The neuronal calcium sensors are a family of EF-hand-containing Ca2+-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin δ, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin δ, we have identified brain cytosolic proteins that bind to neurocalcin δ in a Ca2+-dependent manner. We used immobilized recombinant myristoylated neurocalcin δ combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, α- and β-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin δ indicating that the N-terminal myristoyl group may be important for protein—protein interactions in addition to membrane association. Direct binding of neurocalcin δ to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin δ by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin δ was cytosolic at resting Ca2+ levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca2+ elevation. These data suggest the possibility that neurocalcin δ functions in the control of clathrin-coated vesicle traffic.
Abbreviations used: ARF, ADP-ribosylation factor; DTT, dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IPTG, isopropyl β-d-thiogalactoside; MALDI—TOF, matrix-assisted laser-desorption ionization—time-of-flight; NCS, neuronal calcium sensor; myr-ARF, myristoylated ARF; myr-NCS-1, myristoylated NCS-1; myr-neurocalcin δ, myristoylated neurocalcin δ; NMT, N-myristoyl transferase; TGN, trans-Golgi network; VILIP, visinin-like protein.