are involved in various signalling cascades in neuronal cells. All members of the NCS protein family are EF-hand proteins and share the typical feature of N-terminal myristoylation at the motif M-G-X3-S, as well as the calcium-myristoyl switch. In response to elevated calcium levels, NCS proteins invoke the calcium-myristoyl switch which is the central mechanism of their involvement in cellular calcium signalling. It has been shown that membrane association of proteins by a myristoyl group alone is very weak and requires further stabilising interactions such as hydrogen bonds between the protein and membrane phospholipids or the involvement of another membrane-associated protein. The subcellular membrane localisation of neuronal calcium sensor (NCS) proteins in living cells, such as VILIP-1 and VILIP-3, differs substantially. We have followed the hypothesis that the differential localisation might be due to specific binding capabilities of individual VILIPs for phosphatidylinositol phosphates (PIPs). Several highly conserved lysine residues in the N-terminal region could provide favourable electrostatic interactions. Molecular modelling results support a binding site for phospho-inositides in the N-terminal area of VILIP-1, and the involvement of the conserved N-terminal lysine residues in binding the phosphoinositol head group. Experimentally, the binding of VILIP-1 to inositol derivatives was tested by a PIP strip assay, which showed the requirement of phosphorylation of the inositol group for the interaction of the protein with PIPs. Monolayer adsorption measurements show a clear preference of VILIP-1 binding to PI(4,5)P2 over PI(3,4,5)P3. Moreover, we found co-localisation of VILIP-1 with PI(4,5)P2 at the cell surface membrane in hippocampal neurons, supporting the notion of interactions between VILIP-1 and PIPs in living cells.