title: Differential effects of carboxy-terminal sequence deletions on platelet-derived growth factor receptor signaling activities and interactions with cellular substrates.
abstract: Chimeric receptors composed of the human epidermal growth factor receptor (EGF-R) extracellular domain fused to wild-type and truncated platelet-derived growth factor receptor (PDGF-R) intracellular sequences were stably expressed in NIH 3T3 cells devoid of endogenous EGF-Rs. This experimental system allowed us to investigate the biological activity of PDGF-R cytoplasmic-domain mutants in PDGF-R-responsive NIH 3T3 cells by activating PDGF-specific signaling pathways with EGF. Deletion of 74 carboxy-terminal amino acids severely impaired the ability of the PDGF-R cytoplasmic domain to associate with cellular substrates in vitro. This deletion also inhibited receptor and substrate phosphorylation, reduced the receptor's mitogenic activity, and completely abolished its oncogenic signaling potential. Surprisingly, removal of only six additional amino acids, including Tyr-989, restored substantial receptor and substrate phosphorylation capacity as well as transforming potential and yielded a receptor with wild-type levels of ligand-induced mitogenic activity. However, the ability of this chimera to bind phospholipase C gamma was severely impaired in comparison with the ability of the wild-type receptor, while the association with other cellular proteins was not affected. Further deletion of 35 residues, including Tyr-977, nearly abolished all PDGF-R cytoplasmic-domain biological signaling activities. None of the three C-terminal truncations completely abolished the mitogenic potential of the receptors or had any influence on ligand binding or receptor down regulation. Together, these data implicate the 80 C-terminal-most residues of the PDGF-R, and possibly Tyr-989, in phospholipase C gamma binding, while receptor sequences upstream from Asp-988 appear to be essential for specific interactions with other cellular polypeptides such as ras GTPase-activating protein and phosphatidylinositol 3-kinase. Thus, the mutants described here allow the separation of distinct PDGF-activated signaling pathways and demonstrate that phospholipase C gamma phosphorylation is not required for mitogenesis and transformation.
authors.affiliation: Department of Molecular Biology, Max-Planck-Institut für Biochemie, Martinsried, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Seedorf
authors.forename: B
authors.initials: B
authors.lastname: Millauer
authors.forename: G
authors.initials: G
authors.lastname: Kostka
authors.forename: J
authors.initials: J
authors.lastname: Schlessinger
authors.forename: A
authors.initials: A
authors.lastname: Ullrich
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002455:Cell Division; D003001:Cloning, Molecular; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D016296:Mutagenesis; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; D066246:ErbB Receptors; D017479:Receptors, Platelet-Derived Growth Factor; D010738:Type C Phospholipases