The juxtamembrane region of the epidermal growth factor receptor is required for phosphorylation of Galpha(s).
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.
Poppleton
H M
HM
Department of Pharmacology, University of Tennessee, The Center for Health Sciences, Memphis 38163, USA.
Sun
H
H
Mullenix
J B
JB
Wiepz
G J
GJ
Bertics
P J
PJ
Patel
T B
TB
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
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