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Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function

Significance α1-Adrenergic receptors are important for the regulation of vascular function and are targeted clinically for blood pressure control. Here, we provide evidence that α1A/B-adrenergic receptors (AR) form heteromeric complexes with chemokine (C-X-C motif) receptor 4 (CXCR4) on the cell surface of vascular smooth muscle cells. We show that disruption of α1A/B-AR:CXCR4 heteromeric complexes inhibits α1-AR–mediated functions in vascular smooth muscle cells and that treatment with CXCR4 agonists enhances the potency of the α1-AR agonist phenylephrine to increase blood pressure. These findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of G protein-coupled receptor heteromerization with important functional implications. Compounds targeting the α1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulating blood pressure. Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α1-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α1A/B-AR and CXCR4, and inhibited Ca2+ mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α1-AR activation. CXCR4 silencing reduced α1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca2+ fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC50 of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α1-AR–mediated function in VSMC and that activation of CXCR4 enhances the potency of α1-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α1-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.

Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α 1 -adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α 1A/B -ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α 1A/B -AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α 1A/B -AR and CXCR4, and inhibited Ca 2+ mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α 1 -AR activation. CXCR4 silencing reduced α 1A/B -AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca 2+ fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC 50 of the phenylephrineinduced blood pressure response three-to fourfold. These observations suggest that disruption of the quaternary structure of α 1A/B -AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α 1 -AR-mediated function in VSMC and that activation of CXCR4 enhances the potency of α 1 -AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α 1 -AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α 1A/B -AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.
CXCL12 | ubiquitin | AMD3100 | phenylephrine | blood pressure C hemokine (C-X-C motif) receptor 4 (CXCR4) is a G proteincoupled receptor (GPCR) that is essential during development. Animals lacking CXCR4 are not viable and demonstrate defects of the hematopoietic and cardiovascular system (1). After birth, CXCR4 is expressed in many tissues, including the heart and vasculature, and fulfills multiple functions in the immune system, such as regulation of leukocyte trafficking, stem cell mobilization, and homing (2,3). Moreover, CXCR4 is involved in various disease processes, such as HIV infection, cancer metastasis, and tissue repair (3)(4)(5).
In addition to these established functions, recent observations suggest that CXCR4 also contributes to the regulation of hemodynamics and blood pressure. Treatment with the CXCR4 antagonists AMD3100 and AMD3465 reduced blood pressure in experimental models of pulmonary arterial and systemic hypertension (6,7). We have shown previously that AMD3100 reduces hemodynamic stability and blood pressure during the cardiovascular stress response to traumatic and hemorrhagic shock, whereas selective activation of CXCR4 with the noncognate agonist ubiquitin improves hemodynamic stability and increases systemic blood pressure after traumatic, hemorrhagic, and endotoxic shock (8)(9)(10)(11)(12)(13). Because in vivo pharmacological targeting of CXCR4 did not affect myocardial function, these findings suggested that effects of CXCR4 on hemodynamics and blood pressure are mediated via modulation of vascular function (9). Accordingly, we observed that CXCR4 activation enhances and sensitizes vasoconstriction of isolated mesenteric arteries and veins in response to α 1 -adrenergic receptor (AR) activation with phenylephrine (PE) (9). As these effects were independent of the vascular endothelium, interactions between CXCR4 and α 1 -AR in vascular smooth muscle likely constitute the physiological basis for these observations (9). The molecular mechanisms underlying interactions between CXCR4 and α 1 -AR in vascular smooth muscle, however, remain unknown.
Crosstalk between GPCRs is a widely recognized principle that expands the physiological repertoire of GPCR-mediated Significance α 1 -Adrenergic receptors are important for the regulation of vascular function and are targeted clinically for blood pressure control. Here, we provide evidence that α 1A/B -adrenergic receptors (AR) form heteromeric complexes with chemokine (C-X-C motif) receptor 4 (CXCR4) on the cell surface of vascular smooth muscle cells. We show that disruption of α 1A/B -AR: CXCR4 heteromeric complexes inhibits α 1 -AR-mediated functions in vascular smooth muscle cells and that treatment with CXCR4 agonists enhances the potency of the α 1 -AR agonist phenylephrine to increase blood pressure. These findings extend the current understanding of the molecular mechanisms regulating α 1 -AR and provide an example of G protein-coupled receptor heteromerization with important functional implications. Compounds targeting the α 1A/B -AR:CXCR4 interaction could provide an alternative pharmacological approach to modulating blood pressure.
Here, we provide evidence that heteromeric receptor complexes between α 1A -AR and CXCR4 and between α 1B -AR and CXCR4 are constitutively expressed in rat and human vascular smooth muscle cells (VSMC). We show that disruption of the quaternary structure of the heteromeric receptor complex by targeting transmembrane helix (TM) 2 of CXCR4 and depletion of heteromeric receptor complexes by CXCR4 knockdown inhibit α 1 -AR agonist-induced key signaling events and contraction of VSMC. Furthermore, we show that treatment with CXCR4 agonists increases the potency of the α 1 -AR agonist PE to in-crease blood pressure in vivo. Our observations suggest that α 1 -AR function in VSMC is controlled through the formation of heteromeric α 1A/B -AR:CXCR4 complexes.

Results and Discussion
α 1A/B -AR Associates with CXCR4 on the Cell Surface of Vascular Smooth Muscle Cells. We sought to evaluate whether heteromeric complexes between α 1 -AR and CXCR4 are expressed on the cell surface of VSMC. Thus, we used proximity ligation assays (PLA) to visualize individual receptors and receptorreceptor interactions (39)(40)(41). PLA have been previously used to observe individual proteins and interactions between individual endogenous proteins at a single-molecule resolution (39). We first used PLA to detect CXCR4 and α 1A -AR in a format suited for high sensitivity detection of a single protein. As a positive control, we also tested for atypical chemokine receptor (ACKR) 3 (formerly known as RDC1 and CXCR7), which is known to be able to form heteromeric complexes with CXCR4 (3,28). Tolllike receptor (TLR) 9 was used as a negative control receptor that is unlikely to be associated with α 1A -AR. All individual receptors could be visualized by PLA in the rat vascular smooth muscle cell line A7r5, on freshly isolated aortic rat VSMC, and on primary human aortic VSMC (Fig. 1A). When PLA was used to visualize protein-protein interactions at a single-molecule level, we validated the proximity of CXCR4 and ACKR3 in native cells (28) and also detected signals suggesting close proximity of α 1A -AR:CXCR4 in A7r5 cells and in rat and human VSMC (Fig. 1B). In contrast, PLA signals for α 1A -AR:TLR9 interactions were not detectable (Fig. 1B).
To define whether all α-ARs associate with CXCR4, we then evaluated human VSMC for interactions between α 1 -AR and α 2 -AR subtypes with CXCR4. All α 1 -AR and α 2 -AR subtypes were detectable on human VSMC in PLA when used to detect individual receptors ( Fig. 2A). Quantification of PLA signals per cell suggested that α 1B -ARs were more frequently expressed on human VSMC than all other α-AR subtypes and CXCR4 (Fig.  2C). We then screened human VSMC for possible interactions between CXCR4 and α-AR subtypes and detected PLA signals corresponding to interactions between CXCR4 and α 1A -AR and between CXCR4 and α 1B -AR ( Fig. 2 B and D). Images from PLA signals corresponding to interactions between the receptors at a lower magnification (40×) are shown in Fig. S1. The number of PLA signals per cell corresponding to interactions between all other α-AR subtypes and CXCR4 were not distinguishable from the number of signals obtained in negative control experiments (Fig. 2D). Moreover, 3D reconstruction of the PLA signals from z-stack images confirmed that PLA signals corresponding to α 1A -AR:CXCR4 and α 1B -AR:CXCR4 are localized on the cell surface of VSMC (Fig. 2E).
To confirm direct physical interactions between α 1A/B -AR and CXCR4, we then used a HeLa expression system to perform coimmunoprecipitation analyses of receptor interactions. HeLa cells were cotransfected with FLAG-CXCR4, hemagglutinin (HA)α 1b AR, or HA-ACKR3 (= positive control), followed by immunoprecipitation of cell homogenates with anti-FLAG. HA-α 1b -AR and HA-ACKR3 were detected in FLAG-CXCR4 immunoprecipitates, but not in control samples that expressed either receptor alone (Fig. S2). To further consolidate these observations, we next performed coimmunoprecipitation analyses of endogenous receptor interactions in human VSMC. As shown in Fig. 2F, α 1A -AR and α 1B -AR were detectable in CXCR4 immunoprecipitates, whereas α 2C -AR was not detectable. These findings suggest that α 1A/B -AR and CXCR4 physically interact in human VSMC and that the observed proximity between the receptors corresponds to direct receptor-receptor interactions.  interference with the correct assembly of the target membrane protein (42,43). X4-2-6, a peptide derived from TM2 of CXCR4, has previously been shown to inhibit CXCR4 function (43). Therefore, we then evaluated whether X4-2-6 affects the association of CXCR4 with α 1A/B -AR and ACKR3 on VSMC by PLA. X4-2-6 reduced the association between α 1A -AR and CXCR4 and between α 1B -AR and CXCR4 on the cell surface of human VSMC, compared with R3-2-1, a peptide derived from TM2 of chemokine (C-C motif) receptor 3 ( Fig. 3 A and B). PLA signals corresponding to CXCR4:ACKR3, however, were not affected by X4-2-6 ( Fig. 3 A and B). We observed the same effects of X4-2-6 in PLA experiments with A7r5 cells (Fig. S3 A and B). As assessed by fluorescence-activated cell sorting (FACS) analyses, X4-2-6 did not influence cell-surface expression of the individual receptors, suggesting that X4-2-6 disrupts physical interactions between CXCR4 and α 1A/B -AR in the cell membrane without affecting receptor expression levels (Fig. 3C).
To assess whether disruption of the α 1A/B -AR:CXCR4 association influences α 1 -AR-mediated signaling, we tested the effects of X4-2-6 on PE-induced intracellular Ca 2+ mobilization and myosin light chain (MLC) 2 phosphorylation (Ser-19) in VSMC. As shown in Fig. 3D, X4-2-6 inhibited PE-induced intracellular Ca 2+ mobilization in A7r5 cells. X4-2-6, however, did not affect Ca 2+ mobilization in response to arginine vasopressin (Fig. 3E), suggesting specificity of the observed effects for α 1 -AR. Because pretreatment of A7r5 cells with the selective CXCR4 inhibitor AMD3100 did not affect PE-induced Ca 2+ mobilization (Fig. 3F), effects of X4-2-6 cannot be attributed to the inhibition of CXCR4-mediated signaling. As observed in A7r5 cells, X4-2-6 also attenuated Ca 2+ mobilization in response to PE in human VSMC (Fig. 3G). Furthermore, X4-2-6 inhibited Ca 2+ /calmodulin-dependent MLC2 phosphorylation upon PE stimulation of human VSMC, as assessed by Western blotting (Fig. 3H). To confirm results from Western blotting experiments, we then used PLA to visualize and quantify intracellular phosphorylated MLC2 (Fig. 3I). Consistent with the intracellular localization of phospho-MLC2, we detected PLA signals only when VSMC were permeabilized before incubation with antiphospho MLC2. Quantification of the number of PLA signals per cell showed the same magnitude of PE-induced MLC2 phosphorylation as determined by Western blot analyses and confirmed the inhibition of PE-induced MLC2 phosphorylation by X4-2-6 ( Fig. 3H). As these data indicated that X4-2-6 inhibits key signaling events in the pathway mediating α 1 -AR-induced VSMC contraction, we then tested whether X4-2-6 also influences contraction of freshly isolated rat mesenteric artery smooth muscle cells upon exposure to PE. As shown in Fig. 3J, pretreatment of cells with X4-2-6 reduced the number of cells contracting upon PE stimulation from 71 ± 6% to 46 ± 9%. The observation that the inhibitory effects of X4-2-6 on PEinduced signaling events in human aortic VSMC were more pronounced than effects of X4-2-6 on PE-induced contraction of freshly isolated VSMC from mesenteric arteries could be explained by distinct functional roles of the α 1 -AR subtypes that have been observed among various vascular beds (44).
Because our observations suggested that X4-2-6 functions as an α 1 -AR antagonist, we then evaluated whether X4-2-6 can also interact with α 1A -AR using NMR spectroscopy. We used reductively methylated membranes prepared from cells overexpressing either α 1a -AR or CXCR4 to closely mimic native conditions for receptor folding and interactions with the plasma membrane. A similar approach has recently been used to provide structural insight into ligand regulation of the extracellular surface of the β 2 -AR (45). The overlaid 13  To address the possibility that 13 CH 3 probes on α 1a -AR do not report on X4-2-6 binding, we added 10 μM PE to α 1a -AR in the presence of 10 μM X4-2-6 ( Fig. 4, Lower Left). PE-induced chemical shift changes in α 1a -AR were similar to those in the absence of X4-2-6. When X4-2-6 was added to CXCR4, sig-nificant chemical shift changes in the spectrum of CXCR4 were detected, indicating peptide-receptor interactions (Fig. 4, Lower Right). Because X4-2-6 also caused loss of proximity between α 1A/B -AR and CXCR4 in VSMC, these data suggest that X4-2-6 binding to CXCR4 induces structural rearrangements of the receptor that disrupt the quarternary heteromer interface. Provided that PLA signals for native CXCR4:ACKR3 represent direct receptor interactions, the observation that the proximity between CXCR4 and ACKR3 was not affected by X4-2-6 points toward TM2 of CXCR4 as part of a specific α 1A/B -AR:CXCR4 heteromer interface, which does not participate in the formation of the CXCR4:ACKR3 interface.
Although crystal structures of GPCR heteromers are currently not available, crystallographic structures of GPCR homodimers revealed several different interfaces. The main interface of the CXCR4 homodimer is localized at TM5 and TM6, whereas several other GPCR homodimers form interfaces that also include TM2 (46)(47)(48)(49)(50). Thus, a TM2 contact site in CXCR4 could permit receptor heteromerization without interfering with the constitutive CXCR4 homodimerization (46). Furthermore, as X4-2-6 did not interfere with α 1a -AR and did not affect ligandinduced conformational changes of α 1a -AR in membranes, offtarget effects of X4-2-6 on α 1A/B -AR or PE appear unlikely to account for the inhibitory effects of X4-2-6 on signaling events upon α 1 -AR activation. Thus, these data led to the hypothesis that α 1A/B -AR:CXCR4 heteromers are a prerequisite for α 1 -AR function in VSMC.

CXCR4 Silencing Inhibits α 1 -AR Function in Vascular Smooth Muscle
Cells. To test the hypothesis that CXCR4:α 1A/B -AR heteromeric complexes are required for α 1 -AR function, we aimed to reduce CXCR4:α 1A/B -AR heteromerization by reducing CXCR4 expression in human VSMC with RNA interference. Fig. 5A shows a typical Western blot with anti-CXCR4 with cell homogenates from human VSMC transfected with nontargeting (NT) or CXCR4-targeted siRNA. As expected, anti-CXCR4 recognized multiple bands in human VSMC transfected with NT siRNA, which likely correspond to proteolytically processed, ubiquitylated, or glycosylated forms of CXCR4 (51)(52)(53)(54)(55). The most abundant receptor species after transfection of human VSMC with NT siRNA were detectable at migration positions corresponding to 48-60 kDa. The intensities of these bands were reduced after transfection of the cells with CXCR4 siRNA (Fig.  5A). As estimated by FACS analyses, CXCR4 cell-surface expression was reduced by 69 ± 7% (n = 4) after transfection of human VSMC with CXCR4 siRNA, compared with cells transfected with NT siRNA (Fig. 5B). Cell-surface expressions of α 1A/B -AR were not affected by CXCR4 siRNA (Fig. 5B). When CXCR4 expression was quantified by PLA (Fig. 5C), transfection of human VSMC with CXCR4 siRNA resulted in 62 ± 8% reduction of CXCR4 cell-surface expression, compared with cells transfected with NT siRNA.
Next, we analyzed the expression of α 1A/B -AR:CXCR4 and ACKR3:CXCR4 heteromers by PLA. Compared with human VSMC after transfection with NT siRNA, CXCR4 siRNA silencing reduced the PLA signals for α 1A -AR:CXCR4 (Fig. 5D), α 1B -AR:CXCR4 (Fig. 5E), and ACKR3:CXCR4 (Fig. 5F) heteromers by 90%, 60%, and 59%, respectively. The finding that the degree of reduction of CXCR4 and of the α 1B -AR:CXCR4 and ACKR3:CXCR4 heteromers on the cell surface after siRNA silencing was comparable argues for a 1:1 receptor:receptor stoichiometry. Notably, PLA signals for α 1A -AR:CXCR4 heteromers were almost completely depleted after CXCR4 silencing. Several explanations may account for this observation, such as alteration of the receptor heteromerization equilibrium in the plasma membrane or PLA signals resulting from the association of CXCR4 with other receptors that form heteromeric complexes with α 1A -AR, i.e., during receptor clustering (56). To assess the functional consequences of CXCR4 silencing on α 1 -AR function in human VSMC, we then measured PE-induced Ca 2+ fluxes and MLC2 phosphorylation. We detected that CXCR4 silencing in human VSMC abolished PE-induced Ca 2+ fluxes (Fig. 5G) and MLC2 phosphorylation (Fig. 5 H and I), compared with cells transfected with NT siRNA. Among the α 1 -AR subtypes, α 1A -AR is thought to be most important for mediating vasoconstriction in aortic vascular smooth muscle (44,57). Thus, the large reduction of α 1A -AR:CXCR4 heteromers after CXCR4 silencing in human VSMC (aortic smooth muscle cells) can explain the loss of effect of PE in our experiments, despite only partial reduction of α 1B -AR:CXCR4 heteromers.

CXCR4 Agonists Increase the Potency of PE to Increase Blood
Pressure. To evaluate whether CXCR4 influences α 1 -AR function in vivo, we tested how pharmacological modulation of CXCR4 affects the blood pressure response to PE in rats. Under normal hemodynamic conditions, animals received a single injection of the cognate CXCR4 agonist CXCL12, the noncognate CXCR4 agonist ubiquitin, or the CXCR4 antagonist AMD3100, followed by increasing doses of PE. As a quantifiable marker of the integrated blood pressure response to each dose of PE, we then determined the area under the mean arterial blood pressure (MAP) curve for each dose of PE and generated dose-response curves. As shown in Fig. 6, the EC 50 of PE after vehicle treatment was 664 ng/kg (95% confidence interval: 346-1273 ng/kg), and the maximal area under the MAP curve was 396 mmHg × s (95% confidence interval: 335-458 mmHg × s). CXCL12 and ubiquitin pretreatment reduced the EC 50 of PE 3.9-and 3.5fold, respectively. Whereas AMD3100 pretreatment did not affect the EC 50 of PE, AMD3100 antagonized the effects of CXCL12 and ubiquitin. None of the CXCR4 ligands influenced the efficacy of PE. These findings suggest that CXCR4 activation enhances the potency of PE in vivo.
The observation that AMD3100 did not affect PE-induced effects in normal animals is consistent with the effects of AMD3100 on PE-induced Ca 2+ mobilization in VSMC in the present study and with the previous observation that AMD3100 did not influence PE-induced vasoconstriction in pressure myography experiments (9). As depletion of α 1A/B -AR:CXCR4 heteromers by X4-2-6 and CXCR4 knockdown inhibited PE-induced responses in VSMC, our observations of AMD3100 suggest that heteromeric complex formation per se, independent of ligand occupation or the activation status of CXCR4, controls α 1 -AR function. Similarly, ligand unoccupied ghrelin receptor has been reported to modulate dopamine receptor subtype-2 function via formation of heteromeric ghrelin receptor and dopamine receptor subtype-2 complexes (58). In addition, the finding that CXCR4 agonists enhanced the potency of PE in vivo is in agreement with previous observations from pressure myography experiments with isolated arteries (9). This indicates that ligand activation of CXCR4 further sensitizes α 1 -AR responses and could be explained through allosteric effects of CXCR4 on α 1A/B -AR within the α 1A/B -AR:CXCR4 complex when CXCR4 transitions into an activated configuration upon agonist binding. α 1A/B -AR:CXCR4 and CXCR4:ACKR3 heteromers appear to be constitutively expressed in VSMC, and pharmacological activation of CXCR4 and ACKR3 has been reported to result in opposite effects on PE-induced vasoconstriction in isolated arteries (9). This implies that CXCR4 and also ACKR3 function as modulators of α 1 -AR. Whereas the molecular mechanisms through which ACKR3 influences α 1 -AR function remain to be determined, the observed effects of ubiquitin in the present study are in agreement with the findings that ubiquitin functions as a noncognate CXCR4 agonist that does not bind to ACKR3 (51,52,(59)(60)(61)(62)(63). The observation that CXCL12, which has a much higher affinity for ACKR3 than for CXCR4 (64), also enhanced the potency of PE in normal animals in vivo in the present study, whereas CXCL12 previously desensitized PE-mediated vasoconstriction of isolated arteries and reduced blood pressure during hemorrhagic shock (9), suggests that effects of CXCL12 depend on the relative functional contribution of CXCR4 and ACKR3 within the specific experimental or (patho)physiological environment (65).
Because CXCR4 antagonists have been reported to reduce blood pressure in experimental models of pulmonary arterial and systemic hypertension and during hemorrhagic shock (6,7,9), these findings point toward a pathophysiological role of CXCR4 agonists during blood pressure regulation under disease conditions that are associated with increased catecholamine release, such as shock or hypertension. Interestingly, ubiquitin has been described to be stored along with catecholamines in secretory chromaffin granules in the adrenal gland and to be released into the circulation upon stimulation of chromaffin cells (66), which may reflect a physiological linkage between CXCR4 and adrenergic receptor function.
Conclusively, our data suggest that endogenous α 1A/B -AR: CXCR4 heteromers are constitutively expressed on VSMC, that the heteromeric receptor complex is important for α 1 -AR function, and that ligand activation of CXCR4 further sensitizes α 1 -AR. Such a regulation of α 1 -AR function could be explained through allosteric interactions between α 1A/B -AR and CXCR4 within the heteromeric receptor complex, which could provide the physiological advantage that α 1 -AR, and subsequently vascular function, can be selectively regulated to allow fine-tuning of blood pressure in the systemic circulation or in different vascular beds (67,68). Furthermore, our observations provide an example that signaling events, which have been considered as characteristic intracellular consequences following activation of a homomeric GPCR, reflect a biochemical fingerprint of a GPCR heteromer (69). We believe that our observations extend the current understanding of the molecular mechanisms regulating α 1 -AR function in VSMC and that compounds targeting the α 1A/B -AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate vascular function and blood pressure.

Materials and Methods
Cells and Cell Lines. A7r5 cells (rat aortic vascular smooth muscle cell line) and human aortic VSMC were obtained from the American Type Culture Collection. HeLa cells were as described (70). Rat aortic and mesenteric artery VSMC were isolated from male Lewis and Sprague-Dawley rats as described elsewhere (71)(72)(73). All procedures involving rats were in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee of Loyola University Chicago. A7r5, rat, and human VSMC were cultured in high-glucose Dulbecco's Modified's Eagle Medium, 10 mg/mL sodium pyruvate, 2 mM L-glutamine, 10% (vol/vol) FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.
Proteins, Peptides, and Reagents. PE, arginine vasopressin, and AMD3100 were from Sigma-Aldrich. CXCL12 was as described (9,74) and also obtained from Protein Foundry. Ubiquitin was purchased from R&D Systems. X4-2-6, a peptide derived from the second transmembrane helix of CXCR4, was prepared as described (43); R3-2-1 (LLFLVTLPFWIHYVRGHNWVFGHDDD-PEG 27 -NH 2 ), a peptide derived from the second transmembrane helix of chemokine receptor CCR3, was designed and produced similarly to the X4-2-6 peptide and used as a control. Solid-phase synthesis on a 433A Applied Biosystems Peptide Synthesizer using Fmoc amino acid derivatives was used for the production of the peptides. After cleavage with 87.5% (vol/vol) trifluoroacetic acid containing 5% (vol/vol) water, 5% (vol/vol) thioanisol, and 2.5% (vol/vol) triisopropylsilane, the peptides were purified by reverse phase HPLC using an Atlantis C3 column (Agilent Technologies). The peptide structure and purity was confirmed by ion-spray mass-spectrometry combined with HPLC.
Reductive Methylation of Membrane Preparations. ChemiSCREEN membrane preparations of α 1a AR and CXCR4 were purchased from EMD Millipore. Reductive methylation of the membrane preparations was performed as described previously (75). In brief, 20 μL of 1 M borane-ammonia complex and 4 μL of 13 C formaldehyde were added to 1 mL of membrane preparation. This mixture was incubated with stirring for 2 h at 4°C. The addition of borane-ammonia and formaldehyde was repeated, and the mixture was incubated with stirring for an additional 2 h. Ten microliters of 1 M boraneammonia complex were then added and the mixture was incubated at 4°C overnight with stirring. The reaction was then stopped by adding 110 μL of 2 M Tris·HCl (pH 7.6). Thereafter, the membrane preparations were dialyzed in PBS and used for NMR experiments.
Heteronuclear Single Quantum Coherence NMR. 1 H-13 C HSQC NMR experiments were carried out on a 900-MHz Bruker Avance Spectrometer equipped with a cryogenic probe. Data were processed and analyzed using the NMRPipe software.
Coimmunoprecipitation Analyses of Receptor Interactions. HeLa cells were transiently transfected with DNA encoding FLAG-CXCR4 or empty expression vector pcDNA and pcDNA, HA-ACKR3, or HA-ARα1b, using TransIT-LT1 transfection reagent, similar to previously published protocols (70). Twentyfour hours later, cells were collected in 1.0 mL immunoprecipitation buffer [20 mM Na 2 PO 4 , pH 6.5, 150 mM NaCl, 1% (vol/vol) Triton-X 100, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 10 μg/mL pepstatin A] and incubated at 4°C for 30 min. Cells were sonicated and centrifuged, and 500 μg of the clarified lysates were incubated with an anti-FLAG polyclonal antibody (Sigma) to immunoprecipitate FLAG-CXCR4 followed by immunoblotting to detect bound HA-ACKR3 or HA-α 1b -AR, similar to previously published protocols (76). Coimmunoprecipitation experiments with human VSMC were performed using the Thermo Scientific Pierce coimmunoprecipitation kit according to the manufacturer's protocol. Anti-α 1A/B -AR and anti-α 2C -AR antibodies were purchased from Abcam. Forty micrograms of anti-CXCR4 was incubated with 50 μL Amino LinK Plus coupling resin for 2 h. Three hundred micrograms of cell lysates was precleared with 25 μL of the control agarose resin slurry (30 min at 4°C). Immobilized anti-CXCR4 resin and nonreactive resin (= control) were incubated with precleared lysate overnight at 4°C. After incubation, the resins were washed three times with 200 μL IP lysis/wash buffer, and protein was eluted using 50 μL of elution buffer. Samples were analyzed by Western blotting.
CXCR4 Gene Silencing by RNA Interference. CXCR4 siRNA gene silencing was performed as described previously (51). In brief, VSMC cells were grown in 1 mL Accell siRNA delivery media per well (Thermo Scientific Dharmacon) in 12-well plates (Nunc). Commercially available Accell CXCR4 siRNA was reconstituted with 1× siRNA buffer to a stock concentration of 100 μM. Cells were then transfected with 10 nmol CXCR4 siRNA and incubated for 72 h at 37°C, 5% (vol/vol) CO 2 . Accell NT siRNA pool was used as negative control. After 72 h, cells were assayed for receptor cell-surface expression and used for signaling experiments.
Proximity Ligation Assays. Duolink proximity ligation assays were performed as described previously (40,41). In brief, VSMC and A7r5 cells were grown and fixed on eight-well tissue culture slides. (1:400). The same antibody combinations were used for A7r5 cells, except that ACKR3:α 1A AR interactions were visualized with mouse anti-ACKR3 (LS-C64035, LSBio)/rabbit anti-α 1A AR (ab137123, Abcam) (1:400). For the visualization of phosphorylation of MLC2, slides were treated as described, except that cells were permeabilized in 0.5% Triton-X before blocking. Slides were incubated with mouse anti-phospho (S19) MLC2 (Cell Signaling). Slides were then washed and incubated with secondary anti-rabbit/mouse antibodies conjugated with plus and secondary anti-mouse/goat antibodies conjugated with minus Duolink II PLA probes (1:5). All following steps were as described above for the visualization of individual receptors. As antibody controls, slides were incubated without primary antibodies or without one of the secondary antibodies conjugated with minus Duolink II PLA probes. PLA signals were quantified using the Duolink Image Tool software (Sigma-Aldrich). Images were imported in merged.tiff formats containing both signal and nuclei channels. Merged images were visually verified for analytical quality. Comparisons and statistical analyses were performed only when PLA assays were performed on the same day in parallel experiments and fluorescence microscopy was performed with the identical settings. For each ex-periment and condition, 20-24 randomly selected nonoverlapping vision fields were analyzed, and the mean from all experiments was calculated.
Deconvolution 3D Imaging. Z-stack images were collected (from bottom to top) using identical acquisition parameters with a DeltaVision wide-field fluorescent microscope (Applied Precision, GE) equipped with a digital camera (CoolSNAP HQ; Photometrics), using a 1.4-numerical aperture 100× objective lens. Excitation light was generated using the Insight SSI solid-state illumination module (Applied Precision, GE), and images were deconvolved with the SoftWoRx deconvolution software (Applied Precision, GE). Following deconvolution, images were quantified by Imaris (Bitplane) software using the Surfaces feature function, generating surfaces around red puncta, as described (77). Three-dimensional views of images were generated using Surpass mode of Imaris software (78).
Calcium Assays. Intracellular Ca 2+ in A7r5 cells was measured using the Fluo-4 NW Calcium Assay Kit (Molecular Probes), as described (51,52,79). Intracellular Ca 2+ in human VSMC was measured using the ratiometric Ca 2+ indicator dye Fura-2. VSMC were grown on 15-mm round coverslips for 3 d until 80-100% confluence. Coverslips were washed twice with modified Krebs solution (135 mM NaCl, 5.9 mM KCl, 1.5 mM CaCl 2 , 1.2 mM MgCl 2 , 11.5 mM glucose, 11.6 mM Hepes, pH 7.3) and then incubated in the same solution with 5 μM Fura-2-AM, 0.1% BSA, and 0.02% Pluronic F127 detergent for 60 min at room temperature in the dark. Cells were next washed twice and incubated in the dark in control medium for 0.5-2 h. All experiments were performed at room temperature in the presence of continuous perfusion of bath solution (1× Hanks' balanced salt solution, 20 mM Hepes) at a rate of 2 mL/min. Cell images were acquired using C Imaging System (Compix Inc.) with an Olympus 1× 71 inverted epifluorescence microscope (10× fluorescence objective) and Simple PCI software (Vers.5.3.1.). Fura-2 fluorescence (340 and 380 nm excitation, 510 nm emission) was measured every 2 s for 1-2 min before application of PE, followed by 5 min after PE application. Background fluorescence was determined at the end of each experiment by quenching Fura-2 fluorescence with 2 μM ionomycin and 6 mM MnCl 2 for 2 min and subtracted from individual wavelength measurements before calculating 340:380 nm fluorescence ratios. For Ca 2+ flux analyses, 50 individual cells in each field were continuously monitored, and the corrected 340/380 fluorescence ratio was calculated for each cell and averaged among all cells in the vision field.

Vascular Smooth Muscle Cell Contraction
Assay. Freshly isolated VSMC from mesenteric arteries were dispensed onto a glass coverslip base of the recording chamber and allowed to adhere for at least 15 min at room temperature. All experiments were performed with continuous perfusion of the bath solution containing 140 mM NaCl, 5.36 mM KCl, 1.2 mM MgCl 2 , 2 mM CaCl 2 , 10 mM Hepes, and 10 mM D-glucose (pH 7.3), 298 mOsm/L. Images were acquired using a C Imaging System (Compix Inc.) with an Olympus 1 × 71 inverted epifluorescence microscope (10× objective, phase contrast) and Simple PCI software (Vers.5.3.1.) every 2 s for 1 min before the application of 10 μM PE and for an additional 3 min after PE application. The total number of the cells and the number of the cells that contracted in response to PE were counted in each field. For each experiment, cells from a single animal were tested in triplicate or quadruplicate.
In Vivo Testing of PE Responsiveness. Male Sprague-Dawley rats (300-325 g body weight, Harlan) were anesthetized with 2.5% (vol/vol) isoflurane, and a central venous catheter and an arterial line were placed in the femoral vessels. After stable baseline conditions were achieved, animals received an i.v. bolus injection of the CXCR4 ligands (AMD3100, CXCL12, or ubiquitin; 750 nmol/kg in 0.5 mL of normal saline). Five minutes later, i.v. bolus injections of increasing doses of PE (5 ng/kg-40 μg/kg) in 0.5 mL of normal saline were administered at 5-min intervals. In blocking experiments, AMD3100 was administered 5 min before CXCL12 or ubiquitin treatment. MAP was recorded at 10-s intervals for the duration of the experimental period. At the end of the experimental period, animals were euthanized (isoflurane inhalation, bilateral pneumothorax). For each dose of PE, the area under the MAP curve was calculated with the GraphPad-Prism 6 software, and dose-response curves were generated.
Data Analyses. Data are expressed as mean ± SEM from n independent experiments that were performed on different days. Data were analyzed using the GraphPad-Prism 6 software. Unpaired Student's t test or one-way analyses of variance with Bonferroni's multiple comparison post hoc test for multiple comparisons were used, as appropriate. Dose-response curves were generated using nonlinear regression analyses. Best-fit values were compared with the extra sum-of-squares F test. A two-tailed P < 0.05 was considered significant.