Expression and function of hetereomeric purinergic receptors

异聚嘌呤能受体的表达和功能

• 批准号: 13670109
• 负责人: NAKATA Hiroyasu
• 金额: $ 2.24万
• 依托单位: Tokyo Metropolitan Organization for Medical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670109/
• 关键词: adenosine; purinergic; signal transduction mechanism; heterodimer; cross-talk; P2 receptor; P3 receptor; NECA; 神経伝達

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

腺苷及其内源性前体ATP是嘌呤能系统的主要组成部分，分别通过特定的腺苷和ATP受体调节细胞和组织的功能。虽然腺苷抑制兴奋性，ATP在中枢神经系统中作为兴奋性递质发挥作用，但对腺苷和ATP受体形成新的功能结构的能力知之甚少，例如控制复杂的嘌呤能级联反应的异构体。因此，我们利用一种新的生物发光共振能量转移技术(BRET^2)，结合间接生化或药理学方法，探索了在转基因细胞中G&lt；I/o&gt；蛋白偶联的A1腺苷受体(A_1R)和G&lt；Q&gt；蛋白偶联的P2Y_1受体(P2Y_1R)发生异源齐聚的可能性。用免疫共沉淀法首次证实了共转染HEK293T细胞中存在A_1R/P2Y_1R异源寡聚体。在同一共转染细胞中，腺苷二磷酸βS能够…通过百日咳毒素和A_1R拮抗剂敏感的方式进一步减少A_1R诱导的cAMP积聚，表明A_1R的药理作用在共转染细胞中发生了明显的改变，即腺苷二磷酸βS通过G_I/O&G蛋白结合A_1R并抑制腺苷环化酶活性。激光共聚焦显微镜双重免疫荧光实验显示，A_1R和P_2Y_1R在共转染细胞中有高度的共定位。然后，Bret^2技术在活的HEK293T细胞中发现了A_1R和P2Y_1R之间的结构性异构体齐聚。加入两种受体激动剂后，Bret^2信号也以时间依赖的方式增加，而用P2Y_1R拮抗剂MRS2179预先抑制，表明这一过程是由两种受体同时激活所促进的。这些结果表明，A_1R与P2Y_1R的寡聚结合产生了具有类似于P2Y_1R的激动性药理作用的A_1R，并为增加嘌呤能信号的多样性提供了分子机制。这种混合嘌呤能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。较少

项目成果

K.Yoshioka et al.: "Agonist-promoted heteromeric oligomerization between adenosine A1 and P2Y1 receptors in living cells"FEBS Letters. 523. 147-151 (2002)

K.Yoshioka 等人：“活细胞中腺苷 A1 和 P2Y1 受体之间激动剂促进的异聚寡聚化”FEBS Letters。

K.Yoshioka et al.: "Hetero-oligomerization of adenosine A1 receptors with P2Y1 receptors in rat brains"FEBS Letters. 531. 299-303 (2002)

K.Yoshioka 等人：“大鼠大脑中腺苷 A1 受体与 P2Y1 受体的异源寡聚化”FEBS Letters。

Yoshioka, K., Matsuda, A., Nakata, H.: "Pharmacology of a unique adenosine binding site in rat brain using a selective ligand"Clin. Exp. Pharmacol. Physiol.. 28. 278-284 (2001)

Yoshioka, K.、Matsuda, A.、Nakata, H.：“使用选择性配体的大鼠脑中独特腺苷结合位点的药理学”Clin。

Nakata, H., Yoshioka, K., Saitoh, O.: "Hetero-oligomerization between adenosine A1 and P2Y1 receptors in living cells : formation of ATP-sensitive adenosine receptors"Drug Development Research. In press. (2003)

Nakata, H.、Yoshioka, K.、Saitoh, O.：“活细胞中腺苷 A1 和 P2Y1 受体之间的异源寡聚化：ATP 敏感腺苷受体的形成”药物开发研究。

K.yoshioka et al.: "Pharmacology of a unique adenosine binding site in rat brain using a selective ligand"Clinical Experimental Pharmacology and Physiology. 28. 278-284 (2001)

K.yoshioka 等人：“使用选择性配体的大鼠脑中独特腺苷结合位点的药理学”临床实验药理学和生理学。