MOLECULAR-CLONING AND CHARACTERIZATION OF AN ADENOSINE RECEPTOR - THE A3 ADENOSINE RECEPTOR
MOLECULAR-CLONING AND CHARACTERIZATION OF AN ADENOSINE RECEPTOR - THE A3 ADENOSINE RECEPTOR
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DOI：
10.1073/pnas.89.16.7432
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发表时间：
1992-08-15
影响因子：
11.1
通讯作者：
CIVELLI, O
中科院分区：
文献类型：
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作者：
ZHOU, QY;LI, CY;CIVELLI, O
我们之前报道了几种编码鸟嘌呤核苷酸结合调节蛋白（G蛋白）偶联受体的大鼠纹状体 cDNA 序列的选择性扩增。这些序列之一 (R226) 与先前克隆的两个腺苷受体表现出高序列同一性 (58%)。 R226 的全长 cDNA 克隆已从大鼠脑 cDNA 文库中分离出来。该 cDNA 克隆编码 320 个氨基酸的蛋白质，可以组织成七个跨膜片段。 R226 已在 COS-7 和 CHO 细胞中表达，并且用腺苷受体放射性配体筛选转染细胞的膜。 R226 可以结合非选择性腺苷激动剂氚化 N-乙基腺苷 5'-糖醛酸 ([H-3]NECA) 和 A1 选择性激动剂放射性碘化 N6-2-(4-氨基-3-碘苯基)-乙基腺苷 ([I-125]APNEA)，但不能结合氚化 A1 选择性拮抗剂1,3-二丙基-8-环戊基黄嘌呤 ([H-3]DPCPX) 和 8-{4-[9{[(2-氨基乙基)氨基]羰基}甲基)氧基]-苯基}-1,3-二丙基黄嘌呤 ([H-3]XAC) 或氚化的 A2-选择性激动剂配体 2-[4-(2-羧乙基)苯基]乙基氨基5'-N-乙基甲酰胺腺苷 ([H-3]CGS21680) 和放射性碘化 2-[4-({2-[(4-氨基苯基)甲基羰基氨基]乙氨基羰基}乙基)苯基]乙氨基 5'-N-乙基甲酰胺腺苷。 [I-125]APNEA 的广泛表征表明，R226 以高亲和力结合 [I-125]APNEA (K(d) = 15.5 +/- 2.4 nM），并且特异性 [I-125]APNEA 结合可以被腺苷配体抑制，其效力顺序为 (R)-N6-苯基-2-丙基腺苷 (R-PIA) = NECA > S-PIA > 腺苷 > ATP = ADP，但不是拮抗剂 XAC、异丁基甲基黄嘌呤和 DPCPX。在 R226 稳定转染的 CHO 细胞中，腺苷激动剂 R-PIA、NECA 和 CGS21680 通过百日咳毒素敏感 G 蛋白抑制 40-50% 毛喉素刺激的 cAMP 积累，EC50 分别为 18 +/- 5.6 nM、23 +/- 3.5 nM 和 144 +/- 34 nM。基于这些观察，我们得出结论，R226 编码具有非 A1 和非 A2 特异性的腺苷受体，因此我们将其命名为 A3 腺苷受体。 mRNA分析显示，R226在睾丸中的表达量最高，在肺、肾、心脏以及中枢神经系统的某些部位（例如皮质、纹状体和嗅球）中也发现了低水平的mRNA。睾丸中 A3 受体的高表达水平表明腺苷在生殖中可能发挥作用。
We have previously reported the selective amplification of several rat striatal cDNA sequences that encode guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. One of these sequences (R226) exhibited high sequence identity (58%) with the two previously cloned adenosine receptors. A full-length cDNA clone for R226 has been isolated from a rat brain cDNA library. The cDNA clone encodes a protein of 320 amino acids that can be organized into seven transmembrane stretches. R226 has been expressed in COS-7 and CHO cells and membranes from the transfected cells were screened with adenosine receptor radioligands. R226 could bind the nonselective adenosine agonist tritiated N-ethyladenosine 5'-uronic acid ([H-3]NECA) and A1-selective agonist radioiodinated N6-2-(4-amino-3-iodophenyl)-ethyladenosine ([I-125]APNEA) but not A1-selective antagonists tritiated 1,3-dipropyl-8-cyclopentylxanthine ([H-3]DPCPX) and 8-{4-[9{[(2-aminoethyl)amino]carbonyl}methyl)oxy]-phenyl}-1,3-dipropylxanthine ([H-3]XAC) or the A2-selective agonist ligands tritiated 2-[4-(2-carboxyethyl)phenyl]ethylamino 5'-N-ethylcarboxamidoadenosine ([H-3]CGS21680) and radioiodinated 2-[4-({2-[(4-aminophenyl)methylcarbonyl-amino]ethylaminocarbonyl}ethyl)phenyl]ethylamino 5'-N-ethylcarboxamidoadenosine. Extensive characterization with [I-125]APNEA showed that R226 binds [I-125]APNEA with high affinity (K(d) = 15.5 +/- 2.4 nM) and the specific [I-125]APNEA binding could be inhibited by adenosine ligands with a potency order of (R)-N6-phenyl-2-propyladenosine (R-PIA) = NECA > S-PIA > adenosine > ATP = ADP but not by antagonists XAC, isobutylmethylxanthine, and DPCPX. In R226 stably transfected CHO cells, adenosine agonists R-PIA, NECA, and CGS21680 inhibited by 40-50% the forskolin-stimulated cAMP accumulation through a pertussis toxin-sensitive G protein with an EC50 of 18 +/- 5.6 nM, 23 +/- 3.5 nM, and 144 +/- 34 nM, respectively. Based on these observations we conclude that R226 encodes an adenosine receptor with non-A1 and non-A2 specificity, and we thus name it the A3 adenosine receptor. mRNA analyses revealed that the highest expression of R226 was in the testis and low-level mRNAs were also found in the lung, kidneys, heart, and some parts of the central nervous system such as cortex, striatum, and olfactory bulb. The high-expression level of the A3 receptor in the testis suggests a possible role for adenosine in reproduction.