Intracellular neuronal calcium sensor (NCS) protein VILIP-1 modulates cGMPsignalling pathways in transfected neural cells and cerebellar granule neurones

The family of intracellular neuronal calcium-sensors (NCS) belongs to the s uperfamily of EF-hand proteins. Family members have been shown by in vitro assays to regulate signal cascades in retinal photoreceptor cells. To study the functions of NCS proteins not expressed in photoreceptor cells we exam ined Visinin-like protein-1 (VILIP-1) effects on signalling pathways in liv ing neural cells. Visinin-like protein-1 expression increased cGMP levels i n transfected C6 and PC12 cells. Interestingly, in transfected PC12 cells s timulation was dependent on the subcellular localization of VILIP-1. In cel ls transfected with membrane-associated wild-type VILIP-1 particulate guany lyl cyclase (GC) was stimulated more strongly than soluble GC. In contrast, deletion of the N-terminal myristoylation site resulted in cytosolic local ization of VILIP-1 and enhanced stimulation of soluble GC. To study the mol ecular mechanisms underlying GC stimulation VILIP-1 was examined to see if it can physically interact with GCs. A direct physical interaction of VILIP -1 with the recombinant catalytic domain of particulate GCs-A, B and with n ative GCs enriched from rat brain was observed in GST pull-down as well as in surface plasmon resonance interaction studies. Furthermore, following tr ituration of recombinant VILIP-1 protein into cerebellar granule cells the protein influenced only signalling by GC-B. Together with the observed colo calization of GC-B, but not GCA with VILIP-1 in cerebellar granule cells, t hese results suggest that VILIP-1 may be a physiological regulator of GC-B.