Inhibition by transmembrane peptides of chimeric insulin receptors

Abstract

Receptor tyrosine kinases play essential roles in cell proliferation and differentiation. We have recently shown that peptides corresponding to the transmembrane domains of the epidermal growth factor (EGF) and ErbB2 receptors inhibit their corresponding receptor activation in cancer cell lines. We extend this observation to cells transfected with chimeric insulin receptors where the transmembrane domain has been replaced by that of the EGF receptor or a mutated Erb2 domain. Peptides corresponding to the transmembrane domains of the EGF receptor and ErbB2 are able to inhibit specifically the autophosphorylation of insulin receptors with the corresponding domain. This inhibitory effect is correlated with the propensity of the different transmembrane domains to self-associate in a genetic reporter assay. Thus, our data strengthen the notion that transmembrane domains are involved in erbB receptor activation, and that these receptors can be modulated by inhibiting protein-protein interactions within the membrane.

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.…”

“…When TM domain interactions induced dimerization of this fusion protein, the reporter gene was activated. Here, we used a modified version in which the reporter gene encodes for the luciferase protein (Bennasroune et al, 2005). TM domains of NRP1, mutated NRP1, ErbB2, GPA, and mutated GPA were used for fusion protein construction.…”

“…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.…”

“…The ToxLuc system (Bennasroune et al, 2005) is a modification of the TOXCAT system (Russ and Engelman, 1999) originally designed to screen for TM domains that mediate dimerization; we therefore used this system to assess TM NRP1 dimerization. Different vectors encoding the TM sequences known to mediate dimerization were used as references for dimerization capacity.…”

The Existence of FGFR1–5-HT1A Receptor Heterocomplexes in Midbrain 5-HT Neurons of the Rat: Relevance for Neuroplasticity

“…TM-I peptide consisted of residues 37-62 (VITSLLLGTLIFCAVLGNACVVAAIA); TM-II peptide of residues 74 -98 (LIGSLAVTDLMVSVLVLPMAALYQ V); TM-III peptide of residues 110 -132 (DLFIALDVLCCTSSILHLCAIA L); TM-IV peptide of residues 153-178 (AAALISLTWLIGFLISIPPMLG WRTP); TM-V peptide of residues 192-217 (DHGYTIYSTFGAFYIPLLL MLVLYGR); TM-VI peptide of residues 346 -367 (TLGIIMGTFILCWL PFFIVALV); and TM-VII peptide of residues 379 -403 (TLLGAIINWL GYSNSLLNPVIYAYF). At the C-terminal juxtamembrane sequence of each TM peptide was introduced the tribasic sequence (RKR) as found in many membrane proteins to reduce possible artifact formation through disulfide bridges and to ensure incorporation into the plasma membrane of cells, as has been demonstrated previously (Bennasroune et al, 2004(Bennasroune et al, , 2005. Immediately before use, the peptides were solubilized in dimethyl sulfoxide (DMSO) and diluted in the corresponding cell culture medium to yield a final concentration of 1% DMSO.…”