Wu V, Yang M, McRoberts J A, Ren J, Seensalu R, Zeng N, Dagrag M, Birnbaumer M, Walsh J H
CURE/Digestive Diseases Research Center, Division of Digestive Diseases, Department of Medicine, UCLA School of Medicine, and West Los Angeles Veterans Administration Medical Center, Los Angeles, California 90073,
J Biol Chem. 1997 Apr 4;272(14):9037-42. doi: 10.1074/jbc.272.14.9037.
Cholecystokinin (CCK)-A and CCK-B receptors are highly homologous members of the seven transmembrane domain G-protein-coupled receptor superfamily. Genes of both receptors contain five exons and share a similar exon-intron organization. To determine the structural basis of CCK-A receptor (CCK-AR) functionally coupled to Gs, a series of chimeric mutants were constructed by replacing exons of human CCK-B receptor (CCK-BR), from the second to the fifth (last) exon, with human CCK-AR counterparts. Binding and signal transduction properties of wild-type and chimeric receptors were examined in stably transfected HEK-293 cells. Chimeric receptors that maintained high affinity binding to CCK exhibited dose-dependent increases in intracellular calcium mobilization similar to both wild-type receptors. However, only the wild-type CCK-AR and chimeric mutants containing the second exon of CCK-AR were able to mediate significantly greater increases in intracellular cAMP content and adenylyl cyclase activity compared with wild-type CCK-BR. A CCK-BR mutant was further constructed by replacing five amino acids, Gly-Leu-Ser-Arg-(Arg)-Leu, in the first intracellular loop with the corresponding five CCK-AR specific amino acids, Ile-Arg-Asn-Lys-(Arg)-Met. The resultant receptor maintained high affinity binding to both CCK and gastrin and dose-dependent calcium responses similar to wild-type CCK-BR. However, this first intracellular loop mutant also gained positive cAMP responses to both sulfated CCK-8 and gastrin-17 with EC50 values of 8.5 +/- 1 nM and 23 +/- 7 nM, respectively. These data suggest that the first intracellular loop of CCK-AR is essential for coupling to Gs and activation of adenylyl cyclase signal transduction cascade.
胆囊收缩素（CCK）-A受体和CCK-B受体是七跨膜结构域G蛋白偶联受体超家族中高度同源的成员。两种受体的基因均包含五个外显子，并具有相似的外显子-内含子结构。为了确定与Gs功能性偶联的CCK-A受体（CCK-AR）的结构基础，构建了一系列嵌合突变体，即将人CCK-B受体（CCK-BR）从第二个外显子到第五个（最后一个）外显子替换为人CCK-AR的对应外显子。在稳定转染的HEK-293细胞中检测野生型和嵌合受体的结合及信号转导特性。与两种野生型受体相似，对CCK保持高亲和力结合的嵌合受体在细胞内钙动员方面呈现剂量依赖性增加。然而，与野生型CCK-BR相比，只有野生型CCK-AR和包含CCK-AR第二个外显子的嵌合突变体能够介导细胞内cAMP含量和腺苷酸环化酶活性的显著更大增加。通过将第一个细胞内环中的五个氨基酸Gly-Leu-Ser-Arg-(Arg)-Leu替换为相应的五个CCK-AR特异性氨基酸Ile-Arg-Asn-Lys-(Arg)-Met，进一步构建了一个CCK-BR突变体。所得受体对CCK和胃泌素均保持高亲和力结合，并且具有与野生型CCK-BR相似的剂量依赖性钙反应。然而，这个第一个细胞内环突变体对硫酸化CCK-8和胃泌素-17也获得了阳性cAMP反应，EC50值分别为8.5±1 nM和23±7 nM。这些数据表明，CCK-AR的第一个细胞内环对于与Gs偶联及激活腺苷酸环化酶信号转导级联反应至关重要。
