New Insights into Mechanisms and Functions of Chemokine (C-X-C Motif) Receptor 4 Heteromerization in Vascular Smooth Muscle

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.

Functional and structural consequences of chemokine (C-X-C motif) receptor 4 activation with cognate and non-cognate agonists

“…While the reason for these different dose-responses is unknown, both types have previously been reported in chemotaxis experiments with CXCL12 in human monocytes [23–25]. We then tested whether the chemotactic activities can be inhibited with the selective CXCR4 antagonist AMD3100 and with a TM2 peptide analogue of CXCR4, which has previously been shown to inhibit CXCR4 in the human glioblastoma cell line U87 and in primary human monocytes and vascular smooth muscle cells [10, 15, 18, 26]. As shown in Fig.…”

“…Bovine serum albumin, AMD3100, pertussis toxin and U73122 were purchased from Sigma, LY94002 and U0126 from Cell Signaling and 6bK from Tocris. The peptide analogue of transmembrane helix (TM) 2 of CXCR4 (LLFVITLPFWAVDAVANWYFGNDD-NH 2 ) was as described [15]. …”

“…Cell migration was assessed using the ChemoTx 96-well cell migration system, as described [5, 15]. The chemotactic index (CI) was calculated as the ratio of cells that transmigrated through the filter in the presence versus the absence (= PBS/control) of the test solutions.…”

α 1 ‐Adrenergic Receptors Function Within Hetero‐Oligomeric Complexes With Atypical Chemokine Receptor 3 and Chemokine (C‐X‐C motif) Receptor 4 in Vascular Smooth Muscle Cells

“…Proximity ligation assays (PLAs) were performed as previously described in detail . In brief, cells were grown and fixed on 8‐well chamber slides (Nunc).…”

“…Recently, we reported that CXCR4 activation sensitizes α 1 ‐adrenergic receptor (AR) function in vascular smooth muscle and stabilizes blood pressure during the cardiovascular stress response to hemorrhagic shock, whereas activation of ACKR3 inhibits vascular α 1 ‐AR function and impairs blood pressure . Subsequently, we provided evidence that heteromeric complexes between endogenous CXCR4 and α 1A/B ‐AR are constitutively expressed in human vascular smooth muscle cells (hVSMCs), through which CXCR4 modulates α 1 ‐AR function . The molecular mechanisms underlying cross‐talk between ACKR3 and α 1 ‐AR, however, remain elusive.…”