Chen Chongguang, Li Jin, Bot George, Szabo Imre, Rogers Thomas J, Liu-Chen Lee-Yuan
Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, 3420 North Broad Street, Philadelphia, PA 19140, USA.
Eur J Pharmacol. 2004 Jan 12;483(2-3):175-86. doi: 10.1016/j.ejphar.2003.10.033.
Cross-desensitization between micro-opioid receptor agonists and CC chemokines was shown to occur in immune cells and in the central nervous system. However, these cells do not permit examination of potential mechanisms at cellular levels due to low levels and mixed populations of receptors. In this study, we investigated possible interactions and biochemical mechanisms of cross-desensitization between the mu-opioid and chemokine CCR5 receptors coexpressed in Chinese hamster ovary (CHO) cells. Hemagglutinin (HA)-tagged micro-opioid receptor coimmunoprecipitated with FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-tagged chemokine receptor CCR5 in cells expressing the two receptors, but not in a mixture of cells transfected with one of the two receptors, indicating that the two receptors form heterodimers. Treatment with the mu-opioid receptor agonist DAMGO ([D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin), the chemokine RANTES (Regulated on Activation, Normal T cell-Expressed and -Secreted) (CCL5), or both, did not affect the level of coimmunoprecipitation. DAMGO and RANTES (CCL5) induced chemotaxis in CHO cells coexpressing both receptors, and preincubation with either DAMGO or RANTES (CCL5) profoundly inhibited chemotaxis caused by the other. DAMGO pretreatment enhanced phosphorylation of the chemokine CCR5 receptor and reduced RANTES (CCL5)-promoted [35S]GTP gamma S binding. Conversely, RANTES (CCL5) preincubation slightly increased phosphorylation of the mu-opioid receptor and significantly reduced DAMGO-induced [35S]GTP gamma S binding. These results indicate that activation of either receptor affected G protein coupling of the other, likely due to enhanced phosphorylation of the receptor. Heterodimerization between the two receptors may contribute to the observed cross-desensitization.
微阿片受体激动剂与CC趋化因子之间的交叉脱敏作用已证实在免疫细胞和中枢神经系统中存在。然而，由于受体水平较低且细胞群体混合，这些细胞无法在细胞水平上研究潜在机制。在本研究中，我们调查了在中国仓鼠卵巢（CHO）细胞中共表达的μ-阿片受体和趋化因子CCR5受体之间交叉脱敏的可能相互作用及生化机制。在表达这两种受体的细胞中，带有血凝素（HA）标签的微阿片受体与带有FLAG（天冬氨酸-酪氨酸-赖氨酸-天冬氨酸-天冬氨酸-天冬氨酸-天冬氨酸-赖氨酸）标签的趋化因子受体CCR5共免疫沉淀，但在转染了两种受体之一的细胞混合物中则没有，这表明这两种受体形成了异二聚体。用μ-阿片受体激动剂DAMGO（[D-丙氨酸（2），N-甲基苯丙氨酸（4），甘氨酸（5）-醇]-脑啡肽）、趋化因子RANTES（活化调节正常T细胞表达和分泌）（CCL5）或两者进行处理，均不影响共免疫沉淀水平。DAMGO和RANTES（CCL5）在共表达两种受体的CHO细胞中诱导趋化作用，并预先用DAMGO或RANTES（CCL5）孵育可显著抑制由另一种物质引起的趋化作用。DAMGO预处理增强了趋化因子CCR5受体的磷酸化，并降低了RANTES（CCL5）促进的[35S]GTPγS结合。相反，RANTES（CCL5）预孵育略微增加了μ-阿片受体的磷酸化，并显著降低了DAMGO诱导的[35S]GTPγS结合。这些结果表明，任一受体的激活都会影响另一种受体的G蛋白偶联，这可能是由于受体磷酸化增强所致。两种受体之间的异二聚化可能导致了观察到的交叉脱敏。
