Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin δ: interaction with actin, clathrin and tubulin

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.

S100β is a target protein of neurocalcin δ, an abundant isoform in glial cells

To clarify the function of neurocalcin delta, an isoform found abundantly in glial cells, we attempted to find its target proteins by using neurocalcin delta-affinity chromatography and the 125I-neurocalcin delta gel-overlay method. The 10, 14, 27, 36 and 50 kDa bands found on SDS/PAGE bound to 125I-neurocalcin delta, and 10, 11, 19, 24, 26, 50 and 70 kDa proteins were eluted from a neurocalcin delta-affinity column in a Ca(2+)-dependent manner. Sequence analysis of proteolytic peptides revealed the following identities: S100 beta (10 kDa), S100 alpha (11 kDa), myelin basic protein (19 kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa) and tubulin beta-chain (50 kDa). A zero-length cross-linking study indicated that 1 mol of S100 beta bound to 1 mol of neurocalcin delta. With the gel-overlay method, purified S100 beta protein and calcyclin bound to 125I-neurocalcin delta whereas calgizarrin and calvasculin, other members of the S100 family, did not. These findings suggest that S100 beta is one of the target proteins of neurocalcin delta, and the neurocalcin delta-S100 beta complex may be involved in Ca(2+)-signalling in the glial cell.