title: The role of tyrosine residues in fibroblast growth factor receptor 1 signaling in PC12 cells. Systematic site-directed mutagenesis in the endodomain.
abstract: To assess the contribution of the intracellular domain tyrosine residues to the signaling capacity of fibroblast growth factor receptor 1 (FGFR1), stably transfected chimeras bearing the ectodomain of the platelet-derived growth factor receptor (PDGFR) and the endodomain of FGFR1 were systematically altered by a tyrosine to phenylalanine bloc and individual conversions. The 15 tyrosine residues of the endodomain of this construct (PFR1) were divided into four linear segments (labeled A, B, C, and D) that contained 4, 4, 2, and 5 tyrosine residues, respectively. When stimulated by platelet-derived growth factor, derivatives in which the A, B, or A + B blocs of tyrosines were mutated were about two-thirds as active as the unmodified chimera at 48 h but achieved full activity by 96 h in a neurite outgrowth assay in transfected PC12 cells. Elimination of only the two activation loop tyrosines (C bloc) also inactivated the receptor. All derivatives in which 4 (or 5) of the D bloc tyrosines were mutated were inactive in producing differentiation but showed low levels of kinase activity in in vitro assays. Derivatives in which 1, 2, or 3 tyrosines of the D bloc in different combinations were systematically changed demonstrated that 2 residues (Tyr(677) and Tyr(701), using hFGFR1 numbering) were essential for bioactivity, but the remaining 3 residues, including Tyr(766), the previously identified site for phospholipase C gamma (PLC gamma) activation, were not. Differentiation activity was paralleled by the activation (phosphorylation) of FRS2, SOS, and ERK1/2. PLC gamma activity was dependent on the presence of Tyr(766) but also required Tyr(677) and/or Tyr(701). Although fully active chimeras did not require PLC gamma, the responses of chimeras showing reduced activation of FRS2 were significantly enhanced by this activity. These results establish that PFR1 does not utilize any tyrosine residues, phosphorylated or not, to activate FRS2. However, it does require Tyr(677) and/or Tyr(701), which may function to stabilize the active conformation directly or indirectly.
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, University of California, Irvine, California 92697, USA.
authors.forename: E D
authors.initials: ED
authors.lastname: Foehr
authors.forename: S
authors.initials: S
authors.lastname: Raffioni
authors.forename: J
authors.initials: J
authors.lastname: Murray-Rust
authors.forename: R A
authors.initials: RA
authors.lastname: Bradshaw
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002455:Cell Division; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis; D016501:Neurites / Q000187:drug effects / Q000473:pathology; D016716:PC12 Cells; D010455:Peptides / Q000494:pharmacology; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D051496:Receptor, Fibroblast Growth Factor, Type 1; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C106534:FRS2 protein, human; D007527:Isoenzymes; D008565:Membrane Proteins; D010455:Peptides; D010750:Phosphoproteins; D017468:Receptors, Fibroblast Growth Factor; D014443:Tyrosine; C496350:Fgfr1 protein, rat; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma