Multiplex Detection of Homo- and Heterodimerization of G Protein-Coupled Receptors by Proximity Biotinylation

Details

• Alternative Title:
  PLoS One
• Personal Author:
  Steel, Elisabeth ; Murray, Victoria L. ; Liu, Allen P.
• Description:
  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.
• Subjects:
  Animals Biotinylation Carbon-Nitrogen Ligases Chemokine CXCL12 Endocytosis Escherichia Coli Proteins Fluorescence Resonance Energy Transfer Protein Binding Protein Conformation Protein Multimerization Receptors, CCR2 Receptors, CCR5 Receptors, CXCR4 Receptors, G-Protein-Coupled Repressor Proteins Signal Transduction

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• Source:
  PLoS One. 2014; 9(4).
• Pubmed ID:
  24691126
• Pubmed Central ID:
  PMC3972117
• Document Type:
  Journal Article
• Funding:
  DP2 HL117748/HL/NHLBI NIH HHS/United States ; DP2 HL117748-01/DP/NCCDPHP CDC HHS/United States
• Volume:
  9
• Issue:
  4
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:be1cc51d98e537f196907cbce73e6dbbf8649781184ada9164b17666ed3c9217
• Download URL:
  https://stacks.cdc.gov/view/cdc/23117/cdc_23117_DS1.pdf
• File Type:
  [PDF - 1.21 MB ]