Chemokine receptor hetero-oligomers regulate monocyte chemotaxis

Abstract

It is known that stress influences immune cell function. The underlying molecular mechanisms are unclear. We recently reported that many chemokine receptors (CRs) heteromerize with α₁-adrenoceptors (α₁-ARs) through which CRs are regulated. Here, we show that arginine vasopressin receptor 1A (AVPR1A) heteromerizes with all human CRs, except chemokine (C-X-C motif) receptor (CXCR)1, in recombinant systems and that such heteromers are detectable in THP-1 cells and human monocytes. We demonstrate that ligand-free AVPR1A differentially regulates the efficacy of CR partners to mediate chemotaxis and that AVPR1A ligands disrupt AVPR1A:CR heteromers, which enhances chemokine (C-C motif) receptor (CCR)1-mediated chemotaxis and inhibits CCR2-, CCR8-, and CXCR4-mediated chemotaxis. Using bioluminescence resonance energy transfer to monitor G protein activation and CRISPR/Cas9 gene-edited THP-1 cells lacking AVPR1A or α_1B-AR, we show that CRs that share the propensity to heteromerize with α_1B/D-ARs and AVPR1A exist and function within interdependent hetero-oligomeric complexes through which the efficacy of CRs to mediate chemotaxis is controlled. Our findings suggest that hetero-oligomers composed of CRs, α_1B/D-ARs, and AVPR1A may enable stress hormones to regulate immune cell trafficking.

Figure 1.. Bioluminescence resonance energy transfer (BRET) for the identification of chemokine receptor (CR) heteromerization partners of AVPR1A.

YFP fluorescence and luminescence were read as described in the Materials and Methods section. Net BRET (528/460 nm) was plotted against YFP fluorescence/luminescence (YFP/Lum). (A) HEK293T cells were transfected with AVPR1A-Rluc plus each CR-YFP in triplicate. Net BRET signals are the mean ± SD. Cells transfected with AVPR1A-Rluc and mGlu₁R-YFP at various energy acceptor:donor ratios served as nonspecific controls; nonspecific BRET signals were analyzed by linear regression analysis. The black line shows the regression line, and the dashed lines indicate 99% prediction bands. The gray area indicates the expected distribution of nonspecific BRET signals. BRET signals above the 99% prediction band for nonspecific interactions were considered positive signals for interactions between CRs (colored symbols) and AVPR1A. The graph represents one of three screening experiments. The number of positive BRET signals in three independent screening experiments (0–3/3) is shown for each CR. (B, C, D, E, F, G, H, I, J, K) HEK293T cells were transfected with a fixed amount of AVPR1A-Rluc and with increasing amounts of CR-YFP in duplicate. Figures show saturation BRET signals representative of n = 3 independent experiments per receptor pair. Saturation BRET between AVPR1A and CCR1 (B), CCR2 (C), CCR4 (D), CCR8 (E), CXCR2 (F), CXCR3 (G), CXCR4 (H), CXCR5 (I), CXCR1 (J), and mGlu₁R-YFP (K).

Figure 2.. CR:AVPR1A heteromers are detectable in THP-1 cells and human monocytes.

(A, B) Representative proximity ligation assay images for the detection of individual receptors ((A, B), top rows) and receptor–receptor proximity ((A, B), bottom rows) in THP-1 cells (A) and human monocytes (B). Images show merged DAPI (nuclear counterstain) and proximity ligation assay signals (red, λ_{excitation/emission} 598/634 nm) acquired from z-stack images (n = 10; thickness = 0.5 μm, bottom to top) and are representative of n = 3 independent experiments. As controls, cells were incubated with IgG (A/B, top) or a combination of IgG and anti-AVPR1A (not shown). (A, B) Scale bars, (A) 10 μm and (B) 5 μm.

Figure 3.. AVPR1A ligands modulate chemotaxis mediated by CR heteromerization partners of AVPR1A in THP-1 cells.

(A, B, C, D, E, F, G, H) THP-1 cells were exposed to various concentrations of arginine vasopressin (aVP) (A, B, C, D) or conivaptan (E, F, G, H), and chemotaxis toward CCL2 (10 nmol/liter, (A, E)), CXCL12 (100 nmol/liter, (B, F)), CCL23 (0.1 nmol/liter, (C, G)), or CXCL8 (10 nmol/liter, (D, H)) was tested. CI (%), chemotactic index in the percentage of cells not exposed to AVPR1A ligands. Data are the mean ± SE from n = 3–4 independent experiments. (I, J) Migration of THP-1 cells toward various concentrations of aVP (I) and conivaptan (J). CI, chemotactic index. Data are the mean ± SE from n = 3 independent experiments.

Figure 4.. AVPR1A ligands modulate chemotaxis mediated by CR heteromerization partners of AVPR1A in human monocytes.

(A, B, C, D, E, F) Human monocytes were exposed to various concentrations of aVP (A, B, C) or conivaptan (D, E, F), and chemotaxis toward CCL2 (10 nmol/liter, (A, D)), CCL23 (0.1 nmol/liter, (B, E)), and CXCL8 (10 nmol/liter, (C, F)) was tested. CI (%), chemotactic index in the percentage of cells not exposed to AVPR1A ligands. Data are the mean ± SE from n = 3–4 independent experiments.

Figure 5.. AVPR1A ligands modulate the efficacy of CR heteromerization partners of AVPR1A to mediate chemotaxis.

(A, B, C) Chemotactic dose–responses for CCL2 ((A), n = 3), CCL23 ((B), n = 6), and CXCL12 ((C), n = 3) in THP-1 cells exposed to 10 μmol/liter arginine vasopressin (aVP), 10 μmol/liter conivaptan, or vehicle. Data are the mean ± SE. *P < 0.05 for aVP versus vehicle; ^#P < 0.05 for conivaptan versus vehicle (two-way ANOVA with Dunnett’s multiple comparisons test).

Figure 6.. AVPR1A ligands interfere with heteromerization between AVPR1A and CR partners.

(A, C) THP-1 cells were incubated with vehicle (ctrl., top), 10 μmol/liter arginine vasopressin (aVP, center), or 10 μmol/liter conivaptan (con, bottom) for 30 min at 37°C, and the cell surface expression of individual receptors (A) and receptor–receptor proximity (C) was visualized by the proximity ligation assay (PLA). Images show merged DAPI (nuclear counterstain) and PLA signals (red, λ_{excitation/emission} 598/634 nm) acquired from z-stack images (n = 10; thickness 0.5 μm, bottom to top) and are representative of n = 3 independent experiments. Scale bar, 10 μm. (B) Quantification of PLA signals for AVPR1A, CCR1, CCR2, CCR8, CXCR1, and CXCR4 from n = 3 experiments. Data are the mean ± SE. *P < 0.05 versus ctrl. (D) Quantification of PLA signals for receptor–receptor proximity between AVPR1A and CCR1, CCR2, CCR8, or CXCR4 from n = 3 experiments. Data are the mean ± SE. *P < 0.05 versus ctrl.

Figure 7.. siRNA knockdown of AVPR1A modulates chemotaxis mediated by CR heteromerization partners of AVPR1A.

(A, B, C, D, E, F, G) THP-1 cells were incubated with NT or AVPR1A siRNA. (A) Representative images for the detection of AVPR1A and proximity between AVPR1A and CCR1, CCR2, CCR8, CXCR4, and CXCR1 by the proximity ligation assay (PLA). Images show merged DAPI/PLA signals and are representative of n = 3 independent experiments. Scale bar, 10 μm. (B, C, D, E, F, G) Quantification of the number of PLA signals per cell for AVPR1A and receptor–receptor proximities in THP-1 cells after incubation with NT siRNA (ctrl., white bars) or AVPR1A siRNA (gray bars) (n = 3). Data are the mean ± SE. *P < 0.05 versus cells treated with NT siRNA. (H, I, J, K) THP-1 cells were incubated with NT or AVPR1A siRNA as in (A, B, C, D, E, F, G). Cells were then exposed to various concentrations of aVP, and chemotaxis toward CCL23 (0.1 nmol/liter, (H)), CCL2 (10 nmol/liter, (I)), CXCL12 (100 nmol/liter, (J)), and CXCL8 (10 nmol/liter, (K)) was tested. CI, chemotactic index (mean ± SE, n = 3/condition). *P < 0.05 versus cells incubated with NT siRNA (two-way ANOVA with Dunnett’s multiple comparisons test).

Figure 8.. CRISPR/Cas9 knockout of AVPR1A modulates chemotaxis mediated by CR heteromerization partners of AVPR1A.

(A, B) CRISPR/Cas9 gene editing to generate THP-1 cell lines that lack AVPR1A, with the designation AVPR1A^KO. (A) T7 surveyor assay. Images from agarose gel electrophoresis for the detection of PCR-amplified AVPR1A genomic DNA before (top) and after (bottom) T7EI digestion from puromycin-selected THP-1 cell clones that were transduced with the lentivirus encoding sgRNA targeting AVPR1A and Cas9 (lanes 2 and 3). Lane 1: DNA ladder. (B) Scheme depicting the modified genomic region of AVPR1A in AVPR1A^KO clones from lanes 2 and 3. (C) Detection of individual receptors in a WT THP-1 clone (WT, top) and AVPR1A^KO (bottom) by the proximity ligation assay (PLA). Images show merged DAPI/PLA signals and are representative of n = 3 independent experiments. Scale bars, 10 μm. (D) Quantification of PLA signals per cell for the detection of individual receptors in AVPR1A^KO cells. Data (mean ± SE) are expressed as the percentage of a WT THP-1 cell clone (% WT). *P < 0.05 versus ctrl. (E, F, G) Chemotaxis of AVPR1A^KO and WT THP-1 clones toward various concentrations of CCL2 (E), CCL23 (F), and CXCL8 (G). CI, chemotactic index (mean ± SE, n = 3–4 independent experiments). *P < 0.05 for AVPR1A^KO versus WT THP-1 clones (two-way ANOVA with Dunnett’s multiple comparisons test).

Figure 9.. CRISPR/Cas9 knockout of AVPR1A or ADRA1B does not affect -α_1B/D-AR or AVPR1A expression, respectively.

Expression of individual receptors by the proximity ligation assay (PLA). Images show merged DAPI/PLA signals and are representative of n = 3 independent experiments. Scale bars, 10 μm. (A) PLA images for the detection of α_1B/D-AR in WT and AVPR1A^KO cells. (B) PLA images for the detection of AVPR1A in WT and AVPR1A^KO cells. Quantification of PLA signals for α_1B-AR (C) and α_1D-AR (D) in WT and AVPR1A^KO cells and of PLA signals for AVPR1A in WT and ADRA1B^KO-ARs (E) from n = 3 experiments. Data (mean ± SE) are expressed as % of WT, n = 3.

Figure 10.. CR:AVPR1A and CR:α_1B-AR heteromer expression are interdependent.

(A) Detection of CR:α_1B/D-AR and α_1B-AR:α_1D-AR heteromers in WT THP-1 clones (WT, top) and AVPR1A^KO clones (bottom) by the proximity ligation assay (PLA). Images show merged DAPI/PLA signals and are representative of n = 3 independent experiments. Scale bars, 10 μm. (B) Quantification of PLA signals per cell for the detection of CR:α_1B/D-AR and α_1B-AR:α_1D-AR heteromers in WT THP-1 clones (WT, white bars) and AVPR1A^KO clones (gray bars). Data are the mean ± SE. *P < 0.05 versus WT THP-1 clones. (C) Detection of CR:AVPR1A heteromers in WT THP-1 clones (WT, top) and in ADRA1B^KO clones (bottom) by the PLA. Images show merged DAPI/PLA signals and are representative of n = 3 independent experiments. Scale bars, 10 μm. (D) Quantification of PLA signals per cell for the detection of CR:AVPR1A heteromers in WT THP-1 clones (WT, white bars) and ADRA1B^KO clones (gray bars). Data are the mean ± SE. *P < 0.05 versus WT THP-1 clones.

Figure 11.. CR:AVPR1A and CR:α_1B/D-AR heteromers function interdependently.

(A, B) AVPR1A^KO and WT THP-1 clones were exposed to various concentrations of phenylephrine, and chemotaxis toward CCL23 (0.1 nmol/liter, (A)) and CCL2 (10 nmol/liter, (B)) was tested. CI, chemotactic index, mean ± SE from n = 3 independent experiments. (C, D) ADRA1B^KO and WT THP-1 clones were exposed to various concentrations of aVP, and chemotaxis toward CCL23 (0.1 nmol/liter (C)) and CCL2 (10 nmol/liter, (D)) was tested. CI, chemotactic index, mean ± SE from n = 3 independent experiments. (E, F, G, H, I, J, K) Gαi1 activation assays. Data are the mean ± SE from n = 5 independent experiments. HEK293T cells were transfected with Gαi1-Rlu8, Gβ3, Gγ9-GFP2, and CCR1 together with pcDNA3 (E), α_1b-AR (F, G), AVPR1A (H/I), or α_1b-AR plus AVPR1A (J, K). (E) Cells were exposed to various concentrations of phenylephrine (PE), aVP, or CCL23. (F) Cells were exposed to various concentrations of PE. (G) Cells were exposed to various concentrations of CCL23 plus vehicle or 1 μM PE. (H) Cells were exposed to various concentrations of aVP. (I) Cells were exposed to various concentrations of CCL23 plus vehicle or 0.1 μM aVP. (J) Cells were exposed to various concentrations of phenylephrine (PE), aVP, or CCL23. (K) Cells were exposed to various concentrations of CCL23 plus vehicle, 1 μM PE, or 0.1 μM aVP. *P < 0.05 versus CCL23 plus vehicle (two-way ANOVA with Dunnett’s multiple comparisons test).