Transmembrane Region of the Epidermal Growth Factor Receptor:  Behavior and Interactions via ²H NMR

Abstract

The first wide-line 2H NMR investigation of a receptor tyrosine kinase is reported. Selectively deuterated peptides from the membrane-associated portion of the human epidermal growth factor (EGF) receptor were synthesized for examination in lipid bilayers mimicking certain natural membrane features. The peptide sequence included the 23-amino acid hydrophobic stretch thought to span the membrane (Ile622-Met644 of the EGF receptor), plus the first 10 amino acids of the receptor's cytoplasmic domain (Arg645-Thr654). Dispersion of the peptide with lipid in the lipomimetic solvent, trifluoroethanol (TFE), was found to be a very useful initial step for sample preparation. TFE readily dissolved all components and was then easily removed in vacuo to yield thin films which could be subsequently hydrated to produce bilayers incorporating homogeneously dispersed peptide. Samples extensively studied consisted of 6 mol % peptide in multilamellar liposomes of 1-palmitoyl-2-oleoylphosphatidylcholine and similar liposomes containing cholesterol. 2H NMR spectra of the resulting unsonicated model membranes indicated the existence of peptide monomers undergoing rapid axially symmetric diffusion. It was possible to examine structural and behavioral effects of events often suggested as pivotal in signaling mechanisms and to consider by wide-line NMR for the first time the effect of cholesterol on hydrophobic peptides. When it was incorporated into bilayers by an alternative method involving dialysis of aqueous solutions prepared using a cationic detergent, spectra suggested that the peptide existed primarily as irreversibly aggregated oligomers which were relatively immobile on a time scale of 10(-3)-10(-4) s. For liposomes prepared by hydration of thin films, deuterated methyl groups on the peptide at locations corresponding to Ala623, Met644, and Val650 of the human EGF receptor were individually distinguishable. In highly fluid matrices, spectra suggested the presence of peptide monomers, diffusing symmetrically about axes perpendicular to the membrane. Studied as a function of temperature, 2H NMR spectra of such samples permitted independent consideration of membrane/peptide relationships at separate locations in the receptor tyrosine kinase. None of the locations probed demonstrated significant conformational sensitivity to temperature over a wide range. Effects seen at Ala623 and Met644, at opposite ends of the putative membrane-spanning domain, suggested slight increases in motional order with decreasing temperature. Addition of 33% cholesterol to the membrane caused little apparent conformational change at Val650 or Met644. However, in the presence of the sterol, Met644 and Ala623 exhibited nonaxially symmetric motion at low temperatures, perhaps as a result of peptide oligomerization. Moreover, the presence of cholesterol led to considerable change in spatial arrangement or order at Ala623. There was little evidence to support transmission of conformational changes along the peptide segment probed.

The Transmembrane Domains of ErbB Receptors do not Dimerize Strongly in Micelles

“…While the solid-state NMR experiments are not a direct measure of association and can be difficult to interpret, these studies suggest that the erbB1 TM is largely monomeric in bilayers, with the only signs of interactions being seen at very high concentrations of protein. [57][58][59][60][61] The erbB2 TM appears to exhibit a somewhat greater tendency to associate in the solid-state NMR experiments, but there is still a significant population of molecules that behave as one would expect for an unrestricted monomer. 61 These NMR experiments suggest that the erbB TM peptides are not constitutively associated in bilayers, and thus our inability to detect strong interactions between the erbB TM domains may not be the consequence of the micellar environment required for the sedimentation equilibrium studies.…”

Side Chain and Backbone Dynamics of Phospholamban in Phospholipid Bilayers Utilizing 2H and 15N Solid-State NMR Spectroscopy

“…It has been reported previously that the epidermal growth factor receptor (EGFR tm ) monomer undergoes rapid axial diffusion about its long axes in the membrane (48). Dimerization or oligomerization likely gives rise to a subpopulation exhibiting slower axial rotation (48).…”

“…However, for Leu, the long aliphatic side chain can be isotopically labeled at the δ-and/or ∊-CD 3 sites, and the deuterium NMR powder pattern line shapes will be strongly influenced by the motions about the C γ -C δ bond axis as well as by additional librational motion about the C α -C β and C β -C γ bond axes (see Figure 2B) at various temperatures (40,47). If the CD 3 -methyl probe of the protein undergoes no motion other than those associated with the axial rotation about the C-CD 3 bond in a randomly dispersed sample, the resultant spectra will consist of a Pake pattern with a 40 kHz quadrupolar splitting (48). However, residues located outside the membrane are expected to be more motionally averaged and yield an isotropic peak (49).…”

Structure of the Membrane Reconstituted Transmembrane−Juxtamembrane Peptide EGFR(622−660) and Its Interaction with Ca2+/Calmodulin

“…Grant and co-workers have also characterized the association of EGFR TM peptides using deuterium NMR (32,(37)(38)(39). In their studies, they obtained spectra of the EGFR(621-654) containing deuterated alanine, methionine and valine.…”