Abstract
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.
Highlights
Seven-transmembrane-helix (7TM) receptors, of which the majority are G protein-coupled receptors (GPCRs), play essential roles in many aspects of human physiology and in numerous disease processes
We observed that siRNA knockdown of atypical chemokine receptor 3 (ACKR3) leads to significant increases of CXCR4:arginine vasopressin receptor 1A (AVPR1A) heteromers in the rat aortic smooth muscle cell line A7r5 and in human vascular smooth muscle cells (hVSMCs) [25], which could point toward interdependency of receptor-receptor interactions in the plasma membrane, i.e. that heteromerization between two receptor partners depends on the presence and the expression levels of other partnering receptors
The findings of the present study suggest that the heteromerization patterns among CXCR4, ACKR3, α1A/B/D-adrenergic receptor (AR), and AVPR1A are interdependent and demonstrate plasticity of their heteromerization patterns
Summary
Seven-transmembrane-helix (7TM) receptors, of which the majority are G protein-coupled receptors (GPCRs), play essential roles in many aspects of human physiology and in numerous disease processes. 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. that heteromerization between two receptor partners depends 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 (PLA) to visualize receptor-receptor proximity at single molecule resolution [28] and systematically studied how manipulation of the expression levels of individual protomers affect heteromerization patterns among other interacting receptor partners. The findings of the present study suggest that the heteromerization patterns among CXCR4, ACKR3, α1A/B/D-ARs, and AVPR1A are interdependent and demonstrate plasticity of their heteromerization patterns
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