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

Abstract

Previously, we reported that chemokine (C-C motif) receptor 2 (CCR2) heteromerizes with α_1B-adrenoceptor (α_1B-AR) in leukocytes, through which α_1B-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:α_1b-AR heteromers. Proximity ligation assays detected CCR2:α_1B-AR heteromers in hVSMCs and human mesenteric arteries. CCR2:α_1B-AR heteromerization per se enhanced α_1B-AR-mediated Gα_q-coupling. Chemokine (C-C motif) ligand 2 (CCL2) binding to CCR2 inhibited Gα_q activation via α_1B-AR, cross-recruited β-arrestin to and induced internalization of α_1B-AR in recombinant systems and in hVSMCs. Our findings suggest that CCR2 within CCR2:α_1B-AR heteromers biases α_1B-AR signaling and provide a mechanism for previous observations suggesting a role for CCL2/CCR2 in the regulation of cardiovascular function.

Chemokine (C-C motif) ligand 2 (CCL2) is the principal endogenous agonist of chemokine (C-C motif) receptor 2 (CCR2) and a key driver of the early inflammatory response to traumatic-hemorrhagic shock in animals and humans [1–6]. Systemic CCL2 concentrations have been shown to be associated with hypotension and to segregate surviving from nonsurviving trauma patients [1,7]. In combination with our previous finding that a selective CCR2 antagonist stabilizes hemodynamics in animal models of hemorrhagic shock and fluid resuscitation [8], these data point toward a role of the CCL2/CCR2 axis in the regulation of the cardiovascular stress response to trauma and hemorrhage. The molecular mechanisms underlying these preclinical and clinical observations remain to be determined.

Recently, we provided evidence that most human chemokine receptors can form heteromers with α₁-adrenergic receptors (ARs) in recombinant systems and that such heteromers, including CCR2:α_1B-AR heteromers, are detectable in human leukocytes, through which α_1B-AR regulate chemokine receptor function [9]. Whether CCR2:α_1B-AR heteromerization occurs in human vascular smooth muscle and affects α_1B-AR signaling, however, is unknown. Here, we provide evidence that CCR2:α_1B-AR heteromers are constitutively expressed in human vascular smooth muscle. Moreover, we observed in recombinant systems and in primary human vascular smooth muscle cells (hVSMCs) that ligand-free and agonist-bound CCR2 bias α_1B-AR coupling to its signaling transducers, leading to inhibition of G protein signaling via α_1B-AR, β-arrestin cross-recruitment to and subsequent internalization of α_1B-AR upon CCL2 binding to CCR2.

Materials and methods

Reagents

Phenylephrine (PE) and poly-L-lysine were purchased from Millipore Sigma (St. Louis, MO, USA). CCL2 was purchased from Protein Foundry (Milwaukee, WI, USA). Accell CCR2 siRNA and nontargeting (NT) siRNA were from GE Dharmacon (Lafayette, CO, USA). Luciferase substrate 400a was from Nanolight Technology (Lakeside, AZ, USA) and Bright-Glo from Promega (Madison, WI, USA). Rabbit anti-α_1B-AR (ab169523) and rabbit anti-HA (Abcam 9110) were obtained from Abcam (Cambridge, UK). Mouse anti-CCR2 (MAB150), mouse IgG and rabbit IgG were purchased from R&D Systems (Minneapolis, MN, USA). Mouse anti-FLAG (F1804) was from Millipore Sigma. Goat anti-mouse conjugated with Alexa 488 and goat anti-rabbit conjugated with Alexa 594 antibodies were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

Plasmids

FLAG-tagged Tango plasmid α_1b-AR-TANGO (#66214) was from Addgene (Watertown, MA, USA) deposited by the laboratory of Bryan Roth. FLAG- or human influenza hemagglutinin (HA)-α_1b-AR were generated by PCR amplification of cDNA from α_1b-AR-TANGO and ligation with the FLAG or HA tag. CCR2 cDNA was synthesized by Twist Bioscience (South San Francisco, CA, USA) with optimized human codons encoding the amino acid sequence AAB57792 (Gen-Bank nucleotide sequence database). G protein subunit β1 was from the cDNA Resource Center (Bloomsberg, PA, USA). Gαq118-RluII was kindly provided by Michel Bouvier. Gγ1-GFP2 was from Addgene (#140989). α_1b-AR-RLuc and CCR2-RLuc were generated by ligating RLuc at the C-termini between Age I and Xba I sites. α_1b-AR-YFP and CCR2-YFP were generated by ligating YFP at the C-termini between the Age I and Xba I sites. Metabotropic glutamate receptor 1 (mGlu₁R)-YFP was as described [10,11]. All plasmids were confirmed by sequencing.

Cells and cell culture

Human embryonic kidney 293 T (HEK293T) cells (CRL-3216) and human primary aortic vascular smooth muscle cells (hVSMCs, PCS-100–012) were purchased from American Type Culture Collection. HEK293T cells were cultured in high glucose Dulbecco’s modified Eagle medium supplemented with 10% FBS, 100 U·mL⁻¹ penicillin, and 100 μg·mL⁻¹ streptomycin. hVSMCs were cultured in vascular basal media (ATCC PCS-100–030) supplemented with the vascular smooth muscle growth kit (ATCC PCS-100–042), 100 U·mL⁻¹ penicillin, and 100 μg·mL⁻¹ streptomycin. The HTLA cell line, a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin 2-TEV fusion gene was generously provided by the laboratory of Bryan Roth and maintained in high glucose Dulbecco’s modified Eagle medium supplemented with 10% FBS, 100 U·mL⁻¹ penicillin, 100 μg·mL⁻¹ streptomycin, 100 μg·mL⁻¹ hygromycin B, and 2 μg·mL⁻¹ puromycin. All cells were cultured in a humidified environment at 37 °C, 5% CO₂.

Human mesenteric arteries

The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board of the University of South Florida (Pro00037029). All subjects gave informed consent for inclusion before they participated in the study. Patients undergoing colon resection for noninfectious indications were included. Immediately upon removal of the colon specimen, a small biopsy (0.5 cm) of the mesentery located at the outermost margin of the nondiseased specimen was collected ex vivo in the operating room and submerged in cold 130 mM NaCl, 4.7 mM KCl, 1.18 mM KH₂PO₄, 1.17 mM MgSO₄, 14.9 mM NaHCO₃, 5.5 mM D-glucose, 0.026 mM EDTA and 1.16 mM CaCl₂. After transfer of the biopsy to the laboratory, small arteries were dissected from the biopsy and the surrounding adventitia removed under a dissection microscope. Arteries were then embedded in optimal cutting temperature medium on dry ice and stored at −80 °C until cryosectioning into 5-μm cross-sections. Cryosections were mounted onto gelatin-coated histological slides and air-dried for 30 min. Cryosections were then overlayed with 4% paraformaldehyde for 15 min, washed three times with phosphate-buffered saline and incubated for 30 min with blocking buffer (1% horse serum in phosphate-buffered saline) at room temperature, and then used for proximity ligation assays (PLAs).

Bioluminescence resonance energy transfer

Bioluminescence resonance energy transfer (BRET) assays were performed in HEK293T cells as described previously [10–13]. In brief, HEK293T cells were seeded in 12-well plates and transfected with the indicated plasmids using Lipofectamine 3000 transfection reagent (Thermo Fisher). For BRET titration assays, cells were transfected at a fixed amount of 50 ng of RLuc-tagged plasmids with increasing amounts of YFP or YFP-tagged plasmids (mGlu₁R-YFP, α_1b-AR-YFP or CCR2-YFP). Empty vector pcDNA3.1 was added to keep the total amount DNA for each transfection constant. Cells were incubated overnight and subsequently replated to poly-L-lysine coated 96-well white plates and incubated again overnight. Cells were then washed with PBS and fluorescence was measured in a Biotek Synergy (Winooski, VT, USA) HT4 plate reader (excitation 485 nm, emission 528 nm). For BRET measurements, coelenterazine H was added at a final concentration of 5 μM. After 10 min incubation at room temperature, luminescence was measured at 460 nm and 528 nm. The BRET signal was calculated as the ratio of the relative luminescence units (RLU) measured at 528 nm over RLU at 460 nm. The net BRET is calculated by subtracting the BRET signal detected when α_1b-AR-RLuc or CCR2-RLuc were transfected alone.

Proximity ligation assays

Proximity ligation assays were performed as described in detail previously [13–16]. Mouse anti-CCR2 (1:750 dilution) and rabbit anti-α_1B-AR (1:500 dilution) were used for the detection of individual receptors and receptor:receptor interactions. Mouse and rabbit IgG were used as negative control antibodies. 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 identical settings.

PRESTO-Tango β-arrestin-2 recruitment assay

PRESTO-Tango assays were performed as previously described [11–14,16–19]. HTLA cells were seeded in a six-well plate and transfected with 750 ng of each plasmid (α_1b-AR-Tango plus pcDNA3.1 or HA-CCR2) using Lipofectamine 3000 (Thermo Fisher Scientific). The following day, transfected HTLA cells (75 000 cells/well) were plated in poly-L-lysine pre-coated 96-well plates and allowed to attach to the plate surface for at least 4 h prior to treatment. Cells were treated with receptor agonists for 2 h, then replaced with fresh full medium, and incubated overnight at 37 °C, 5% CO₂ in a humidified chamber. After overnight culture, media was replaced with a 100 μL 1 : 10 mixture of Bright-Glo and 1× HBSS, 20 mM HEPES solution. Plates were then incubated at room temperature for 10 min before measuring luminescence on a Biotek Synergy HT4 plate reader.

Flow cytometry

Flow cytometry was used to evaluate receptor expression, as described [10,11,20]. At least 10 000 cells/sample were recorded on LSRII flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with the FLOWJO software (Flowjo LLC, Ashland, OR, USA). To detect the expression levels of α_1b-AR-Tango and HA-CCR2 in transfected HTLA cells, cells were probed with mouse anti-FLAG antibody (Sigma F1804) or rabbit anti-HA antibody (Abcam 9110), followed by labeling with corresponding secondary Alexa 488 or Alexa 594 antibody. To examine receptor internalization in hVSMCs, cells were incubated with 200 nM CCL2 for 30 min at 37 °C. Cells were then washed with 50 mM glycine buffer, pH 7.2, 150 mM NaCl, followed by washing with ice-cold PBS. Cell surface α_1B-AR and CCR2 receptors were probed with anti-α_1B-AR or anti-CCR2 and corresponding secondary Alexa 488- or Alexa 594-conjugated antibodies. Receptor cell surface expression levels are expressed as the median fluorescence intensity (MFI) of CCL2 stimulated cells in percent of the MFI measured in unstimulated cells (=100%).

Gαq activation

HEK293T cells were transfected with 0.2 μg each of α_1b-AR, Gαq118-RlucII, Gβ1, Gγ1-GFP2 together with pcDNA3.1 or CCR2. After 48 h incubation, cells were detached with 5 mM EDTA/PBS, resuspended in 0.1% glucose/PBS and replated to a 96-well plate. Cells were incubated with luciferase substrate 400a at room temperature for 1 min. Subsequently, cells were treated with vehicle, 100 nM CCL2, 1 μM PE or CCL2 plus PE at room temperature in duplicate. One minute after treatment with vehicle or agonists, luminescence was measured in a plate reader (Cytation 1 Cell Imaging Multi-Mode Reader, BioTek, Ex 410 nm, Em 515 nm) four times in 30 s intervals in each independent experiment and the mean of the four measurements was calculated. The BRET signal was calculated as the ratio of RLU measured at 515 nm over RLU at 410 nm. The ligand-induced BRET change was calculated by subtracting the BRET signal of the control wells treated with vehicle.

Gene silencing by RNA interference

Human vascular smooth muscle cells were transfected with NT siRNA or CCR2 siRNA at a final concentration of 1 μM using Accell Delivery Media (GE Dharmacon) as previously described [9,14,16,20].

Calcium flux assays

Intracellular calcium was measured using the FLIPR Calcium 6 assay kit (Molecular Devices, San Jose, CA, USA). HEK293T cells were plated in a six-well plate and transfected with 1 μg each of α_1b-AR together with pcDNA3.1 or CCR2. The next day, cells were replated to a black clear bottom 96-well plate. After overnight incubation, cells were loaded with FLIPR Calcium 6 dye following the manufacturer’s instructions. Calcium flux was read in a Biotek Synergy HT4 plate reader (excitation 485 nm, emission 528 nm) at room temperature. After 45 s of basal reading, cells were treated with 1 μM PE with or without 100 nM CCL2 and read for 2 min.

Inositol trisphosphate measurements

Inositol trisphosphate (IP3) was measured with an IP3 enzyme-linked immunoassay (ELISA) kit following the manufacturer’s protocol (LS BIO, F10644, Seattle, WA, USA). In brief, hVSMCs were cultured in six-well plates and incubated with NT siRNA or CCR2 siRNA for 3 days for gene silencing. On day 3, cells were replaced with hVSMC culture medium. After overnight incubation, cells were treated with PE (1 μM) together with or without CCL2 (200 nM) for 5 min. Cells were then washed with cold PBS, harvested in 250 μL of cold PBS, and lysed by sonication. The cell lysate was centrifuged for 10 min at 4 °C at 1500 g to remove cellular debris. The supernatants were loaded to ELISA plate wells for IP3 measurements according to the manufacturer’s protocol. The amounts of IP3 were normalized to protein levels. Protein concentrations were measured with the Bio-Rad (Hercules, CA, USA) DC Protein Assay kit.

Data analyses

Data are expressed as mean ± standard error. Titration curves were analyzed with nonlinear regression analyses. Best-fit values were compared with the extra-sum-of-squares F test. Student’s t-test and one-way analyses of variance (ANOVA) with Dunnett’s multiple comparison post hoc test for multiple comparisons were used to assess statistical significance, as appropriate. A two-tailed P < 0.05 was considered significant. All analyses were calculated with the GRAPHPAD PRISM 8, Version 8.4.0 software.

Results and Discussion

CCR2 and α_1B-AR heteromerize in human vascular smooth muscle

To confirm that recombinant CCR2 heteromerizes with α_1b-AR (upper and lower case subscripts are used to denote endogenous and recombinant α₁-AR subtypes, respectively [21]), we performed intermolecular saturation BRET measurements to assess receptor interactions in HEK293T cells when each receptor serves as the energy donor and energy acceptor, respectively. Saturation BRET between α_1b-AR-RLuc or CCR2-RLuc and mGlu₁R-YFP and between α_1b-AR-RLuc or CCR2-RLuc and YFP were used as negative controls and performed in parallel experiments. Fig. 1A,B show the BRET between α_1b-AR-RLuc and CCR2-YFP (Fig. 1A) and between CCR2-RLuc and α_1b-AR-YFP (Fig. 1B), respectively. Consistent with our previous observations, BRET between α_1b-AR and CCR2 showed hyperbolic progressions with increasing energy acceptor: donor ratios in each energy acceptor: donor configuration, suggesting constitutive heteromerization [9,22]. As expected, BRET between α_1b-AR-RLuc or CCR2-RLuc and mGlu₁R-YFP or YFP was low and increased linearly with increasing energy acceptor: donor ratios, which is consistent with nonspecific bystander BRET [9–11].

Fig. 1.

Recombinant and vascular smooth muscle CCR2 form heteromers with α_1b/B-AR. (A,B) BRET between CCR2 and α_1b-AR in HEK293T cells. Cells were transfected with a fixed amount of α_1b-AR-RLuc and increasing amounts of CCR2-YFP (circles), mGlu₁R-YFP (squares), or YFP (triangles) (A), or with a fixed amount of CCR2-RLuc and increasing amounts of α_1b-AR-YFP (circles), mGlu₁R-YFP (squares), or YFP (triangles) (B). Forty-eight hours after transfection, YFP fluorescence and luminescence were read as described in Materials and methods. Net BRET (528/460 nm) was plotted against YFP fluorescence/luminescence (YFP/Lum). The graphs are representative of three independent experiments. (C) PLA to detect (top to bottom) CCR2, α_1B-AR and CCR2:α_1B-AR heteromers in hVSMCs (center) and human mesenteric arteries (right). As controls, cells were incubated with mouse or rabbit IgG (m/rIgG) (left). Images show merged 4′,6-diamidino-2-phenylindole (DAPI, nuclear counterstain) and PLA signals (red, λexcitation/emission 598/634 nm) and are representative of n = 3 independent experiments with hVSMCs and human arteries from 3 different donors. Scale bars: left and center – 20 μm; right – 0.5 mm.

To test whether such heteromers can be detected in isolated hVSMCs and in small human mesenteric arteries in situ, we performed PLAs to visualize individual receptors and receptor–receptor proximity at single molecule resolution [23]. As shown in Fig. 1C (center), we observed positive PLA signals for α_1B-AR and CCR2 individually, and for interactions between α_1B-AR and CCR2 in hVSMCs. Control experiments with mouse and rabbit IgG did not result in PLA signals (Fig. 1C left). Moreover, PLA signals for α_1B-AR, CCR2 and α_1B-AR:CCR2 interactions were detectable in the tunica media of human mesenteric arteries (Fig. 1C, right). These findings confirm our previous observations that recombinant and endogenously expressed α_1B-AR and CCR2 constitutively heteromerize, and suggest that such heteromers are also expressed in human vascular smooth muscle [9].

Recombinant CCR2 modulates α_1b-AR coupling to signaling transducers

To gain initial insight into the roles of CCL2/CCR2 in the regulation of α_1B-AR signaling, we utilized BRET in HEK293T cells to monitor α_1b-AR-induced Gαq activation by measuring dissociation of the heterotrimeric G proteins. As shown in Fig. 2A, PE stimulation of cells transfected with α_1b-AR and BRET G protein biosensors resulted in a significant decrease of BRET, which was not affected by CCL2. In cells co-transfected with α_1b-AR, CCR2 and the BRET G protein biosensors, CCL2 inhibited the PE-induced BRET change, as compared with cells stimulated with PE alone (Fig. 2B). Moreover, we noted that the PE-induced BRET change was more pronounced in cells co-transfected with α_1b-AR and CCR2, as compared with cells transfected with α_1b-AR alone (BRET change: α_1b-AR alone, −0.017 ± 0.0007; α_1b-AR plus CCR2, −0.02 ± 0.001, P < 0.05). Because we did not confirm that each individual recombinant protein was expressed at comparable levels in these experiments, this observation should be interpreted with caution. While the increased PE-induced BRET change in cells co-transfected with α_1b-AR and CCR2 may suggest that the presence of CCR2 enhances coupling of α_1b-AR to Gαq, the observed phenomenon could also be explained by different expression levels of α_1b-AR or the BRET G protein biosensors under our experimental conditions. The latter, however, does not affect interpretation of the effects of CCL2 on α_1b-AR coupling to Gαq in the absence and presence of CCR2.

Fig. 2.

CCL2/CCR2 modulate Gαq coupling and calcium fluxes upon activation of α_1b-AR. (A,B) BRET assays to detect Gαq activation. Cells were transfected with α_1b-AR, Gαq-118RlucII, Gβ1, and Gγ1-GFP2 together with pcDNA3 (A) or CCR2 (B). Forty eight hours after transfection, cells were treated with 100 nM CCL2, 1 μM PE, or both. BRET signals were read as described in Materials and methods. BRET change: BRET in the presence of agonists minus BRET after vehicle treatment. *: P < 0.05 vs. vehicle treatment; ^#: P < 0.05 vs. PE treatment. N = 5. (C) Flow cytometry for the detection of FLAG-α_1b-AR with anti-FLAG (left) and of HA-CCR2 with anti-HA (right). Cells were transfected with FLAG-α_1b-AR plus pcDNA3 (red line) or HA-CCR2 (green line). Gray area: unstained cells. (D,E) Calcium flux assays. HEK293T were transfected with FLAG-α_1b-AR plus pcDNA3 (D) or HA-CCR2 (E), as in (C). Cells were then treated with 1 μM PE (arrows) plus vehicle or 100 nM CCL2. RFU (%): relative fluorescence units, expressed as % of baseline (=100%). N = 6. (F). Areas under curves (AUC) of Ca²⁺-fluxes, as in D and E. *: P < 0.05 vs. all other conditions.

Because α_1b-AR coupling to Gαq leads to phospholipase C-mediated intracellular Ca²⁺ fluxes, we then measured Ca²⁺ fluxes in HEK293T cells transfected with α_1b-AR alone or with α_1b-AR plus CCR2. In these experiments, we confirmed by flow cytometry that expression of α_1b-AR was comparable between the experimental conditions and that CCR2 was expressed in cells co-transfected with both receptors (Fig. 2C). In cells transfected with α_1b-AR alone, PE induced robust Ca²⁺-fluxes, which were not affected by CCL2 (Fig. 2D,F). In cells co-transfected with α_1b-AR and CCR2, PE-induced Ca²⁺-fluxes were higher than in cells transfected with α_1b-AR alone and could be inhibited by CCL2 (Fig. 2E,F). In combination with our observations on α_1b-AR coupling to Gαq, these findings indicate that agonist-bound CCL2 inhibits Gαq-mediated signaling of α_1b-AR, and favor the assumption that ligand free CCR2 enhances Gαq-mediated signaling of α_1b-AR from the α_1b-AR:CCR2 heteromer.

Next, we employed the PRESTO-Tango cell system to assess whether CCR2 also modulates β-arrestin recruitment to α_1b-AR-Tango [17]. We confirmed comparable expression of α_1b-AR-Tango and expression of CCR2 by flow cytometry under our experimental conditions (Fig. 3A). PE-induced β-arrestin recruitment to α_1b-AR was not affected by the presence of CCR2 (Fig. 3B, open symbols). While CCL2 stimulation did not induce β-arrestin recruitment to α_1b-AR in cells transfected with α_1b-AR-Tango alone, CCL2 stimulation in cells transfected with α_1b-AR-Tango plus CCR2 resulted in β-arrestin recruitment to α_1b-AR in a dose-dependent manner (Fig. 3B, black symbols). As CCL2 induces β-arrestin recruitment to CCR2 [24], our findings suggest cross-recruitment of β-arrestin to α_1b-AR within the CCL2 stimulated α_1b-AR:CCR2 heteromeric complex. The EC₅₀ of CCL2 to induce cross-recruitment of β-arrestin to α_1b-AR was 85 ± 64 nM. The maximal efficacy of CCL2 to induce β-arrestin recruitment to α_1b-AR was comparable to an EC₃₅ of PE. Fig. 3C shows β-arrestin recruitment to α_1b-AR when cells were co-stimulated with increasing concentrations of PE plus 100 nM of CCL2, a dose that resembles the EC₅₀ of CCL2 to induce cross-recruitment of β-arrestin recruitment to α_1b-AR. While CCL2 did not affect β-arrestin recruitment to α_1b-AR in cells transfected with α_1b-AR alone, co-stimulation with CCL2 in cells transfected with α_1b-AR and CCR2 increased the efficacy of PE to induce β-arrestin recruitment to α_1b-AR by 31 ± 4% (P < 0.01 vs. cells transfected with α_1b-AR alone).

Fig. 3.

Ligand binding to CCR2 cross recruits β-arrestin to ligand free α_1b-AR and increases β-arrestin recruitment to activated α_1b-AR. (A) Flow cytometry for the detection of FLAG-α_1b-AR-Tango with anti-FLAG (top) and of HA-CCR2 with anti-HA (bottom). HTLA cells were transfected with FLAG-α_1b-AR-Tango plus pcDNA3 (red line) or HA-CCR2 (green line). Gray area: unstained cells. (B,C) α_1b-AR PRESTO-Tango β-arrestin recruitment assays. HTLA cells were transfected with α_1b-AR-Tango plus pcDNA3 (circles) or with α_1b-AR-Tango plus CCR2 (squares), as in (A). RLU (%): relative luminescence units (RLU) subtracted by the RLU of unstimulated cells and expressed as % RLU measured in cells transfected with α_1b-AR-Tango plus pcDNA3 and stimulated with the highest concentration of phenylephrine (PE). (B) Cells were stimulated with increasing concentrations of PE (open symbols) or CCL2 (black symbols). (C) Cells were stimulated with increasing concentrations of PE plus vehicle (open symbols) or 100 nM CCL2 (black symbols).

CCR2 regulates signaling and internalization of α_1B-AR in hVSMCs

To evaluate the roles of endogenously expressed CCR2 in the regulation of α₁-AR signaling in hVSMCs, we utilized siRNA to deplete CCR2 from the cell surface and measured IP3 concentrations upon PE stimulation. Typical PLA images for the detection of individual receptors and α_1b-AR:CCR2 heteromers in cells after incubation with NT and CCR2 siRNA are shown in Fig. 4A and the quantification of PLA signals from three independent experiments is shown in Fig. 4B. As compared with hVSMCs incubated with NT-siRNA, PLA signals corresponding to CCR2 and to α_1b-AR:CCR2 heteromers were reduced by 75–80% in cells after incubation with CCR2 siRNA. Expression of α_1b-AR was not affected by CCR2 siRNA. The PE-induced IP3 concentrations in hVSMCs after incubation with NT and CCR2 siRNA are shown in Fig. 4C. In hVSMCs incubated with NT siRNA, PE stimulation resulted in a significant increase in IP3 concentrations, whereas IP3 concentrations after stimulation with CCL2 were indistinguishable from unstimulated cells. Co-stimulation with CCL2 reduced the PE-induced increase in IP3 concentrations by more than 60%. In hVSMCs after CCR2 siRNA knockdown, the PE-induced increase in IP3 concentrations was reduced by 50%, as compared with hVSMCs incubated with NT siRNA, and co-stimulation with CCL2 did not affect PE-induced IP3 generation. These findings are consistent with our observations on the effects of CCL2/CCR2 on Gαq coupling of α_1b-AR and subsequent G protein-mediated downstream signaling in the recombinant system. The observation that the effects of endogenous CCR2 on α_1B-AR signaling were more pronounced than in the recombinant system could be explained by a reduced efficacy of recombinant receptors to couple to downstream signaling transducers, when compared with endogenously expressed receptors and signaling transducers in a physiological cellular environment.

Fig. 4.

CCL2/CCR2 regulate Gαq signaling and internalization of α_1B-AR from the CCR2:α_1B-AR heteromer in hVSMCs. (A–C) hVSMCs were transfected with nontarget-siRNA (NT siRNA) or CCR2 siRNA. (A) PLA to visualize individual receptors and receptor interactions, as in Fig. 1C. Images show merged 4′,6-diamidino-2-phenylindole (DAPI, nuclear counterstain) and PLA signals (red, λexcitation/emission 598/634 nm) and are representative of n = 3 independent experiments. Scale bars: 20 μm. (B) Quantification of PLA signals from n = 3 independent experiments. PLA signals (% NT siRNA): PLA signals in % of PLA signals in cells incubated with NT siRNA (=100%). *: P < 0.05 vs. cells incubated with NT siRNA. (C) hVSMCs were treated with vehicle, 1 μM PE, 200 nM CCL2 or both for 5 min. IP3 production was measured by ELISA. N = 3 independent experiments. *: P < 0.05 vs. cells incubated with NT siRNA and stimulated with vehicle. ^#: P < 0.05 vs. cells incubated with NT siRNA and stimulated with PE alone. (D,E) hVSMCs were stimulated with 200 nM CCL2 at 37 °C for 30 min. (D) Flow cytometry to detect CCR2 (top) and α_1B-AR (bottom). Red lines: unstimulated cells. Green lines: cells after CCL2 treatment. Gray area: unstained cells. (E) Quantification of receptor expression levels from n = 3 independent experiments, as in (D). Receptor expression is expressed as % of unstimulated cells. *: P < 0.05 vs. unstimulated cells.

Because β-arrestin recruitment to α_1b-AR is intimately involved in the internalization of the receptor and our findings on recombinant receptors suggested β-arrestin cross-recruitment to α_1b-AR by agonist-bound CCR2, we exposed hVSMCs to CCL2 and measured cell surface expression of CCR2 and α_1B-AR by flow cytometry. Fig. 4D shows a representative analysis of receptor expression levels before and after 30 min of CCL2 stimulation by flow cytometry and Fig. 4E shows the quantification of receptor expression levels from 3 independent experiments. In agreement with CCL2-induced cross-recruitment of β-arrestin to α_1b-AR, CCL2 stimulation reduced CCR2 expression by 64 ± 6% and α_1B-AR expression by 51 ± 7% (Fig. 4E).

Based on these observation, we propose the following molecular mechanisms by which α_1B-AR coupling to its signaling transducers is regulated within α_1B-AR:CCR2 heteromers in human vascular smooth muscle (Fig. 5). Agonist binding to the α_1B-AR proto- or homo-oligomer results in balanced coupling to its signaling transducers (Fig. 5A). Heteromerization of α_1B-AR with ligand-free CCR2 biases agonist activated α_1B-AR coupling toward Gαq, which enhances G protein-mediated signaling (Fig. 5B). Binding of CCL2 to CCR2 within the α_1B-AR:CCR2 heteromer results in β-arrestin cross-recruitment to ligand free α_1B-AR and subsequent co-internalization of the α_1B-AR:CCR2 heteromer (Fig. 5C). Upon binding of CCL2 to CCR2, agonist-induced coupling of α_1B-AR to Gαq and G protein-mediated downstream signaling is inhibited and β-arrestin recruitment to α_1B-AR enhanced (Fig. 5D).

Fig. 5.

Schematic of the proposed mechanisms by which α_1B-AR within CCR2:α_1B-AR heteromers is regulated in human vascular smooth muscle. PE: Phenylephrine. β-ARR: β-arrestin. (A) Balanced agonist-induced coupling of the α_1B-AR proto- or homomer to Gαq and β-arrestin. (B). Within the CCR2:α_1B-AR heteromer, agonist-induced coupling of α_1B-AR to its signaling transducers is biased toward Gαq. (C) Agonist activated CCR2 cross-recruits β-arrestin to ligand free α_1B-AR within the CCR2:α_1B-AR heteromer. (D) Agonist bound CCR2 inhibits agonist-induced coupling of α_1B-AR to Gαq and enhances agonist-induced β-arrestin recruitment to α_1B-AR within the CCR2:α_1B-AR heteromer.

In conclusion, our findings provide evidence for the existence and functional relevance of α_1B-AR:CCR2 heteromers in hVSMCs. The proposed molecular mechanisms by which α_1B-AR:CCR2 heteromerization regulates α_1B-AR function in hVSMCs imply that CCL2 binding to CCR2 within α_1B-AR:CCR2 heteromers impairs the responsiveness of VSMC to endogenous catecholamines, leading to insufficient vascular tone and impaired blood pressure during the cardiovascular stress response in disease processes. The latter assumption is consistent with clinical and preclinical observations suggesting that CCL2/CCR2 contribute to the development of hemodynamic instability and development of hypotension during the early cardiovascular stress response to trauma and hemorrhage [1,7,8]. At least 13 of the 23 members of the CR family have been detected in vascular smooth muscle during homeostasis or in disease processes [6,25–34]. Because we showed that most of these chemokine receptors can form heteromers with α₁-ARs, it is likely that other chemokine receptors and their agonists are also involved in the regulation of vascular tone in diverse inflammatory disease processes. We believe that a better understanding of the mechanisms by which chemokines/chemokine receptors contribute to the regulation of α₁-AR function in VSMC provides the potential to discover novel therapeutic approaches to modulate blood pressure in the future.

Abbreviations

AR

adrenergic receptor

BRET

bioluminescence resonance energy transfer

CCL

chemokine (C-C motif) ligand

CCR

chemokine (C-C motif) receptor

HEK

human embryonic kidney

hVSMC

human vascular smooth muscle cell

IP3

inositol trisphosphate

mGlu₁R

metabotropic glutamate receptor 1

NT

nontargeting

PE

phenylephrine

PLA

proximity ligation assays

RLuc

Renilla luciferase

YFP

enhanced yellow fluorescent protein