Roettger B F, Ghanekar D, Rao R, Toledo C, Yingling J, Pinon D, Miller L J
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota 55905, USA.
Mol Pharmacol. 1997 Mar;51(3):357-62.
Receptor-mediated endocytosis has been observed after agonist occupation of several G protein-coupled receptors, which contributes to the desensitization response to agonist stimulation; however, the cellular signals required to initiate this process are unclear. In this study, we developed and characterized a new antagonist analogue of cholecystokinin (D-Tyr-Gly-[(Nle28,31,D-Trp30)cholecystokinin-26-32]-phen eth yl ester) that can be tagged with a fluorescent rhodamine and radioiodinated. This has permitted us to demonstrate that antagonist occupation of the cholecystokinin receptor also results in receptor internalization, which dissociates this response from second messenger signaling activities and receptor phosphorylation. Immunolocalization of this receptor after occupation with an established nonpeptidyl antagonist confirmed this phenomenon. Antagonist-induced receptor internalization probably results from stabilization of the receptor in a conformation that exposes a domain critical to directing it into the clathrin-dependent endocytic pathway. This work provides evidence for a new and independent mechanism for receptor internalization, provides a mechanism for the rarely observed phenomenon of antagonist-induced desensitization, and raises important issues regarding the approach to establish optimal treatment regimens for antagonist drugs.
在激动剂占据几种G蛋白偶联受体后，已观察到受体介导的内吞作用，这有助于对激动剂刺激产生脱敏反应；然而，启动这一过程所需的细胞信号尚不清楚。在本研究中，我们开发并鉴定了一种新的胆囊收缩素拮抗剂类似物（D-酪氨酸-甘氨酸-[（Nle28,31，D-色氨酸30）胆囊收缩素-26-32]-苯乙酯），它可以用荧光罗丹明标记并进行放射性碘化。这使我们能够证明，拮抗剂占据胆囊收缩素受体也会导致受体内化，从而使这种反应与第二信使信号传导活动和受体磷酸化分离。用已确立的非肽类拮抗剂占据该受体后进行免疫定位证实了这一现象。拮抗剂诱导的受体内化可能是由于受体稳定在一种构象中，该构象暴露了一个对将其引导至网格蛋白依赖性内吞途径至关重要的结构域。这项工作为受体内化提供了一种新的独立机制的证据，为很少观察到的拮抗剂诱导的脱敏现象提供了一种机制，并提出了关于确定拮抗剂药物最佳治疗方案方法的重要问题。
