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 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 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 α_1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulating blood pressure.

Recent evidence suggests that C-X-C chemokine receptor type 4 (CXCR4) heteromerizes with α_1A/B-adrenoceptors (AR) and atypical chemokine receptor 3 (ACKR3) and that CXCR4:α_1A/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:α_1A/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:α_1A-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:α_1A/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 α_1A-AR via TM2 and with ACKR3 via TM4 is presented. Our findings further imply that CXCR4:α_1A-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.

Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α1-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α1-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α1-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca(2+) channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α1-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α1-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure.

Background: Recently, we provided evidence that α₁‐adrenergic receptors (ARs) in vascular smooth muscle are regulated by chemokine (C‐X‐C motif) receptor (CXCR) 4 and atypical chemokine receptor 3 (ACKR3). While we showed thatCXCR4 controls α₁‐ARsthrough formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross‐talk betweenACKR3 and α₁‐ARsis unknown.Methods and Results: We show thatACKR3 agonists inhibit inositol trisphosphate production inhVSMCson stimulation with phenylephrine. In proximity ligation assays and co‐immunoprecipitation experiments, we observed that recombinant and endogenousACKR3 form heteromeric complexes with α_1A/B/D‐AR. While small interfering RNA knockdown ofACKR3 inhVSMCsreduced α_1B/D‐AR:ACKR3,CXCR4:ACKR3, and α_1B/D‐AR:CXCR4 complexes, small interfering RNA knockdown ofCXCR4 reduced α_1B/D‐AR:ACKR3 heteromers. Phenylephrine‐induced inositol trisphosphate production fromhVSMCswas abolished afterACKR3 andCXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 ofACKR3 showed differential effects on heteromerization betweenACKR3, α_1A/B/D‐AR,andCXCR4. While the transmembrane domain 2 peptide interfered with α_1B/D‐AR:ACKR3 andCXCR4:ACKR3 heteromerization, it increased heteromerization betweenCXCR4 and α_1A/B‐AR. The transmembrane domain 2 peptide inhibitedACKR3 but did not affect α_1b‐ARin β‐arrestin recruitment assays. Furthermore, the transmembrane domain 2 peptide inhibited phenylephrine‐induced inositol trisphosphate production in hVSMCs and attenuated phenylephrine‐induced constriction of mesenteric arteries.Conclusions: α₁‐ARsform hetero‐oligomeric complexes with theACKR3:CXCR4 heteromer, which is required for α_1B/D‐ARfunction, and activation ofACKR3 negatively regulates α₁‐ARs. G protein–coupled receptor hetero‐oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α₁‐ARs function within a network of hetero‐oligomeric receptor complexes.

Proteasomes appear to be involved in the pathophysiology of various acute and chronic lung diseases. Information on the human lung proteasome in health and disease, however, is sparse. Therefore, we studied whether end-stage pulmonary diseases are associated with alterations in lung 20S/26S proteasome content, activity and 20S subunit composition. Biopsies were obtained from donor lungs (n=7) and explanted lungs from patients undergoing lung transplantation because of end stage chronic obstructive pulmonary disease (COPD; n=7), idiopathic pulmonary fibrosis (IPF, n=7) and pulmonary sarcoidosis (n=5). 20S/26S proteasomes in lung extracts were quantified by ELISA, chymotrypsin-like proteasome peptidase activities measured and 20S proteasome β subunits analyzed by Western blot. As compared with donor lungs, proteasome content was increased in IPF and sarcoidosis, but not in COPD. The relative distribution of free 20S and 26S proteasomes was similar; 20S proteasome was predominant in all extracts. Proteasome peptidase activities in donor and diseased lungs were indistinguishable. All extracts contained a mixed composition of inducible 20S β immuno-subunits and their constitutive counterparts; a disease associated distribution could not be identified. A higher content of lung proteasomes in IPF and pulmonary sarcoidosis may contribute to the pathophysiology of human fibrotic lung diseases.

Recent observations suggest that atypical chemokine receptor (ACKR)3 and chemokine (C-X-C motif) receptor (CXCR)4 regulate human vascular smooth muscle function through hetero-oligomerization with α₁-adrenoceptors. Here, we show that ACKR3 also regulates arginine vasopressin receptor (AVPR)1A. We observed that ACKR3 agonists inhibit arginine vasopressin (aVP)-induced inositol trisphosphate (IP₃) production in human vascular smooth muscle cells (hVSMCs) and antagonize aVP-mediated constriction of isolated arteries. Proximity ligation assays, co-immunoprecipitation and bioluminescence resonance energy transfer experiments suggested that recombinant and endogenous ACKR3 and AVPR1A interact on the cell surface. Interference with ACKR3 : AVPR1A heteromerization using siRNA and peptide analogues of transmembrane domains of ACKR3 abolished aVP-induced IP₃production. aVP stimulation resulted in β-arrestin 2 recruitment to AVPR1A and ACKR3. While ACKR3 activation failed to cross-recruit β-arrestin 2 to AVPR1A, the presence of ACKR3 reduced the efficacy of aVP-induced β-arrestin 2 recruitment to AVPR1A. AVPR1A and ACKR3 co-internalized upon agonist stimulation in hVSMC. These data suggest that AVPR1A : ACKR3 heteromers are constitutively expressed in hVSMC, provide insights into molecular events at the heteromeric receptor complex, and offer a mechanistic basis for interactions between the innate immune and vasoactive neurohormonal systems. Our findings suggest that ACKR3 is a regulator of vascular smooth muscle function and a possible drug target in diseases associated with impaired vascular reactivity.