Peptide Antisera to Human Colony-Stimulating Factor 1 Receptor Detect Ligand-Induced Conformational Changes and a Binding Site for Phosphatidylinositol 3-Kinase

Abstract

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 (Ptdlns 3-K) from CSF-1-stimulated cells, whereas anti-KI₂ serum did not. In an in vitro assay, binding of CSF-1R to Ptdlns 3-K required receptor tyrosine phosphorylation but not CSF-lR-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 Ptdlns 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.