Dh. Jones et al., THE EGF RECEPTOR TRANSMEMBRANE DOMAIN - H-2 NMR-STUDY OF PEPTIDE PHOSPHORYLATION EFFECTS IN A BILAYER ENVIRONMENT, Biochemistry, 37(20), 1998, pp. 7504-7508
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.