The dynamic assembly of multi-protein complexes is a central regulatory mechanism for many cell signalling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive response to rapidly changing cellular conditions. Accordingly, many oncogenes can promote tumourigenesis through the formation of aberrant multi-protein complexes. However, there is still a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signalling complexes.

Here we describe a novel approach that overcomes many of these existing limitations and allows the specific isolation and downstream proteomic characterisation of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners (Croucher et al., Science Signaling, 2016). This novel isolation technique is achieved through the combination of a protein-fragment complementation assay and affinity purification with a conformation specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). We also demonstrate the utility of this approach through characterisation of the specific interactome of the breast cancer oncogene ERBB2, in the form of a homodimer or a heterodimer with either EGFR or ERBB3. Through this analysis we have observed dimer-specific interaction patterns for key adaptor proteins that direct the differential signalling capacity of these dimers, and also identified a number of novel interacting partners. Functional analysis for one novel interaction has also led to the identification of a non-canonical mechanism of ERK activation that is specific to the ERBB2:ERBB3 heterodimer, which acts through the adaptor protein FAM59A to induce ERK activation in breast cancer cells.