Expression and function of hetereomeric purinergic receptors

腺苷及其内源性前体ATP是嘌呤能系统的主要成分，分别通过特异性腺苷和ATP受体调节细胞和组织功能。虽然腺苷抑制兴奋性和ATP功能作为一种兴奋性递质在中枢神经系统中，很少有人知道腺苷和ATP受体的能力，形成新的功能结构，如异聚体控制复杂的嘌呤级联反应。因此，我们在间接的<q>生化或药理学方法的基础上，通过一种新的生物发光共振能量转移技术（BRET），探索了转染培养细胞中G_&lt;i/o&gt;蛋白偶联的A_1腺苷受体（A_1R）和G_蛋白偶联的P2Y_1受体（P2Y_1R）之间异源寡聚化的可能性。用免疫共沉淀法首次证实了A_1R/P2Y_1R异源寡聚体在HEK 293 T细胞中的存在。在相同的共转染细胞中，ADPβS能够 ...更多信息 结果表明，ADPβS可通过G_（i/O）蛋白抑制腺苷酸环化酶活性，并与A_1R拮抗剂敏感，从而抑制Forskolin诱导的cAMP的积累。激光共聚焦显微镜双免疫荧光实验显示A_1R和P2Y_1R在共转染细胞中高度共定位。然后，BRET^2技术揭示了在活HEK 293 T细胞中A_1R和P2Y_1R之间的组成性异源寡聚体化。在加入两种受体的激动剂后，BRET^2信号也以时间依赖性方式增加，这被P2Y_1R拮抗剂MRS 2179预处理所抑制，表明这一过程被两种受体的同时激活所促进。这些结果表明A_1R与P2Y_1R的寡聚结合产生了A_1R与P2Y_1R类似的激动药理学作用，并为嘌呤能信号的多样性增加提供了分子机制。这种杂合嘌呤受体的存在可能解释了腺嘌呤核苷酸对突触传递的有争议的抑制作用。少

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors, respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of adenosine and ATP receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Therefore we have explored the possibility of hetero-oligomerization between G_<i/o> protein-coupled A_1 adenosine receptor (A_1R) and G_<q> protein-coupled P2Y_1 receptor (P2Y_1R) in transfected cultured cells by a new bioluminescence resonance energy transfer technology (BRET^2) in addition to indirect biochemical or pharmacological methods. The existence of A_1R/P2Y_1R hetero-oligomers in co-transfected HEK293T cells was first shown using co-immunoprecipitation methods. In the same co-transfected cells, ADPβS was able to … More reduce forskolin-evoked cAMP accumulation with pertussis toxin- and A_1R antagonist-sensitive manner, indicating that pharmacology of A_1R was significantly modified in the co-transfected cells, i.e. ADPβS binds A_1R and inhibits adenylyl cyclase activity via G_<i/O> proteins. Also, a high degree of A_1R and P2Y_1R co-localization was demonstrated in co-transfected cells by double immunofluorescence experiments with confocal laser microscopy. Then, the BRET^2 technique revealed constitutive heteromeric oligomerization between A_1R and P2Y_1R in living HEK293T cells. The BRET^2 signal also increased in a time-dependent manner upon addition of agonists for both receptors, which was inhibited by pretreatment with the P2Y_1R antagonist MRS2179, indicating that this process is promoted by the simultaneous activation of both receptors. These results suggest that the oligomeric association of A_1R with P2Y_1R generates A_1R with P2Y_1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purinergic signaling. Existence of this hybrid purinergic receptor may explain the controversial inhibition of synaptic transmission by adenine nucleotides. Less