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Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation

Arvidsson, AK; Rupp, E; Nånberg, E; Downward, J; Rönnstrand, Lars LU ; Wennström, S; Schlessinger, J; Heldin, Carl-Henrik and Claesson-Welsh, Lena (1994) In Molecular and Cellular Biology 14(10). p.6715-6726
Abstract
Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated,... (More)
Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor. (Less)
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@misc{b1a8a12e-847b-4405-9152-25058d473cc4,
  abstract     = {Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.},
  author       = {Arvidsson, AK and Rupp, E and Nånberg, E and Downward, J and Rönnstrand, Lars and Wennström, S and Schlessinger, J and Heldin, Carl-Henrik and Claesson-Welsh, Lena},
  issn         = {0270-7306},
  keyword      = {Platelet-Derived Growth Factor/genetics/*metabolism
Sequence Analysis
Signal Transduction
Structure-Activity Relationship,*Adaptor Proteins,Cultured
DNA Mutational Analysis
GRB2 Adaptor Protein
Guanosine Triphosphate/metabolism
Humans
Ligands
Molecular Sequence Data
Peptide Fragments/metabolism
Phosphorylation
Platelet-Derived Growth Factor/*metabolism
Protein Binding
Proteins/*metabolism
Proto-Oncogene Proteins p21(ras)/*metabolism
Receptor Protein-Tyrosine Kinases/genetics/*metabolism
Receptors,Signal Transducing
Amino Acid Sequence
Animals
Base Sequence
Cell Division
Cells},
  language     = {eng},
  number       = {10},
  pages        = {6715--6726},
  publisher    = {ARRAY(0x98cb260)},
  series       = {Molecular and Cellular Biology},
  title        = {Tyr-716 in the platelet-derived growth factor beta-receptor kinase insert is involved in GRB2 binding and Ras activation},
  volume       = {14},
  year         = {1994},
}