CALBINDIN(28KDA) AND CALMODULIN ARE HYPERABUNDANT IN RAT DENTAL ENAMEL CELLS - IDENTIFICATION OF THE PROTEIN PHOSPHATASE CALCINEURIN AS A PRINCIPAL CALMODULIN TARGET AND OF A SECRETION-RELATED ROLE FOR CALBINDIN(28KDA)
Mj. Hubbard, CALBINDIN(28KDA) AND CALMODULIN ARE HYPERABUNDANT IN RAT DENTAL ENAMEL CELLS - IDENTIFICATION OF THE PROTEIN PHOSPHATASE CALCINEURIN AS A PRINCIPAL CALMODULIN TARGET AND OF A SECRETION-RELATED ROLE FOR CALBINDIN(28KDA), European journal of biochemistry, 230(1), 1995, pp. 68-79
Enamel cells are likely to experience heavy demands for intracellular
calcium homeostasis during the secretion and hypermineralization of de
ntal enamel. Here, the two major high-affinity calcium-binding protein
s in rat enamel epithelium were identified as calbindin(28 kDa) and ca
lmodulin, using a microscale approach. Both proteins were hyperabundan
t, totalling up to 2% of the soluble protein and surpassing the amount
s in cerebellum the benchmark tissue. Calbindin(28 kDa) and calmodulin
accounted for 26% of the total calcium-binding capacity in enamel cel
l cytosol, under near physiological conditions. Numerous calmodulin-bi
nding proteins were detected with an overlay assay, indicating that ca
lmodulin has multiple major targets in enamel cells. The calcium/calmo
dulin-regulated protein phosphatase, calcineurin, was identified as a
principal calmodulin target constituting 0.1% of the soluble protein.
Calmodulin and calcineurin were expressed constitutively, implying con
tinued heavy usage of calcium/calmodulin-based and phosphorylation-bas
ed signalling events throughout enamel cell development. Calbindin(28
kDa), in contrast, was expressed at fourfold higher levels in secretio
n-phase cells than during the calcium-intensive hypermineralization ph
ase, unexpectedly pointing to an important role associated with secret
ion. Supporting this notion, immunoblots revealed that 33% of total (S
DS-soluble) calbindin(28 kDa), was in the particulate fraction and pre
dominantly associated with the Triton-insoluble cytoskeleton. Solubili
sation of cytoskeletal calbindin(28 kDa) required high concentrations
of NaCl or urea, indicating the existence of a high-affinity target li
gand. The unusual abundance of calmodulin, calbindin(28 kDa) and calci
neurin demonstrated here provides the first molecular evidence that en
amel cells possess a strong capability for intracellular calcium homeo
stasis. Since none of these proteins was up-regulated during enamel hy
permineralization, it appears that other calcium-binding proteins are
primarily involved in the putative transcellular passage of calcium.