Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin delta: interaction with actin, clathrin and tubulin.
The neuronal calcium sensors are a family of EF-hand-containing Ca(2+)-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin delta, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin delta, we have identified brain cytosolic proteins that bind to neurocalcin delta in a Ca(2+)-dependent manner. We used immobilized recombinant myristoylated neurocalcin delta combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, alpha- and beta-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin delta indicating that the N-terminal myristoyl group may be important for protein-protein interactions in addition to membrane association. Direct binding of neurocalcin delta to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin delta by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin delta was cytosolic at resting Ca(2+) levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca(2+) elevation. These data suggest the possibility that neurocalcin delta functions in the control of clathrin-coated vesicle traffic.
Ivings
Lenka
L
The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, U.K.
Pennington
Stephen R
SR
Jenkins
Roz
R
Weiss
Jamie L
JL
Burgoyne
Robert D
RD
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019921:Biotinylation; D001923:Brain Chemistry; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002850:Chromatography, Gel; D002966:Clathrin / Q000378:metabolism*; D004533:Egtazic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051596:Neurocalcin; D010449:Peptide Mapping; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D044169:Receptors, Calcium-Sensing; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D014404:Tubulin / Q000378:metabolism*
D000199:Actins; D002135:Calcium-Binding Proteins; D002966:Clathrin; C496685:NCALD protein, human; C496686:Ncald protein, mouse; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D044169:Receptors, Calcium-Sensing; D014404:Tubulin; C496690:Vsnl1 protein, rat; D004533:Egtazic Acid; D002118:Calcium