Deletion or Substitution within the a Platelet-Derived Growth Factor Receptor Kinase Insert Domain: Effects on Functional Coupling with Intracellular Signaling Pathways

Abstract

The tyrosine kinase domains of the platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-l)/c-fms receptors are interrupted by kinase inserts (ki) which vary in length and amino acid sequence. To define the role of the ki in the human αPDGF receptor (αPDGFR), we generated deletion mutants, designated αRΔki-1 and αRΔki-2, which lacked 80 (710 to 789) and 95 (695 to 789) amino acids of the 104-amino-acid ki region, respectively. Their functional characteristics were compared with those of the wild-type αPDGFR following introduction into a naive hematopoietic cell line, 32D. Biochemical responses, including PDGF-stimulated PDGFR tyrosine phosphorylation, phosphatidylinositol (PI) turnover, and receptor-associated PI-3 kinase activity, were differentially impaired by the deletions. Despite a lack of any detectable receptor-associated PI-3 kinase activity, 32D cells expressing αRΔki-1 showed only partially impaired chemotactic and mitogenic responses and were capable of sustained proliferation in vitro and in vivo under conditions of autocrine stimulation by the c-sis product. 32D transfectants expressing the larger ki deletion (αRΔki-2) showed markedly decreased or abolished biochemical and biological responses. However, insertion of the highly unrelated smaller cfms (685 to 750) ki domain into αRΔki-2 restored each of these activities to wild-type αPDGFR levels. Since the CSF-1R does not normally induce PI turnover, the ability of the c-fms ki domain to reconstitute PI turnover in the αRΔki-2 transfectant provides evidence that the ki domain of the αPDGFR does not directly couple with this pathway. Taken together, all of these findings imply that their ki domains have evolved to play very similar roles in the known signaling functions of PDGF and CSF-1 receptors.