MECHANISM OF KIT-LIGAND, PHORBOL ESTER, AND CALCIUM-INDUCED DOWN-REGULATION OF C-KIT RECEPTORS IN MAST-CELLS

The proto-oncogene c-kit is allelic with the white spotting locus (W) on mouse chromosome 5 and it encodes a transmembrane protein tyrosine kinase which belongs to the platelet-derived growth factor and macroph age-colony stimulating factor (CSF-1) receptor subfamily. In an effort to study the function of the c-kit receptor, specifically the physiol ogical mechanism of controlling the signal induced by the ligand, the effect and mechanism of down-regulation of the c-kit receptor by the k it ligand (KL) was investigated in mast cells. Following preincubation with KL, the capacity of mast cells to bind kit antibody was reduced and binding of radiolabeled KL to mast cells decreased with similar ki netics, suggesting that KL stimulates the loss of c-kit receptor from the cell surface. After binding to the c-kit receptor, KL was rapidly internalized, and degradation of the receptor was accelerated. The c-k it receptor was transmodulated by the protein kinase C activator 12-O- tetradecanoylphorbol-13-acetate (TPA) and by the calcium ionophore ion omycin. TPA- and ionomycin-induced down-regulation of the c-kit recept or was accompanied by release of the extracellular domain of the recep tor, presumably by proteolytic cleavage near the transmembrane domain. Release of the extracellular domain of the c-kit receptor occurred al so in untreated cells but at a slow rate. In addition, ionomycin induc ed shedding of the intact c-kit receptor. In mast cells depleted of pr otein kinase C, the c-kit receptor remained sensitive to down-regulati on induced by KL and ionomycin, but not by treatment with TPA. Therefo re, the down-regulation of the c-kit receptor induced by KL, activated protein kinase C, and an increased level of intracellular calcium is mediated through independent mechanisms.