Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α1-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays

Gao, Xianlong; Enten, Garrett; DeSantis, Anthony J.; Volkman, Brian F.; Gaponenko, Vadim; Majetschak, Matthias

doi:10.1016/j.bbrc.2020.02.094

Cited by 9 publications

(25 citation statements)

References 32 publications

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“…2C/D). Similar to our previous observation that the CXCR4 homodimer forms heterotrimers with a 1a -AR [14], we observed hyperbolic progressions of saturation BiLC/BiFC BRET signals for interactions between dimeric CXCR4 and AVPR1A (Fig. 2E) or ACKR3 (Fig.…”

Section: Resultssupporting

confidence: 91%

“…Intermolecular bioluminescence resonance energy transfer (BRET) to measure receptor-receptor interactions BRET assays were performed as described previously [12,14,19,20]. In brief, HEK293T cells were seeded in 12well plates and transfected with the plasmids indicated using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA).…”

Section: Cell Culturementioning

confidence: 99%

“…Bimolecular luminescence complementation assays (BiLC), bimolecular fluorescence complementation assays (BiFC), and BiLC/BiFC BRET were performed as previously described [14]. For BiLC, two GPCRs, of which one is fused with L1 and the other with L2, were cotransfected in HEK293T cells, with one GPCR at a fixed amount and the other at increasing amounts.…”

Section: Bimolecular Luminescence and Fluorescence Complementation Bretmentioning

confidence: 99%

“…Flow cytometry was used to evaluate receptor expression, as described [9,14]. Cells were incubated with mouse anti-CXCR4 (R&D Systems, Minneapolis, MN, USA, MAB172), mouse anti-AVPR1A (LSBio, Seattle, WA, USA, LS-C196728), rabbit anti-a 1A -AR (Abcam, Cambridge, MA, USA, Ab137123), or mouse anti-FLAG antibody (Sigma, F1804, for FLAG-ACKR3) followed by incubation with Alexa Fluor 488-conjugated goat antimouse or anti-rabbit antibody (Thermo Fisher Scientific, 1 : 50 dilution).…”

Section: Flow Cytometrymentioning

confidence: 99%

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Class A G protein‐coupled receptors assemble into functional higher‐order hetero‐oligomers

et al. 2021

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Although class A seven-transmembrane helix (7TM) receptor heterooligomers have been proposed, information on the assembly and function of such higher-order hetero-oligomers is not available. Utilizing bioluminescence resonance energy transfer (BRET), bimolecular luminescence/fluorescence complementation (BiLC/BiFC), and BiLC/BiFC BRET in HEK293T cells, we provide evidence that chemokine (C-X-C motif) receptor 4, atypical chemokine receptor 3, a 1a -adrenoceptor, and arginine vasopressin receptor 1A form hetero-oligomers composed of 2-4 different protomers. We show that hetero-oligomerization per se and ligand binding to individual protomers regulate agonist-induced coupling to the signaling transducers of interacting receptor partners. Our findings support the concept that receptor hetero-oligomers form supramolecular machineries with molecular signaling properties distinct from the individual protomers. These findings provide a mechanism for the phenomenon of context-dependent receptor function.

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Section: Resultssupporting

confidence: 91%

Section: Cell Culturementioning

confidence: 99%

Section: Bimolecular Luminescence and Fluorescence Complementation Bretmentioning

confidence: 99%

Section: Flow Cytometrymentioning

confidence: 99%

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Class A G protein‐coupled receptors assemble into functional higher‐order hetero‐oligomers

et al. 2021

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“…Moreover, CXCR4 has been shown to form heteromers with multiple other GPCRs, such as chemokine (C-C motif) receptor 2 (CCR2), CCR5, CXCR3, atypical chemokine receptor (ACKR) 3, chemerin receptor 23, β 2 -adrenergic receptor (AR), δ-opioid receptor, cannabinoid receptor 2, protease-activated receptor 1, or the virally-encoded GPCR of Herpesvirus saimiri, which is thought to alter the pharmacological properties of the receptor partners [7,[12][13][14][15][16][17][18][19][20][21]. We showed previously that CXCR4 and ACKR3 also heteromerize with α 1A/B/D -ARs and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs), through which the receptors cross-talk [9,[22][23][24][25][26][27]. Furthermore, we observed that siRNA knockdown of ACKR3 leads to significant increases of CXCR4:AVPR1A heteromers in the rat aortic smooth muscle cell line A7r5 and in hVSMCs [25], which could point toward interdependency of receptor-receptor interactions in the plasma membrane, i.e.…”

Section: Introductionmentioning

confidence: 99%

Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

2021

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Recently, we reported that the chemokine (C-X-C motif) receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3) heteromerize with α1A/B/D-adrenoceptors (ARs) and arginine vasopressin receptor 1A (AVPR1A) in recombinant systems and in rodent and human vascular smooth muscle cells (hVSMCs). In these studies, we observed that heteromerization between two receptor partners may depend on the presence and the expression levels of other partnering receptors. To test this hypothesis and to gain initial insight into the formation of these receptor heteromers in native cells, we utilized proximity ligation assays in hVSMCs to visualize receptor-receptor proximity and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. We confirmed subtype-specific heteromerization between endogenously expressed α1A/B/D-ARs and detected that AVPR1A also heteromerizes with α1A/B/D-ARs. siRNA knockdown of CXCR4 and of ACKR3 resulted in a significant re-arrangement of the heteromerization patterns among α1-AR subtypes. Similarly, siRNA knockdown of AVPR1A significantly increased heteromerization signals for seven of the ten receptor pairs between CXCR4, ACKR3, and α1A/B/D-ARs. Our findings suggest plasticity of seven transmembrane helix (7TM) receptor heteromerization in native cells and could be explained by a supramolecular organization of these receptors within dynamic clusters in the plasma membrane. Because we previously observed that recombinant CXCR4, ACKR3, α1a-AR and AVPR1A form hetero-oligomeric complexes composed of 2–4 different protomers, which show signaling properties distinct from individual protomers, re-arrangements of receptor heteromerization patterns in native cells may contribute to the phenomenon of context-dependent GPCR signaling. Furthermore, these findings advise caution in the interpretation of functional consequences after 7TM receptor knockdown in experimental models. Alterations of the heteromerization patterns among other receptor partners may alter physiological and pathological responses, in particular in more complex systems, such as studies on the function of isolated organs or in in vivo experiments.

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Heteromerization between α_1B‐adrenoceptor and chemokine (C‐C motif) receptor 2 biases α_1B‐adrenoceptor signaling: Implications for vascular function

et al. 2022

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Previously, we reported that chemokine (C‐C motif) receptor 2 (CCR2) heteromerizes with α1B‐adrenoceptor (α1B‐AR) in leukocytes, through which α1B‐AR controls CCR2. Whether such heteromers are expressed in human vascular smooth muscle cells (hVSMCs) is unknown. Bioluminescence resonance energy transfer confirmed formation of recombinant CCR2:α1b‐AR heteromers. Proximity ligation assays detected CCR2:α1B‐AR heteromers in hVSMCs and human mesenteric arteries. CCR2:α1B‐AR heteromerization per se enhanced α1B‐AR‐mediated Gαq‐coupling. Chemokine (C‐C motif) ligand 2 (CCL2) binding to CCR2 inhibited Gαq activation via α1B‐AR, cross‐recruited β‐arrestin to and induced internalization of α1B‐AR in recombinant systems and in hVSMCs. Our findings suggest that CCR2 within CCR2:α1B‐AR heteromers biases α1B‐AR signaling and provide a mechanism for previous observations suggesting a role for CCL2/CCR2 in the regulation of cardiovascular function.

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Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α1-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays

Cited by 9 publications

References 32 publications

Class A G protein‐coupled receptors assemble into functional higher‐order hetero‐oligomers

Class A G protein‐coupled receptors assemble into functional higher‐order hetero‐oligomers

Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

Heteromerization between α_1B‐adrenoceptor and chemokine (C‐C motif) receptor 2 biases α_1B‐adrenoceptor signaling: Implications for vascular function

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Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α1-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays

Cited by 9 publications

References 32 publications

Class A G protein‐coupled receptors assemble into functional higher‐order hetero‐oligomers

Class A G protein‐coupled receptors assemble into functional higher‐order hetero‐oligomers

Plasticity of seven-transmembrane-helix receptor heteromers in human vascular smooth muscle cells

Heteromerization between α1B‐adrenoceptor and chemokine (C‐C motif) receptor 2 biases α1B‐adrenoceptor signaling: Implications for vascular function

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Heteromerization between α_1B‐adrenoceptor and chemokine (C‐C motif) receptor 2 biases α_1B‐adrenoceptor signaling: Implications for vascular function