Phosphatidylinositol 3-kinase and Ca2+ influx dependence for ligand-stimulated internalization of the c-Kit receptor.
We have evaluated the role of phosphatidylinositol 3-kinase (PI3-kinase) and Ca2+ influx in ligand-stimulated internalization of the c-Kit receptor. The wild type (wt) c-Kit receptor and YF719, a mutant receptor in which the SH2-mediated binding site for the p85 subunit of PI3-kinase is disrupted, were expressed in DA-1 cells. YF719 internalized with similar kinetics as wt c-Kit although the receptor remained localized close to the plasma membrane. However, in the absence of extracellular Ca2+, or in the presence of the competitive Ca2+ influx blocker Ni2+, the YF719 mutant failed to internalize. Failure to internalize in the absence of Ca2+ was also observed for the wt c-Kit receptor in cells that were pretreated with the PI3-kinase inhibitor, wortmannin. Following 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 failed to co-immunoprecipitate with c-Kit. Our results demonstrate that both Ca2+ influx and PI3-kinase activity influence c-Kit endocytosis, and inhibition of these two signals disrupts the earliest stages of ligand-mediated internalization.
Gommerman
J L
JL
Wellesley Hospital Research Institute and Department of Immunology, University of Toronto, Toronto, Ontario, Canada M4Y 1J3. Berger@WHRI.ON.CA
Rottapel
R
R
Berger
S A
SA
D016475:3T3 Cells; D019943:Amino Acid Substitution; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002966:Clathrin / Q000502:physiology; D015536:Down-Regulation; D004705:Endocytosis; D008024:Ligands; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D000077191:Wortmannin
D000730:Androstadienes; D002966:Clathrin; D008024:Ligands; D019869:Phosphatidylinositol 3-Kinases; D019009:Proto-Oncogene Proteins c-kit; D002118:Calcium; D000077191:Wortmannin