Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α(1)-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays.
Characterization of heteromeric complexes between chemokine (C-X-C motif) receptor 4 and α(1)-adrenergic receptors utilizing intermolecular bioluminescence resonance energy transfer assays.
复制标题
DOI：
10.1016/j.bbrc.2020.02.094
复制
发表时间：
2020-07-23
影响因子：
3.1
通讯作者：
Majetschak M
中科院分区：
文献类型：
--
作者：
Gao X;Enten GA;DeSantis AJ;Volkman BF;Gaponenko V;Majetschak M
最近，我们报道了趋化因子(C-X-C基序)受体4(CXCR4)与α1肾上腺素能受体(AR)在血管平滑肌细胞表面异构化，并通过受体相互作用。然而，CXCR4：α1-AR异构体的直接生物物理证据尚不清楚。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)分析来评估CXCR4：α1a/b/d-AR异构化。以非典型趋化因子受体3(ACKR3)和代谢性谷氨酸受体1(MGlu1R)作为对照。CXCR4-RLuc(肾型)与增强型黄色荧光蛋白(EYFP)标记的ACKR3或α1a/b/d-Ars之间的Bret满足构成异构化的标准。CXCR4-RLuc与EYFP或mGlu1R-EYFP之间的Bret是非特异性的。CXCR4：ACKR3和CXCR4：α1a/b/d-AR异构体的BRET50具有可比性。苯肾上腺素刺激增加CXCR4：α1a/b/d-AR异构体的BRETmax而不影响BRET50，CXCl12刺激降低CXCR4：α1a-AR异构体的BRETmax，但不影响CXCR4：α1b/d-AR的BRETmax或BRETmax/50。CXCR4跨膜区(TM)2的多肽类似物降低了CXCR4：α1a/b/d-AR异构体的BRETmax，增加了CXCR4：α1a/b-AR相互作用的BRET50。CXCR4的TM4类似物不改变Bret。我们用BIFC/BiLC观察到CXCR4、α1a-AR和mGlu1R的均二聚化，用BIFC/BiLC Bret观察到CXCR4和α1a-AR的异构化。同源二聚体CXCR4与同源二聚体mGlu1R相互作用的BIFC/BiLC Bret是非特异性的。我们的研究结果表明，CXCR4对α受体3和α1-Ars的异构化亲和力是相当的，为受体复合体在激动剂结合时的构象变化提供了证据，并支持了原核和寡聚体CXCR4和CXCR4-Ars构成高阶杂寡体受体簇的概念。
Recently, we reported that chemokine (C-X-C motif) receptor 4 (CXCR4) heteromerizes with α1-adrenergic receptors (AR) on the cell surface of vascular smooth muscle cells, through which the receptors cross-talk. Direct biophysical evidence for CXCR4:α1-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:α1a/b/d-AR heteromerization. Atypical chemokine receptor 3 (ACKR3) and metabotropic glutamate receptor 1 (mGlu1R) were utilized as controls. BRET between CXCR4-RLuc (Renilla reniformis) and enhanced yellow fluorescent protein (EYFP)-tagged ACKR3 or α1a/b/d-ARs fulfilled criteria for constitutive heteromerization. BRET between CXCR4-RLuc and EYFP or mGlu1R-EYFP were nonspecific. BRET50 for CXCR4:ACKR3 and CXCR4:α1a/b/d-AR heteromers were comparable. Stimulation of cells with phenylephrine increased BRETmax of CXCR4:α1a/b/d-AR heteromers without affecting BRET50; stimulation with CXCL12 reduced BRETmax of CXCR4:α1a-AR heteromers, but did not affect BRET50 or BRETmax/50 for CXCR4:α1b/d-AR. A peptide analogue of transmembrane domain (TM) 2 of CXCR4 reduced BRETmax of CXCR4:α1a/b/d-AR heteromers and increased BRET50 of CXCR4:α1a/b-AR interactions. A TM4 analogue of CXCR4 did not alter BRET. We observed CXCR4, α1a-AR and mGlu1R homodimerization by BiFC/BiLC, and heteromerization of homodimeric CXCR4 with proto- and homodimeric α1a-AR by BiFC/BiLC BRET. BiFC/BiLC BRET for interactions between homodimeric CXCR4 and homodimeric mGlu1R was nonspecific. Our findings suggest that the heteromerization affinity of CXCR4 for ACKR3 and α1-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 α1-ARs constitutively form higher-order hetero-oligomeric receptor clusters.
登录
查看更多内容
影响因子：
14.9
作者：
Sharman JL;Benson HE;Pawson AJ;Lukito V;Mpamhanga CP;Bombail V;Davenport AP;Peters JA;Spedding M;Harmar AJ;NC-IUPHAR
通讯作者：
NC-IUPHAR
影响因子：
4.8
作者：
Stanasila, L;Perez, JB;Cotecchia, S
通讯作者：
Cotecchia, S
DOI：
10.1073/pnas.1014598108
发表时间：
2011-06-14
影响因子：
11.1
作者：
Tarasov, Sergey G.;Gaponenko, Vadim;Tarasova, Nadya I.
通讯作者：
Tarasova, Nadya I.
DOI：
10.1146/annurev-pharmtox-011613-135952
发表时间：
2016
影响因子：
12.5
作者：
Gomes I;Ayoub MA;Fujita W;Jaeger WC;Pfleger KD;Devi LA
通讯作者：
Devi LA
影响因子：
3.7
作者：
Szalai B;Hoffmann P;Prokop S;Erdélyi L;Várnai P;Hunyady L
通讯作者：
Hunyady L