Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.
Molecular cloning and functional expression of a sheep A3 adenosine receptor with widespread tissue distribution.
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发表时间：
1993-09
影响因子：
3.6
通讯作者：
Joel Linden;H. Taylor;Anna S. Robeva;Amy L. Tucker;Jörg H. Stehle;S. Rivkees;J. Fink;S. Reppert
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文献类型：
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作者：
Joel Linden;H. Taylor;Anna S. Robeva;Amy L. Tucker;Jörg H. Stehle;S. Rivkees;J. Fink;S. Reppert
利用聚合酶链反应，从绵羊垂体结节中克隆了一个A3腺苷受体。该克隆体编码一种317个氨基酸的蛋白质，与大鼠A3腺苷受体的72%相同。与大鼠不同，大鼠主要在睾丸中发现丰富的A3 mRNA转录物，绵羊的转录物在肺、脾脏和松果体中最丰富，在脑、肾和睾丸中也有中等水平的存在。激动剂n6 -氨基[125I]碘苄腺苷与重组A3腺苷受体结合，具有高亲和力（Kd值约为6 nm）和特异性，在COS-1细胞中短暂表达，在CHO K1细胞中稳定表达。激动剂的效价顺序为n6 -氨基碘苄基腺苷> n -乙基羧腺苷>或= (R)-苯异丙基腺苷>>环戊基腺苷。很少或没有检测到嘌呤核苷酸的结合。拮抗剂的效价顺序为：3-（3-碘-4-氨基苯基）-8-（4-氧乙酸酯）苯基-1-丙基黄嘌呤（I-ABOPX） （Ki = 3 nM）； > -1,3-二丙基-8-（4-丙烯酸酯）苯基黄嘌呤（BW-A1433）； > 1,3-二丙基-8-磺胺基黄嘌呤=黄嘌呤胺原物>> 8-环戊基-1,3-二丙基黄嘌呤。Enprofylline不结合。这些数据表明，与A1腺苷受体相比，A3腺苷受体优先结合腺嘌呤n6位和黄嘌呤c8位的芳基环配体。在拮抗剂中，A3腺苷受体优先结合8-苯基黄嘌呤与酸性或碱性对取代基（I-ABOPX > BW-A1433 > 1,3-二丙基-8-磺苯基黄嘌呤=黄嘌呤胺原）。受体激动剂稳定降低福斯克林诱导的重组羊A3腺苷受体介导的中国仓鼠卵巢细胞cAMP积累8-环戊基-1,3-二丙基黄嘌呤可阻断还原反应，而BW-A1433可阻断还原反应。这些数据表明：(i) A3腺苷受体在物种同源物中表现出不同寻常的结构多样性，（ii）与大鼠相比，绵羊A3腺苷受体具有广泛的组织分布，（iii）一些酸性侧链的黄嘌呤与A3腺苷受体结合具有高亲和力。
Using the polymerase chain reaction, an A3 adenosine receptor has been cloned from the hypophysial par tuberalis of sheep. The clone encodes a 317-amino acid protein that is 72% identical to the rat A3 adenosine receptor. In contrast to rat, where abundant A3 mRNA transcript is found primarily in testis, the sheep transcript is most abundant in lung, spleen, and pineal gland and is present in moderate levels in brain, kidney, and testis. The agonist N6-amino[125I]iodobenzyladenosine binds with high affinity (Kd congruent to 6 nm) and specificity to recombinant A3 adenosine receptors expressed transiently in COS-1 cells or stably in CHO K1 cells. The potency order of agonists is N6-aminoiodobenzyladenosine > N-ethylcarboxamidoadenosine > or = (R)-phenylisopropyladenosine >> cyclopentyladenosine. Little or no binding of purine nucleotides was detected. The potency order of antagonists is 3-(3-iodo-4-aminobenzyl)-8-(4-oxyacetate)phenyl-1- propylxanthine (I-ABOPX) (Ki = 3 nM) > 1,3-dipropyl-8-(4-acrylate)phenylxanthine (BW-A1433) > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener >> 8-cyclopentyl-1,3-dipropylxanthine. Enprofylline does not bind. These data indicate that, in contrast to A1 adenosine receptors, A3 adenosine receptors preferentially bind ligands with aryl rings in the N6-position of adenine and in the C8-position of xanthine. Among antagonists, the A3 adenosine receptor preferentially binds 8-phenylxanthines with acidic versus basic para-substituents (I-ABOPX > BW-A1433 > 1,3-dipropyl-8-sulfophenylxanthine = xanthine amine cogener). Agonists reduce forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells stably transfected with recombinant sheep A3 adenosine receptors; the reduction is blocked by BW-A1433 but not by 8-cyclopentyl-1,3-dipropylxanthine. These data suggest that (i) A3 adenosine receptors display unusual structural diversity for species homologs, (ii) in contrast to rat, sheep A3 adenosine receptors have a broad tissue distribution, and (iii) some xanthines with acidic side chains bind with high affinity to A3 adenosine receptors.