Roettger B F, Pinon D I, Burghardt T P, Miller L J
Center for Basic Research in Digestive Diseases and Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Am J Physiol. 1999 Mar;276(3):C539-47. doi: 10.1152/ajpcell.1999.276.3.C539.
Partial agonists are effective tools for advancing development of highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of a partial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in native pancreatic acinar cells and Chinese hamster ovary cells expressing CCK receptor (CHO-CCKR). We developed and characterized a novel fluorescent partial agonist, rhodamine-Gly-[(Nle28, 31)CCK-26-32]-phenethyl ester, that binds specifically and with high affinity to CCK receptors. Such analogs are fully efficacious pancreatic acinar cell secretagogues without supramaximal inhibition that mobilize intracellular calcium with little or no increase in phospholipase C (PLC) activity. Despite minimal phosphorylation of CCK receptors in response to this partial agonist, receptor trafficking was the same as that observed with full agonist (CCK). This included normal internalization via clathrin-dependent endocytosis in CHO-CCKR cells and insulation on the surface of pancreatic acinar cells. Also, as with CCK-occupied receptor, fluorescence recovery after photobleaching of partial agonist-occupied receptor on the acinar cell surface demonstrated a marked temperature-dependent slowing of its rate of diffusion. This was similarly associated with resistance to acid-induced dissociation of ligand. Thus some key molecular regulatory mechanisms for CCK receptor internalization and insulation may be initiated by cellular signaling cascades that are not dependent on PLC activation or receptor phosphorylation.
部分激动剂是推动高选择性药物研发以及深入了解细胞功能分子调控的有效工具。在此，我们探讨了一种部分激动剂对天然胰腺腺泡细胞和表达胆囊收缩素（CCK）受体的中国仓鼠卵巢细胞（CHO-CCKR）中CCK受体调控的关键方面、其侧向移动性和细胞转运的影响。我们研发并表征了一种新型荧光部分激动剂，罗丹明 - 甘氨酸 - [（Nle28, 31）CCK - 26 - 32] - 苯乙酯，它能特异性且高亲和力地结合CCK受体。这类类似物是完全有效的胰腺腺泡细胞促分泌剂，无超最大抑制作用，能动员细胞内钙，而磷脂酶C（PLC）活性几乎无增加。尽管该部分激动剂刺激下CCK受体的磷酸化极少，但受体转运与完全激动剂（CCK）刺激时观察到的情况相同。这包括CHO-CCKR细胞中通过网格蛋白依赖的内吞作用进行正常内化，以及胰腺腺泡细胞表面的隔离。此外，与CCK占据的受体一样，胰腺腺泡细胞表面部分激动剂占据的受体光漂白后的荧光恢复显示其扩散速率明显受温度影响而减慢。这同样与对酸诱导的配体解离的抗性相关。因此，CCK受体内化和隔离的一些关键分子调控机制可能由不依赖PLC激活或受体磷酸化的细胞信号级联反应启动。
