Peptide antisera to human colony-stimulating factor 1 receptor detect ligand-induced conformational changes and a binding site for phosphatidylinositol 3-kinase.
A peptide antiserum (anti-A) directed to the intracellular, juxtamembrane region (residues 552 to 574) of the human colony-stimulating factor 1 receptor (CSF-1R) precipitated only ligand-activated, native receptors from solution but bound to unstimulated forms after their denaturation. Two peptide antisera (anti-KI1 and -KI2), directed to residues 679 to 700 and 701 to 721, respectively, in the CSF-1R kinase insert (KI) domain and including mapped sites of ligand-induced phosphorylation at Tyr-699 and Tyr-708, bound at least 80% of the receptor molecules expressed in either CSF-1-stimulated or unstimulated cells. Immune complexes formed with anti-KI1, anti-A, or a peptide antiserum to the CSF-1R carboxyl terminus (anti-C-ter) coprecipitated CSF-1R complexed to a phosphatidylinositol 3-kinase (PtdIns 3-K) from CSF-1-stimulated cells, whereas anti-KI2 serum did not. In an in vitro assay, binding of CSF-1R to PtdIns 3-K required receptor tyrosine phosphorylation but not CSF-1R-mediated phosphorylation of the lipid kinase, and the association was specifically blocked by anti-KI2 or antibodies to phosphotyrosine. Neither anti-KI1, anti-A, nor anti-C-ter serum inhibited binding. We conclude that (i) only a minority of ligand-activated receptors form a stable complex with PtdIns 3-K in vivo, (ii) efficient binding of the lipid kinase requires receptor tyrosine phosphorylation within the CSF-1R KI domain, and (iii) a region within the KI domain defined by residues 701 to 721 at least partially overlaps the PtdIns 3-K binding site.
Downing
J R
JR
Department of Tumor Cell Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105.
Shurtleff
S A
SA
Sherr
C J
CJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D000939:Epitopes / Q000032:analysis; D006801:Humans; D007106:Immune Sera; D007074:Immunoglobulin G; D007700:Kinetics; D008024:Ligands; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D019869:Phosphatidylinositol 3-Kinases; D010770:Phosphotransferases / Q000378:metabolism*; D011487:Protein Conformation; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection
D000939:Epitopes; D007106:Immune Sera; D007074:Immunoglobulin G; D008024:Ligands; D010455:Peptides; D016173:Macrophage Colony-Stimulating Factor; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor