title: Probing homodimer formation of epidermal growth factor receptor by selective crosslinking.
abstract: Ligand binding promotes conformational rearrangement of the epidermal growth factor receptor (EGFR) leading to receptor autophosphorylation and downstream signaling. However, transient interactions between unstimulated EGFR molecules on the cell surface are not fully understood. In this report, we describe the investigation of homodimer formation of EGFR by means of an SNAP-tag based selective crosslinking approach (S-CROSS). EGFR homodimers were selectively captured in living cells and utilized for analysis of protein receptor interactions on the plasma membrane and ligand-induced activation. We showed that EGFR forms homodimers in unstimulated cells with efficiencies similar to those seen in cells treated with the epidermal growth factor ligand (EGF) supporting the existence of constitutive transient receptor-receptor interactions. EGFR crosslinked homodimers displayed a substantially increase in kinase activation upon ligand stimulation. Interestingly, in unstimulated cells the levels of spontaneous phosphorylation were found to correlate with the yields of the crosslinked homodimers species. In addition, we demonstrated that this crosslinking approach can be applied to interrogate the effect of small molecule inhibitors on receptor dimerization and kinase activity. Our crosslinking assay provides a new tool to dissect ligand-independent dimerization and activation mechanisms of receptor tyrosine kinases, many of which are important anticancer drug targets.
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Sun
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Florence
authors.initials: F
authors.lastname: Dusserre-Bresson
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Brenda
authors.initials: B
authors.lastname: Baker
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Aihua
authors.initials: A
authors.lastname: Zhang
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Patrick
authors.initials: P
authors.lastname: Xu
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Cassandra
authors.initials: C
authors.lastname: Fibbe
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Noren
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA. Electronic address: correa@neb.com.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.affiliation: New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA. Electronic address: xum@neb.com.
authors.forename: Ming-Qun
authors.initials: MQ
authors.lastname: Xu
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002470:Cell Survival; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D055503:Protein Multimerization / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship
chemical_list: D003432:Cross-Linking Reagents; D008024:Ligands; D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; D004815:Epidermal Growth Factor; D066246:ErbB Receptors
keywords: Phosphorylation; Receptor tyrosine kinase; S-CROSS; SNAP-tag