Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, USA.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA.
Biochem Biophys Res Commun. 2020 Jul 23;528(2):368-375. doi: 10.1016/j.bbrc.2020.02.094. Epub 2020 Feb 19.
Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with α-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:α-AR heteromers, however, is lacking. Here we utilized bimolecular luminescence/fluorescence complementation (BiLC/BiFC) combined with intermolecular bioluminescence resonance energy transfer (BRET) assays in HEK293T cells to evaluate CXCR4:α-AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGluR) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or α-ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGluR-EYFP were nonspecific. BRET for CXCR4:ACKR3 and CXCR4:α-AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRET of CXCR4:α-AR heteromers without affecting BRET; stimulation with CXCL12 reduced BRET of CXCR4:α-AR heteromers, but did not affect BRET or BRET for CXCR4:α-AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRET of CXCR4:α-AR heteromers and increased BRET of CXCR4:α-AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, α-AR and mGluR homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric α-AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGluR was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and α-ARs is comparable, provide evidence for conformational changes of the receptor complexes upon agonist binding and support the concept that proto- and oligomeric CXCR4 and α-ARs constitutively form higher-order hetero-oligomeric receptor clusters.
最近，我们报道趋化因子（C-X-C 基序）受体 4（CXCR4）与血管平滑肌细胞表面的α-肾上腺素能受体（AR）异源二聚化，通过该受体发生串扰。然而，直接的生物物理证据表明 CXCR4：α-AR 异源二聚体缺乏。在这里，我们利用双分子发光/荧光互补（BiLC/BiFC）结合分子间生物发光共振能量转移（BRET）测定法在 HEK293T 细胞中评估 CXCR4：α-AR 异源二聚体。利用非典型趋化因子受体 3（ACKR3）和代谢型谷氨酸受体 1（mGluR）作为对照。CXCR4-RLuc（圆斑蝰）与增强型黄色荧光蛋白（EYFP）标记的 ACKR3 或 α-ARs 之间的 BRET 满足组成型异源二聚化的标准。CXCR4-RLuc 与 EYFP 或 mGluR-EYFP 之间的 BRET 是非特异性的。CXCR4：ACKR3 和 CXCR4：α-AR 异源二聚体的 BRET 相当。用苯肾上腺素刺激细胞会增加 CXCR4：α-AR 异源二聚体的 BRET，而不会影响 BRET；用 CXCL12 刺激会降低 CXCR4：α-AR 异源二聚体的 BRET，但不会影响 BRET 或 CXCR4：α-AR 的 BRET。CXCR4 跨膜结构域（TM）2 的肽类似物降低了 CXCR4：α-AR 异源二聚体的 BRET，并增加了 CXCR4：α-AR 相互作用的 BRET。CXCR4 的 TM4 类似物不会改变 BRET。我们通过 BiFC/BiLC 观察到 CXCR4、α-AR 和 mGluR 同源二聚体，通过 BiFC/BiLC BRET 观察到同源二聚体 CXCR4 与原同源二聚体和同源二聚体 α-AR 的异源二聚体。BiFC/BiLC 用于同源二聚体 CXCR4 与同源二聚体 mGluR 之间相互作用的 BRET 是非特异性的。我们的发现表明，CXCR4 与 ACKR3 和 α-AR 的异源二聚化亲和力相当，为激动剂结合后受体复合物构象变化提供了证据，并支持原和寡聚体 CXCR4 和 α-AR 组成型形成更高阶异源寡聚体受体簇的概念。
