Structural Determinants in the Platelet-derived Growth Factor α-Receptor Implicated in Modulation of Chemotaxis

Koutaro Yokote,Seijiro Mori,Agneta Siegbahn,Lars Rönnstrand,Christer Wernstedt,Carl-Henrik Heldin,Lena Claesson-Welsh

doi:10.1074/jbc.271.9.5101

Abstract

Activation of the platelet-derived growth factor (PDGF) beta-receptor leads to cell growth and chemotaxis. The PDGF alpha-receptor also mediates a mitogenic signal, but fails to induce cell migration in certain cell types. To examine this difference in signal transduction, a series of point-mutated PDGF alpha-receptors were analyzed. Porcine aortic endothelial cells expressing mutant PDGF alpha-receptors, in which tyrosine residues 768, 993, or 1018 were changed to phenylalanine residues migrated toward PDGF, whereas wild-type alpha-receptors and mutant alpha-receptors changed at tyrosine residues 720, 944, or 988 failed to migrate. All mutant receptors were mitogenically active and their capacity to activate phosphatidylinositol 3'-kinase and phospholipase C-gamma was not different from that of the wild-type receptor. Tyr-768 was found to be phosphorylated in PDGF-stimulated cells; in the Y768F mutant, there was a considerable increase in phosphorylation of Ser-767. Tyr-993 was not phosphorylated, but mutation of this tyrosine residue to a phenylalanine residue resulted in increased efficiency of phosphorylation on Tyr-988. Tyr-1018 is known to be an autophosphorylation site. Phosphorylated Tyr-768 and Tyr-1018 may bind signal transduction molecules involved in negative modulation of the chemotactic signaling capacity, whereas phosphorylated Tyr-988 may mediate increased chemotaxis. Thus our data indicate that the PDGF alpha-receptor has an intrinsic ability to transduce a chemotactic signal, and that this signal is counteracted by overriding negative signals.

Highlights

Activation of the platelet-derived growth factor (PDGF) ␤-receptor leads to cell growth and chemotaxis
Porcine aortic endothelial cells expressing mutant PDGF ␣-receptors, in which tyrosine residues 768, 993, or 1018 were changed to phenylalanine residues migrated toward PDGF, whereas wild-type ␣-receptors and mutant ␣-receptors changed at tyrosine residues 720, 944, or 988 failed to migrate
Characterization of porcine aortic endothelial (PAE) Cell Lines Expressing Y720F, Y768F, Y944F, Y988F, Y993F, and Y1018F Mutants of the PDGF ␣-Receptors—Signal transduction by receptor tyrosine kinases is dependent on interactions between autophosphorylated tyrosine residues in the receptors and Src homology 2 (SH2)-domain containing intracellular signaling proteins

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Mutagenesis—Site-directed mutagenesis was performed on a cDNA encoding the full-length human PDGF ␣-receptor [24] using the Altered sites in vitro Mutagenesis System (Promega Corp.). Immunoprecipitation with rabbit antiserum or monoclonal antibody was for 2 h at 4 °C, followed by incubation with protein A-Sepharose CL-4B (Pharmacia) for 30 min at 4 °C. PI 3-Kinase Assay—Serum-starved cells were treated with 50 ng/ml PDGF-BB at 37 °C for 5 min, rinsed with an ice-cold buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, and 100 ␮M Na3VO4 and lysed in the same buffer supplemented with 10% glycerol, 1% Nonidet P-40, 5 mM EDTA, 1% aprotinin, and 1 mM phenylmethylsulfonyl fluoride. In case of immunoprecipitation of tryptic fragments, lyophilized tryptic digests were dissolved in 50 mM ammonium bicarbonate and incubated for 2 h at 4 °C with YSD-antiserum covalently coupled to protein A-Sepharose. Peptides were hydrolyzed in 6 M hydrochloric acid for 1 h at 110 °C, separated by two-dimensional electrophoresis on a cellulose plate, and examined by autoradiography

RESULTS

Receptor type

DISCUSSION