SEQUENCE-RELATED BEHAVIOR OF TRANSMEMBRANE DOMAINS FROM CLASS-I RECEPTOR TYROSINE KINASES

H-2 NMR spectroscopy and freeze-fracture elect:ron microscopy were use d 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, EG FR(tm), was synthesised to contain the 23 amino acid hydrophobic stret ch (Ile(622) to Met(644)) thought to span the membrane of the human EG F receptor, plus the first 10 amino acids (Arg(645) to Thr(654)) of th e cytoplasmic domain. Deuterium probes replaced selected H-1 nuclei at sites corresponding to Ala(623), Met(644), and Val(650). The 38-resid ue 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 cyto plasmic domain. Deuterium probes replaced selected H-1 nuclei at Ala(6 61), Leu(667), and Val(676). A third peptide, Neu(tm), was also prepa red, corresponding to the transmembrane domain of a constitutively-act ivating Neu/erbB-2 transformant in which Val(664) is replaced by Glu: it was deuterated in a manner identical to Neu(tm). Peptides were stud ied by H-2 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 degrees to 65 degre es C. Overall motion was found to be different for each of the three p eptides under a given set of conditions. EGFR(tm) spectra were charact eristic of axially symmetric motion in membranes of POPC alone, and in POPC/cholesterol at 35 degrees C and above. In contrast, spectra of t he transmembrane peptides, Neu(tm) and Neu(tm) , were characteristic of significantly axially asymmetric motion under all conditions studie d (and regardless of sample preparation method). Addition of 33% chole sterol to membranes was accompanied by spectral changes consistent wit h increased formation of peptide dimers/oligomers in all cases. The tr ansformant peptide, Neu(tm) , showed greater spectral evidence of imm obilisation than did the wild type - probably reflecting a greater ten dency to form large oligomers. Sequence-related details within the tra nsmembrane domains of Class I receptor tyrosine kinases appear to exer t important control over their associations within membranes. Freeze-f racture electron microscopy of the NMR samples demonstrated their lipo somal nature. Peptide-related intramembranous particles (IMPss) were p resent 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 s pectroscopy reflect structures smaller than the 2 nm resolution limit of freeze-fracture EM and peptide relationships within its 20 nm accur acy of identifying lateral position. (C) 1998 Elsevier Science B.V. Al l rights reserved.