Sequence-related behaviour of transmembrane domains from class I receptor tyrosine kinases

Abstract

2 H NMR spectroscopy and freeze-fracture electron microscopy were used to compare the transmembrane domains of two Class I protein receptor tyrosine kinases (the EGF receptor and Neu/erbB-2) regarding overall behaviour in fluid lipid bilayer membranes. The 34-residue peptide, EGFR tm, was synthesised to contain the 23 amino acid hydrophobic stretch (Ile 622 to Met 644) thought to span the membrane of the human EGF receptor, plus the first 10 amino acids (Arg 645 to Thr 654) of the cytoplasmic domain. Deuterium probes replaced selected 1 H nuclei at sites corresponding to Ala 623, Met 644, and Val 650. The 38-residue peptide, Neu tm, was synthesised having the 21 residue hydrophobic stretch (Ile 660 to Ile 680) calculated to span the membrane in rat Neu/erbB-2, plus residues Lys 681 to Thr 691 of the contiguous cytoplasmic domain. Deuterium probes replaced selected 1 H nuclei at Ala 661, Leu 667, and Val 676. A third peptide, Neu tm*, was also prepared, corresponding to the transmembrane domain of a constitutively-activating Neu/erbB-2 transformant in which Val 664 is replaced by Glu: it was deuterated in a manner identical to Neu tm. Peptides were studied by 2 H NMR spectroscopy at 1 mol% and 6 mol% in unsonicated fluid bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and in POPC containing 33 mol% cholesterol, over the range 12° to 65°C. Overall motion was found to be different for each of the three peptides under a given set of conditions. EGFR tm spectra were characteristic of axially symmetric motion in membranes of POPC alone, and in POPC/cholesterol at 35°C and above. In contrast, spectra of the transmembrane peptides, Neu tm and Neu tm*, were characteristic of significantly axially asymmetric motion under all conditions studied (and regardless of sample preparation method). Addition of 33% cholesterol to membranes was accompanied by spectral changes consistent with increased formation of peptide dimers/oligomers in all cases. The transformant peptide, Neu tm*, showed greater spectral evidence of immobilisation than did the wild type - probably reflecting a greater tendency to form large oligomers. Sequence-related details within the transmembrane domains of Class I receptor tyrosine kinases appear to exert important control over their associations within membranes. Freeze-fracture electron microscopy of the NMR samples demonstrated their liposomal nature. Peptide-related intramembranous particles (IMPs) were present which likely represent oligomers of the transmembrane peptide. IMP size and distribution were similar under a given set of conditions for all three peptides, suggesting that the differences seen by NMR spectroscopy reflect structures smaller than the 2 nm resolution limit of freeze-fracture EM and peptide relationships within its 20 nm accuracy of identifying lateral position.