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.
Highlights
G protein-coupled receptors (GPCRs) mediate the majority of our physiological responses to neurotransmitters, hormones, and environmental stimulants by their capacity to engage in diverse signaling pathways [1]
Characterization of proximity biotinylation assay for chemokine receptor dimerization To demonstrate the concept of using proximity biotinylation for receptor dimerization, several receptors fusions were created in an adenovirus vector (Figure 1)
Using flow cytometry and a cell line that expresses CXCR4 or CCR5 as positive controls, we found that retinal pigment epithelial (RPE) cells contain undetectable levels of CXCR4, CCR2, and CCR5 and are deemed suitable for the current study (Figure S1)
Summary
GPCRs mediate the majority of our physiological responses to neurotransmitters, hormones, and environmental stimulants by their capacity to engage in diverse signaling pathways [1]. Dimerization/oligomerization between GPCRs is recognized to modulate the pharmacological characteristics of the receptors and influence their coupling to G proteins [2]. Recent studies have shown that GPCRs can exist as dimers or as part of larger oligomeric complexes [3,4]; the functional significance of dimerization remains poorly understood. There is increasing evidence that homoand heterodimerization of activated GPCRs represents a means to control the specificity and increase the diversity of signaling events [5]. While Class B GPCR are obligate dimers [6], most class A GPCRs are capable of functioning as single units or form homoor heterodimers [7,8]
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