title: The juxtamembrane region of the epidermal growth factor receptor is required for phosphorylation of Galpha(s).
abstract: We have previously demonstrated that Galpha(s) associates with the juxtamembrane region of the epidermal growth factor (EGF) receptor (EGFR) and that the EGFR can phosphorylate and activate this G protein (H. Poppleton et al., 1996, J. Biol. Chem. 271, 6947-6951; H. Sun et al., 1995, Proc. Natl. Acad. Sci. USA 92, 2229-2233). In this report, we have employed peptides EGFR-13 and EGFR-14 (corresponding to amino acids 645-657 and 679-692 in the EGFR, respectively) which disrupt the association of Galpha(s) with the EGFR to investigate whether or not this region of the EGFR is required for phosphorylation of the G protein. EGFR-13 increased the tyrosine phosphorylation of G(alpha)s by two-fold whereas EGFR-14 decreased the phosphorylation of the G protein. Phosphorylation of EGFR-13 on the threonine residue corresponding to Thr654 of the EGFR obliterated the ability of the peptide to increase Galpha(s) phosphorylation. EGFR-13 and EGFR-14, but not phospho-EGFR-13, competed for the association of the EGFR with Galpha(s). A peptide betaIII-2 corresponding to amino acids Arg259-Lys273 in the beta2-adrenergic receptor which competes for association of Galpha(s) with the EGFR and increases protein tyrosine kinase activity of the EGFR could mimic the effects of EGFR-13. Among the three peptides (EGFR-13, EGFR-14, and betaIII-2) that interfere with association of Galpha(s) to the EGFR, only EGFR-13 and betaIII-2 have been shown to activate the G protein. Polylysine which increases EGFR tyrosine kinase activity but does not interfere with association of Galpha(s) and EGFR also augmented phosphorylation of Galpha(s) by the EGFR. Phosphopeptide mapping demonstrated that EGFR-13 and polylysine increased phosphorylation of Galpha(s) by the EGFR on the same additional sites. Collectively, these data suggest that the interaction of Galpha(s) with residues 645-657 of the EGFR, or a peptide corresponding to this sequence alters the conformation of the G protein and/or the EGFR such that Galpha(s) is readily phosphorylated by the EGFR. The peptide EGFR-14, which does not activate Galpha(s), does not allow for the efficient phosphorylation of the G protein even though it does elevate the intrinsic tyrosine kinase activity of the EGFR. The hyperphosphorylation of Galpha(s) by EGFR is likely to require the contact of the G protein with EGFR-13 region (aa 645-657 in the EGFR) as well as augmentation of EGFR kinase activity.
authors.affiliation: Department of Pharmacology, University of Tennessee, The Center for Health Sciences, Memphis 38163, USA.
authors.forename: H M
authors.initials: HM
authors.lastname: Poppleton
authors.forename: H
authors.initials: H
authors.lastname: Sun
authors.forename: J B
authors.initials: JB
authors.lastname: Mullenix
authors.forename: G J
authors.initials: GJ
authors.lastname: Wiepz
authors.forename: P J
authors.initials: PJ
authors.lastname: Bertics
authors.forename: T B
authors.initials: TB
authors.lastname: Patel
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry; D008954:Models, Biological; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000596:Amino Acids; D010455:Peptides; D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins; D011107:Polylysine; D013912:Threonine; D014443:Tyrosine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D020962:Heterotrimeric GTP-Binding Proteins