THE EGF RECEPTOR TRANSMEMBRANE DOMAIN - H-2 NMR-STUDY OF PEPTIDE PHOSPHORYLATION EFFECTS IN A BILAYER ENVIRONMENT

Phosphorylation events are considered to be key control points in rece ptor tyrosine kinase function. We have used wide-line H-2 NMR spectros copy to look for physical effects of phosphorylating a threonine resid ue within the cytoplasmic domain of the human EGF receptor, as sensed at a distant site in the transmembrane portion. Modifications were mad e to Thr(654) (a cytoplasmic residue suggested to be involved in regul ation of EGF binding and of cytoplasmic domain function), and effects were sought at Ala(623) (near the extracellular membrane surface but w ithin the membrane spanning region). The study was carried out on synt hetic peptides corresponding to the EGF receptor transmembrane domain plus 10 or 11 residues of the cytoplasmic domain, assembled into lipid bilayer membranes. Three peptides were compared that differed only at Thr(654). This residue was alternately: nonphosphorylated but left as a (-)-charged C-terminus (-Thr(654)COO(-)), nonphosphorylated and wit h a neighboring amidated glycine residue as the C-terminus (-Thr(654)G lyCONH(2)), or phosphorylated and with a neighboring amidated glycine residue as the C-terminus (-Thr(654)PO(4)(-)GlyCONH(2)), Bilayer membr anes were composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) o r 2:1 POPC/cholesterol, containing 6 mol % peptide relative to phospho lipid. The deuterated site, Ala(623), was intrinsically conformational ly sensitive; yet spatial orientation and motional order of the probe location were found not to be obviously influenced by phosphorylation.