title: Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers.
abstract: The dynamic assembly of multiprotein complexes is a central mechanism of many cell signaling pathways. This process is key to maintaining the spatiotemporal specificity required for an accurate, yet adaptive, response to rapidly changing cellular conditions. We describe a technique for the specific isolation and downstream proteomic characterization of any two interacting proteins, to the exclusion of their individual moieties and competing binding partners. We termed the approach bimolecular complementation affinity purification (BiCAP) because it combines the use of conformation-specific nanobodies with a protein-fragment complementation assay with affinity purification. Using BiCAP, we characterized the specific interactome of the epidermal growth factor receptor (EGFR) family member ERBB2 when in the form of a homodimer or when in the form of a heterodimer with either EGFR or ERBB3. We identified dimer-specific interaction patterns for key adaptor proteins and identified a number of previously unknown interacting partners. Functional analysis for one of these newly identified partners revealed a noncanonical mechanism of extracellular signal-regulated kinase (ERK) activation that is specific to the ERBB2:ERBB3 heterodimer and acts through the adaptor protein FAM59A in breast cancer cells.
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia. St. Vincent's Hospital Clinical School, University of New South Wales, Sydney, New South Wales 2052, Australia. School of Medicine, University College Dublin, Belfield, Dublin D4, Ireland. d.croucher@garvan.org.au d.saunders@unsw.edu.au.
authors.forename: David R
authors.initials: DR
authors.lastname: Croucher
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.
authors.forename: Mary
authors.initials: M
authors.lastname: Iconomou
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.
authors.forename: Jordan F
authors.initials: JF
authors.lastname: Hastings
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia. Systems Biology Ireland, University College Dublin, Belfield, Dublin D4, Ireland.
authors.forename: Sean P
authors.initials: SP
authors.lastname: Kennedy
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.
authors.forename: Jeremy Z R
authors.initials: JZ
authors.lastname: Han
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Shearer
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.
authors.forename: Jessie
authors.initials: J
authors.lastname: McKenna
authors.affiliation: Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada.
authors.forename: Adrian
authors.initials: A
authors.lastname: Wan
authors.affiliation: Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada.
authors.forename: Joseph
authors.initials: J
authors.lastname: Lau
authors.affiliation: Department of Molecular Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia V5Z 1L3, Canada. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada.
authors.forename: Samuel
authors.initials: S
authors.lastname: Aparicio
authors.affiliation: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia. School of Medical Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia. d.croucher@garvan.org.au d.saunders@unsw.edu.au.
authors.forename: Darren N
authors.initials: DN
authors.lastname: Saunders
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D066246:ErbB Receptors / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D061986:MCF-7 Cells; D055503:Protein Multimerization; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism; D020893:Receptor, ErbB-3 / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D066246:ErbB Receptors; D018719:Erb-b2 Receptor Tyrosine Kinases; D020893:Receptor, ErbB-3; C512478:EGFR protein, human; C508053:ERBB2 protein, human; C581292:ERBB3 protein, human