Dufresne M, Escrieut C, Clerc P, Le Huerou-Luron I, Prats H, Bertrand V, Le Meuth V, Guilloteau P, Vaysse N, Fourmy D
Institut National de la Santé et de la Recherche Médicale, Unité 151, Institut Louis Bugnard, Centre Hospitalier Universitaire de Rangueil, Toulouse, France.
Eur J Pharmacol. 1996 Feb 15;297(1-2):165-79. doi: 10.1016/0014-2999(95)00737-7.
We have cloned the calf predominant pancreatic cholecystokinin B (CCKB)/gastrin receptor cDNA. It encodes a 454 amino acid protein with 90% identity with the CCKB/gastrin receptor cloned in other species and tissues. However, the calf pancreatic CCKB/gastrin receptor contains a pentapeptide cassette within the third intracellular loop which is absent in the cloned human brain and stomach receptor. Quantification of the CCKB/gastrin receptor mRNA levels by reverse transcription polymerase chain reaction demonstrated the same level of transcripts at birth, +7 and +28 days. On the other hand, binding study with pancreatic membranes showing a dramatic increase (600-fold) in the number of CCKB/gastrin receptor sites between at birth and +28 days indicates that the development of the calf pancreatic CCKB/gastrin receptor occurs during the first 4 weeks of post-natal life. COS monkey cells (COS-7 cells) transiently transfected by the cloned cDNA exhibit binding of 125I-Bolton-Hunter-[Thr28,Ahx31]CCK-(25-33) and 125I-Bolton-Hunter-[Leu15]human gastrin-(2-17) to two affinity classes of sites. Kd values of the high affinity binding components indicate a 4-fold higher affinity of the receptor for sulfated gastrin than for CCK. Finally, the recombinant receptor is coupled to G proteins and [Ca2+]i mobilization, and is expressed as a glycoprotein of 82 kDa.
我们克隆了小牛主要的胰腺胆囊收缩素B（CCKB）/胃泌素受体cDNA。它编码一种由454个氨基酸组成的蛋白质，与在其他物种和组织中克隆的CCKB/胃泌素受体具有90%的同一性。然而，小牛胰腺CCKB/胃泌素受体在第三个细胞内环中含有一个五肽盒，而在克隆的人类脑和胃受体中则不存在。通过逆转录聚合酶链反应对CCKB/胃泌素受体mRNA水平进行定量分析，结果显示在出生时、出生后第7天和第28天转录本水平相同。另一方面，用胰腺膜进行的结合研究表明，出生时到出生后第28天之间CCKB/胃泌素受体位点数量急剧增加（600倍），这表明小牛胰腺CCKB/胃泌素受体的发育发生在出生后的前4周。用克隆的cDNA瞬时转染的COS猴细胞（COS-7细胞）表现出125I-博尔顿-亨特-[苏氨酸28，Ahx31]CCK-（25-33）和125I-博尔顿-亨特-[亮氨酸15]人胃泌素-（2-17）与两类亲和力位点的结合。高亲和力结合成分的Kd值表明，该受体对硫酸化胃泌素的亲和力比对CCK高4倍。最后，重组受体与G蛋白偶联并介导[Ca2+]i动员，并且作为一种82 kDa的糖蛋白表达。
