Department of Surgery, Burn and Shock Trauma Research Institute, Loyola University Chicago Stritch School of Medicine, Maywood, IL.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI.
J Am Heart Assoc. 2017 Aug 17;6(8):e006575. doi: 10.1161/JAHA.117.006575.
Recently, we provided evidence that α-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 that CXCR4 controls α-ARs through formation of heteromeric receptor complexes in human vascular smooth muscle cells (hVSMCs), the molecular basis underlying cross-talk between ACKR3 and α-ARs is unknown.
We show that ACKR3 agonists inhibit inositol trisphosphate production in hVSMCs on stimulation with phenylephrine. In proximity ligation assays and co-immunoprecipitation experiments, we observed that recombinant and endogenous ACKR3 form heteromeric complexes with α-AR. While small interfering RNA knockdown of ACKR3 in hVSMCs reduced α-AR:ACKR3, CXCR4:ACKR3, and α-AR:CXCR4 complexes, small interfering RNA knockdown of CXCR4 reduced α-AR:ACKR3 heteromers. Phenylephrine-induced inositol trisphosphate production from hVSMCs was abolished after ACKR3 and CXCR4 small interfering RNA knockdown. Peptide analogs of transmembrane domains 2/4/7 of ACKR3 showed differential effects on heteromerization between ACKR3, α-AR, and CXCR4. While the transmembrane domain 2 peptide interfered with α-AR:ACKR3 and CXCR4:ACKR3 heteromerization, it increased heteromerization between CXCR4 and α-AR. The transmembrane domain 2 peptide inhibited ACKR3 but did not affect α-AR in 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.
α-ARs form hetero-oligomeric complexes with the ACKR3:CXCR4 heteromer, which is required for α-AR function, and activation of ACKR3 negatively regulates α-ARs. G protein-coupled receptor hetero-oligomerization is a dynamic process, which depends on the relative abundance of available receptor partners. Endogenous α-ARs function within a network of hetero-oligomeric receptor complexes.
最近，我们提供的证据表明，血管平滑肌中的α-肾上腺素能受体（ARs）受趋化因子（C-X-C 基序）受体（CXCR）4 和非典型趋化因子受体 3（ACKR3）调节。虽然我们表明，在人血管平滑肌细胞（hVSMCs）中，CXCR4 通过形成异源受体复合物来控制 α-ARs，但 ACKR3 和 α-AR 之间的串扰的分子基础尚不清楚。
我们表明，ACKR3 激动剂在刺激苯肾上腺素时抑制 hVSMCs 中三磷酸肌醇的产生。在接近连接测定和共免疫沉淀实验中，我们观察到重组和内源性 ACKR3 与 α-AR 形成异源复合物。虽然 hVSMCs 中的 ACKR3 小干扰 RNA 敲低减少了 α-AR:ACKR3、CXCR4:ACKR3 和 α-AR:CXCR4 复合物，但 CXCR4 的小干扰 RNA 敲低减少了 α-AR:ACKR3 异源二聚体。ACKR3 和 CXCR4 小干扰 RNA 敲低后，苯肾上腺素诱导的 hVSMCs 三磷酸肌醇的产生被消除。ACKR3 的跨膜结构域 2/4/7 的肽类似物对 ACKR3、α-AR 和 CXCR4 之间的异源二聚体化表现出不同的影响。虽然跨膜结构域 2 肽干扰了 α-AR:ACKR3 和 CXCR4:ACKR3 异源二聚体化，但它增加了 CXCR4 和 α-AR 之间的异源二聚体化。跨膜结构域 2 肽抑制 ACKR3，但在β-arrestin 募集测定中不影响 α-AR。此外，跨膜结构域 2 肽抑制苯肾上腺素诱导的 hVSMCs 三磷酸肌醇的产生，并减弱苯肾上腺素诱导的肠系膜动脉收缩。
α-AR 与 ACKR3:CXCR4 异源三聚体形成异源寡聚体复合物，这是 α-AR 功能所必需的，ACKR3 的激活负调节 α-ARs。G 蛋白偶联受体异源寡聚化是一个动态过程，取决于可用受体伴侣的相对丰度。内源性 α-ARs 作用于异源寡聚体受体复合物网络内。
