PHOSPHATIDYLINOSITOL 3-KINASE AND CA2-STIMULATED INTERNALIZATION OF THE C-KIT RECEPTOR( INFLUX DEPENDENCE FOR LIGAND)

We have evaluated the role of phosphatidylinositol 3-kinase (PI3-kinas e) and Ca2+ influx in ligand-stimulated internalization of the c-Kit r eceptor. The wild type (wt) c-Kit receptor and YF719, a mutant recepto r in which the SH2-mediated binding site for the p85 subunit of PI3-ki nase is disrupted, were expressed in DA-1 cells. YF719 internalized wi th similar kinetics as wt c-Kit although the receptor remained localiz ed close to the plasma membrane. However, in the absence of extracellu lar Ca2+, or in the presence of the competitive Ca2+ influx blocker Ni 2+, the YF719 mutant failed to internalize, Failure to internalize in the absence of Ca2+ was also observed for the wt c-Kit receptor in cel ls that were pretreated with the PI3-kinase inhibitor, wortmannin. Fol lowing stimulation with ligand, clathrin heavy chains were found to co -immunoprecipitate with c-Kit, However, under conditions in which PI3- kinase activity is inhibited and Ca2+ influx is blocked, clathrin fail ed to co-immunoprecipitate with c-Kit, Our results demonstrate that bo th Ca2+ influx and PI3-kinase activity influence c-Kit endocytosis, an d inhibition of these two signals disrupts the earliest stages of liga nd-mediated internalization.