title: Multiplex detection of homo- and heterodimerization of g protein-coupled receptors by proximity biotinylation.
abstract: Dimerization of G protein-coupled receptors (GPCRs) represents a potential mechanism by which GPCR functions are regulated. Several resonance energy transfer (RET)-based methods have revealed GPCR homo- and heterodimerization. However, interpretation of an increase in FRET efficiency could be attributed to either dimerization/oligomerization events or conformational changes within an already dimerized/oligomerized receptor complex. Furthermore, RET-based methods can only measure pairwise dimerization, and cannot easily achieve multiplex detection. In this study, we applied proximity-based biotinylation for detecting receptor dimerization by utilizing a specific enzyme-substrate pair that are fused to GPCRs. The biotin ligase BirA is fused to CXCR4 and site-specifically biotinylates an acceptor peptide (AP) in the presence of biotin. As a test case for our newly developed assay, we have characterized the homo-dimerization of chemokine receptor CXCR4 and heterodimerization of CXCR4 with CCR2 or CCR5. The degree of biotinylation varies with the amount of GPCR-AP as well as biotinylation time. Using enzyme/substrate receptor pairs and measuring receptor biotinylation, we demonstrate that CXCR4 can homo-dimerize and hetero-dimerize with CCR2 and CCR5. The effect of CXCL12, agonist for CXCR4, was found to decrease surface biotinylation of CXCR4-AP. This effect is due to a combination of CXCR4 endocytosis and stabilization of CXCR4 homodimers. Finally, when CXCR4-AP, CCR2-AP, and CCR5-AP were expressed together, we observed CXCR4-CXCR4 homodimers and CXCR4-CCR2 and CXCR4-CCR5 heterodimers. The newly developed assay opens new opportunity for multiplex detection for GPCR homo- and heterodimerization within the same cellular context.
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Steel
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Victoria L
authors.initials: VL
authors.lastname: Murray
authors.affiliation: Department of Mechanical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America; Department of Biomedical Engineering, University of Michigan, Ann Arbor, Michigan, United States of America; Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, Michigan, United States of America; Biophysics Program, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Allen P
authors.initials: AP
authors.lastname: Liu
mesh_terms: D000818:Animals; D019921:Biotinylation; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry; D054377:Chemokine CXCL12 / Q000737:chemistry; D004705:Endocytosis; D029968:Escherichia coli Proteins / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D054390:Receptors, CCR2 / Q000737:chemistry; D019713:Receptors, CCR5 / Q000737:chemistry; D019718:Receptors, CXCR4 / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D012097:Repressor Proteins / Q000737:chemistry; D015398:Signal Transduction
chemical_list: C515334:CCR2 protein, human; C584933:CCR5 protein, human; C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D029968:Escherichia coli Proteins; D054390:Receptors, CCR2; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D043562:Receptors, G-Protein-Coupled; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli