Lateral dimerization of transmembrane (TM) α-helical domains plays an important role in receptor tyrosine kinase (RTK)-mediated signal transduction1–3
. Since RTK dimers are active while monomers are inactive, the dimerization process controls RTK activity. The TM domains are known to contribute as much as −3 kcal/mol to the dimerization energetics4–7
, and defects in dimerization due to single residue mutations in RTK TM domains are known to cause human pathologies, including cancer and dwarfism. TM helix dimerization is believed to be driven by particular sequence motifs8–10
. Experiments have successfully identified a few dimerization motifs for TM helix dimers using a variety of model systems, including SDS-PAGE gels, detergent micelles and bicelles, lipid vesicles, bacterial membranes and mammalian membranes (reviewed in8
). Yet, despite these successes, we currently do not know the interaction motifs for most RTK TM domains, and more importantly we cannot predict if a particular TM sequence will form dimers in membranes. Even when dimerization has been demonstrated experimentally, a prediction for the TM dimer interaction interface cannot be made with certainty. Finally, we cannot yet design dimerization motifs de novo
, underscoring the importance of the work that is yet to be done in this field. The successful de novo
design of TM sequences that interact strongly with RTK TM domains would be an important achievement, as it will allow for the development of novel RTK inhibitors that could be used in the clinic.
Information on TM helix dimerization has been derived from synthetic peptides, peptide-protein chimeras and full-length membrane proteins. Numerous methods exist to measure dimerization and such measurements have been made in many different hydrophobic “membrane mimetic” environments11
. In detergent micelles, TM helix dimerization has been measured using FRET, analytical ultracentrifugation, and cysteine cross-linking12–14
. In lipid bilayers, TM helix dimerization has been characterized using FRET, cysteine cross-linking, and recently, an elegant novel steric trap approach15–17
. In bacterial membranes, TM helix dimerization has been studied using genetic reporter assays such as TOXCAT, ToxR and GALLEX18–20
. Finally, interactions between TM helices have been probed in mammalian cell membranes using FRET-based assays5,21
One of the earliest methods used to study TM helix dimerization, SDS-PAGE, is fast, simple, and is used in most laboratories. SDS-PAGE is often the method of choice for initial characterization of TM helix interactions22,23
and has been used to define the most well understood dimerization motif: the GxxxG motif that drives the dimerization of the TM domain of glycophorin A (GpA)24
. While often reliable for assaying TM helix dimerization, SDS-PAGE has been shown sometimes to yield misleading results25,26
. Thus, the extent of SDS validity is an open question. Here we investigate if SDS-PAGE can be used as a simple high-throughput screening method to identify strongly interacting TM sequences that can activate an RTK better than its own TM domain in mammalian membranes. Furthermore, in this work we address the following questions. (1) How potent and how promiscuous are the known dimerization motifs, as detected by SDS-PAGE? (2) What is the correlation between RTK TM domain dimerization in SDS-PAGE and RTK activation in mammalian membranes? (3) Do small changes in TM domain sequences affect RTK activation? (4) What is the importance of the GxxxG dimerization motif in RTK activation? and (5) How much can RTK activation be varied by changing the TM domain sequence?
The model RTK that we use for this work is rat Neu. Neu (ErbB2 or HER2) belongs to the Epidermal Growth Factor (EGF) receptor family and is unique because it has no ligand. Thus, its activation is regulated exclusively by lateral dimerization. A single residue mutation in the Neu TM domain, V664E, is oncogenic due to increased dimerization and increased activation compared with wild-type27–29
. It has been proposed that the effect results from the formation of Glu-mediated hydrogen bonds between the two helices30,31
. While this mutation has not been identified in humans thus far, the mutation is activating when engineered into the human sequence.
In this work we used the wild-type Neu TM domian, Neu/WT, which is monomeric in SDS-PAGE, and Neu/V664E TM domain, which is dimeric in SDS-PAGE, as the standards to which we compare the dimerization and activation driven by novel Neu TM analog sequences. We designed a peptide library based on the Neu TM domain sequence in which we varied five residues that are likely involved in the interaction interface (). The 3,888-member library contained several thousand members with at least one known dimerization motif, including the GxxxG motif identified to be important for GpA dimerization. The library included other motifs of two small residues spaced by three residues, also believed to drive TM helix dimerization and referred to as SmxxxSm motifs (where Sm is a small amino-acid such as Ala, Ser or Thr, in addition to Gly)32
. The library also included polar and charged residues, believed to stabilize TM helix dimers via hydrogen bonds or salt-bridges8,33,34
Rational combinatorial library design
We used SDS-PAGE-based screening to identify sequences within the library that homodimerize more strongly than Neu/WT. A very small number of homodimeric sequences were identified in the SDS-PAGE-based screen, demonstrating that the presence of known dimerization motifs do not guarantee strong interactions. Next, we hypothesized that, if the SDS-PAGE environment is relevant to interactions in mammalian membranes, the selected homodimeric sequences would enhance Neu activation in mammalian membranes. Therefore we measured the activation of chimeric Neu receptors containing these selected TM domain sequences. Some, but not all, of the selected TM domains increased Neu activation in mammalian cell membranes. These results provide answers for the five questions posed above and suggest that SDS-PAGE may be a useful, but not ideal, high-throughput tool for identifying sequences that alter the biological function of RTKs.