Characterization of the A2B Adenosine Receptor from Mouse, Rabbit, and Dog
Characterization of the A2B Adenosine Receptor from Mouse, Rabbit, and Dog
复制标题
DOI：
10.1124/jpet.108.148270
复制
发表时间：
2009-04-01
影响因子：
3.5
通讯作者：
Maas, Garren E.
中科院分区：
文献类型：
--
作者：
Auchampach, John A.;Kreckler, Laura M.;Maas, Garren E.
我们已经从狗、兔和小鼠中克隆并鉴定了A（2B）腺苷受体（AR）。用逆转录-聚合酶链反应（RT-PCR）扩增了狗和小鼠A（2B）AR的全编码区，并从兔脑cDNA文库中筛选获得兔A（2B）AR cDNA。值得注意的是，通过文库筛选分离出了另外一个克隆，其序列与全长兔A（2B）AR相同，除了编码氨基酸103至111（A（2B）AR（103-111））区域中的27个碱基对缺失。该9个氨基酸缺失位于A（2B）AR唯一已知的剪接点处的第二个胞内环中，似乎是由于使用了在兔和犬中发现的额外5'供体位点，而在人、大鼠或小鼠序列中未发现。[H-3]3-异丁基-8-吡咯烷基黄嘌呤和8-[4-[（（4-氰基-[2，6 - 3 H]-苯基）氨基甲酰基甲基）氧基]苯基]-1，3-二（正丙基）黄嘌呤（[H-3] MRS 1754）以高亲和力与由表达小鼠、兔和犬A（2B）AR的人胚肾（HEK）293细胞制备的膜结合。用一组激动剂（腺苷和2-氨基-3，5-二氰基-4-苯基吡啶类似物）和拮抗剂配体进行的竞争结合研究确定了A（2B）AR直系同源物的相似效力顺序，尽管与来自兔和小鼠的A（2B）AR相比，大多数黄嘌呤拮抗剂对犬A（2B）AR显示出较低的结合亲和力。用表达兔A（2B）AR（103-111）变体的HEK 293细胞制备的膜未检测到特异性结合。此外，该变体未能刺激腺苷酸环化酶或钙动员。我们得出结论，A（2B）AR的拮抗剂药理学在物种之间存在显着差异，一些物种由于“泄漏”剪接而表达A（2B）AR的非功能性变体。
We have cloned and pharmacologically characterized the A(2B) adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A(2B)AR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A(2B)AR cDNA was obtained by screening a rabbit brain cDNA library. It is noteworthy that an additional clone was isolated by library screening that was identical in sequence to the full-length rabbit A(2B)AR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A(2B)AR(103-111)). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A(2B)AR and seems to result from the use of an additional 5' donor site found in the rabbit and dog but not in the human, rat, or mouse sequences. [H-3]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-3H]-phenyl) carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl) xanthine ([H-3] MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A(2B)ARs. Competition binding studies performed with a panel of agonist (adenosine and 2-amino-3,5-dicyano-4-phenylpyridine analogs) and antagonist ligands identified similar potency orders for the A(2B)AR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A(2B)AR compared with A(2B)ARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A(2B)AR(103-111) variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A(2B)AR exist between species and that some species express nonfunctional variants of the A(2B)AR due to "leaky" splicing.