Fiorentini Chiara, Busi Chiara, Gorruso Emanuela, Gotti Cecilia, Spano Pierfranco, Missale Cristina
Section of Pharmacology, Department of Biomedical Sciences and Biotechnology, University of Brescia, Viale Europa 11, 25124 Brescia, Italy.
Mol Pharmacol. 2008 Jul;74(1):59-69. doi: 10.1124/mol.107.043885. Epub 2008 Apr 18.
Colocalization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations suggests that their functional cross-talk might involve direct interactions. Here we report that the D1R coimmunoprecipitates with the D3R from striatal protein preparations, suggesting that they are clustered together in this region. Using bioluminescence resonance energy transfer (BRET(2)), we further suggest the existence of a physical interaction between D1R and D3R. Tagged D1R and D3R cotransfected in human embryonic kidney (HEK) 293 cells generated a significant BRET(2) signal that was insensitive to agonist stimulation, suggesting that they form a constitutive heterodimer. D1R and D3R regulate adenylyl cyclase (AC) in opposite ways. In HEK 293 cells coexpressing D1R and D3R, dopamine stimulated AC with higher potency and displaced [3H]R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) binding with higher affinity than in cells expressing the D1R. In HEK 293 cells individually expressing D1R or D3R, agonist stimulation induces internalization of D1R but not of D3R. Heterodimerization with D3R abolishes agonist-induced D1R cytoplasmic sequestration induced by selective D1R agonists and enables internalization of the D1R/D3R complex in response to the paired stimulation of both D1R and D3R. This mechanism involves beta-arrestin binding because it was blocked by mutant beta-arrestinV53D. These data suggest that as a result of dimerization, the D3R is switched to the desensitization mechanisms typical of the D1R. These data give a novel insight into how D1R and D3R may function in an integrated way, providing a molecular mechanism by which to converge D1R- and D3R-related dysfunctions.
多巴胺D1（D1R）和D3受体（D3R）在特定神经元群体中的共定位表明，它们的功能相互作用可能涉及直接相互作用。在此我们报告，D1R与纹状体蛋白质制剂中的D3R进行共免疫沉淀，这表明它们在该区域聚集在一起。使用生物发光共振能量转移（BRET(2)），我们进一步表明D1R和D3R之间存在物理相互作用。在人胚肾（HEK）293细胞中共转染的标记D1R和D3R产生了显著的BRET(2)信号，该信号对激动剂刺激不敏感，表明它们形成了组成型异二聚体。D1R和D3R以相反的方式调节腺苷酸环化酶（AC）。在共表达D1R和D3R的HEK 293细胞中，多巴胺刺激AC的效力更高，并且与表达D1R的细胞相比，以更高的亲和力取代[3H]R-(+)-7-氯-8-羟基-3-甲基-1-苯基-2,3,4,5-四氢-1H-3-苯并氮杂卓（SCH23390）结合。在单独表达D1R或D3R的HEK 293细胞中，激动剂刺激诱导D1R内化，但不诱导D3R内化。与D3R异二聚化消除了选择性D1R激动剂诱导的激动剂诱导的D1R细胞质隔离，并使D1R/D3R复合物能够响应D1R和D3R的配对刺激而内化。该机制涉及β-抑制蛋白结合，因为它被突变体β-抑制蛋白V53D阻断。这些数据表明，由于二聚化，D3R转变为D1R典型的脱敏机制。这些数据为D1R和D3R如何以整合方式发挥作用提供了新的见解，提供了一种使D1R和D3R相关功能障碍汇聚的分子机制。
