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

Abstract

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.