Transmembrane Peptides as Inhibitors of ErbB Receptor Signaling

Abstract

Receptor tyrosine kinases have a single transmembrane (TM) segment that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, mutations within some of these receptors, and recent studies with the epidermal growth factor (EGF) and ErbB2 receptors have indicated that interactions between TM domains do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimerization and activation. One consequence of the importance of these interactions is that short hydrophobic peptides corresponding to these domains should act as specific inhibitors. To test this hypothesis, we constructed expression vectors encoding short fusion peptides encompassing native or mutated TM domains of the EGF, ErbB2, and insulin receptors. In human cell lines overexpressing the wild-type EGF receptor or ErbB2, we observed that the peptides are expressed at the cell surface and that they inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor. Identical results were obtained with peptides chemically synthesized. Mechanism of action involves inhibition of dimerization of the receptors as shown by the lack of effects of mutant nondimerizing sequences, completed by density centrifugation and covalent cross-linking experiments. Our findings stress the role of TM domain interactions in ErbB receptor function, and possibly for other single-spanning membrane proteins.

Transmembrane Domain Interactions Control Biological Functions of Neuropilin-1

“…Such a motif has been shown to be important for the dimerization of TM segments of ErbB receptors (Mendrola et al, 2002). Consistently with previous work by Marchesi and colleagues (Bormann et al, 1989), we have also recently demonstrated that synthetic peptides mimicking TM domains are able to inhibit specifically kinase activity and cell signaling by EGF (erbB1) and erbB2 receptors in cultured cells (Bennasroune et al, 2004) as well as chimeric insulin receptors containing these domains (Bennasroune et al, 2005). This inhibition indeed relates to the presence of the GxxxG-type sequence.…”

“…For example, interactions between TM domains contribute to receptor dimerization and thus play a role in the activation of tyrosine kinase receptors of the ErbB family (Mendrola et al, 2002;Bennasroune et al, 2004), the erythropoietin receptor (Constantinescu et al, 2001), alphaIIb integrin (Li et al, 2004), or the T-cell receptor (Teixeiro et al, 2002). These interactions depend on the presence of sequence motifs that govern the packing of the TM helices into homo-or hetero-oligomers (Gordon-Weeks et al, 1989;Arkin, 2002;Curran and Engelman, 2003;Senes et al, 2004).…”

“…The original C-terminal juxtamembrane sequence of the NRP1 TM peptide (CTC) was replaced by a more classical tribasic sequence (RKR) as found in many membrane proteins in order to reduce possible artifactual formation of disulfide bridges. We have previously shown that other TM peptides containing such a tribasic Cterminus do readily incorporate into the plasma membrane of cultured cells (Bennasroune et al, 2004(Bennasroune et al, , 2005. The first peptide corresponding to the TM sequence of NRP1: ILITIIAMSALGVLLGAVCGVVLYRKR (in one-letter code, amino acids I 857 to Y 880 plus RKR according to Swissprot entry P97333) will be referred to as pTM-NRP1.…”

“…Peptide purity estimated by reverse phase-HPLC (RP-HPLC, Beckman Gold apparatus, Vydac 219-TP510 diphenyl columns) was higher than 90%. Peptides were routinely prepared in a micellar form in 7 mM LDS (lithium dodecylsulfate), and administered to cells in a small volume so that detergent concentration fell well below its critical micelle concentration (CMC), ensuring partitioning of the peptides in cell membranes (Bennasroune et al, 2004).…”

“…This inhibitory effect requires the binding of Sema3A to NRP1 (Castellani and Rougon, 2002). Previous work in our laboratory has demonstrated that synthetic peptides mimicking the TM domains are useful tools for investigating the biological function of such domains (Bennasroune et al, 2004). We therefore performed a growth cone collapse assay to test the ability of pTM-NRP1 to block the activity of Sema3A.…”