Abstract
Recent evidence suggests that C-X-C chemokine receptor type 4 (CXCR4) heteromerizes with α1A/B-adrenoceptors (AR) and atypical chemokine receptor 3 (ACKR3) and that CXCR4:α1A/B-AR heteromers are important for α1-AR function in vascular smooth muscle cells (VSMC). Structural determinants for CXCR4 heteromerization and functional consequences of CXCR4:α1A/B-AR heteromerization in intact arteries, however, remain unknown. Utilizing proximity ligation assays (PLA) to visualize receptor interactions in VSMC, we show that peptide analogs of transmembrane-domain (TM) 2 and TM4 of CXCR4 selectively reduce PLA signals for CXCR4:α1A-AR and CXCR4:ACKR3 interactions, respectively. While both peptides inhibit CXCL12-induced chemotaxis, only the TM2 peptide inhibits phenylephrine-induced Ca2+-fluxes, contraction of VSMC and reduces efficacy of phenylephrine to constrict isolated arteries. In a Cre-loxP mouse model to delete CXCR4 in VSMC, we observed 60% knockdown of CXCR4. PLA signals for CXCR4:α1A/B-AR and CXCR4:ACKR3 interactions in VSMC, however, remained constant. Our observations point towards TM2/4 of CXCR4 as possible contact sites for heteromerization and suggest that TM-derived peptide analogs permit selective targeting of CXCR4 heteromers. A molecular dynamics simulation of a receptor complex in which the CXCR4 homodimer interacts with α1A-AR via TM2 and with ACKR3 via TM4 is presented. Our findings further imply that CXCR4:α1A-AR heteromers are important for intrinsic α1-AR function in intact arteries and provide initial and unexpected insights into the regulation of CXCR4 heteromerization in VSMC.
Highlights
G protein-coupled receptors (GPCR) comprise the largest group of cell surface receptors and mediate a multitude of important cell functions
To test whether transmembrane helix (TM) peptide analogs may permit selective targeting of CXCR4 heteromers, we sought to evaluate the effects of TM peptide analogs of CXCR4 in proximity ligation assays (PLA) with human VSMC (hVSMC)
TM5 and TM7 peptide analogs had no or little effects on CXCR4 function and crystallographic structures revealed that CXCR4 exists as a homodimer in which TM5 and TM6 form the main interface between the receptor protomers, which make TM5/6 of CXCR4 as interaction sites with other GPCRs unlikely [31,32]
Summary
G protein-coupled receptors (GPCR) comprise the largest group of cell surface receptors and mediate a multitude of important cell functions. The structural determinants of GPCR heteromerization, are largely unknown, and the biological relevance of heteromeric receptor complexes is still under debate. We provided evidence that C-X-C chemokine receptor 4 (CXCR4) forms heteromeric complexes with atypical chemokine receptor 3 (ACKR3) and α1A/B-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells (VSMC). As interference with CXCR4:α1A/B-AR heteromerization inhibited α1-AR mediated responses of VSMC, these data suggested that CXCR4:α1A/B-AR heteromerization is important for normal α1-AR function. Whether it is possible to interfere with CXCR4:ACKR3 heteromerization without affecting CXCR4:α1A/B-AR heteromers, is unknown and the effects of CXCR4:α1A/B-AR heteromerization in VSMC on the function of intact arteries or on blood pressure regulation remain to be determined
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