Bhuiyan Mohiuddin Ahmed, Nagatomo Takafumi
Department of Pharmacy, The University of Asia Pacific, Dhaka, Bangladesh.
Methods Enzymol. 2010;484:165-77. doi: 10.1016/B978-0-12-381298-8.00009-5.
Constitutively active mutants (CAMs) of G-protein-coupled receptors mimic the active conformation of the receptor in their ability to activate second messenger systems in the absence of agonist. They have revealed novel properties of drugs that reverse the basal levels of constitutive activity, indicating that the drugs have the inverse agonist activity. Internalization plays an important role in receptor endocytosis and signal transduction. The present chapter provides the investigation of the internalization behavior of CAM N111G of Angiotensin II type 1 (AT(1)) receptor and correlates the result with the mechanism of constitutive activity of the mutant. Both wild-type (WT) and N111G mutant receptors were transiently expressed in COS-7 cells and total inositol phosphate production was measured in presence and absence of the angiotensin II receptor blockers (ARBs). The binding affinities toward agonist and ARBs were also determined. We found that the ARBs have the inverse agonist activity in CAM N111G of AT(1) receptor. The internalization of the mutant, which was much lower than WT receptor, was significantly increased in presence of the ARBs. The results indicate that internalization of CAM N111G of AT(1) receptor is induced by the ARBs, which may be an important characteristic of inverse agonist activities of the ARBs in N111G.
G蛋白偶联受体的组成型活性突变体（CAMs）在无激动剂的情况下激活第二信使系统的能力方面，模拟了受体的活性构象。它们揭示了逆转组成型活性基础水平的药物的新特性，表明这些药物具有反向激动剂活性。内化作用在受体胞吞作用和信号转导中起重要作用。本章对血管紧张素II 1型（AT(1)）受体的CAM N111G的内化行为进行了研究，并将结果与该突变体的组成型活性机制相关联。野生型（WT）和N111G突变体受体均在COS-7细胞中瞬时表达，并在有和无血管紧张素II受体阻滞剂（ARBs）的情况下测量总肌醇磷酸生成量。还测定了对激动剂和ARBs的结合亲和力。我们发现ARBs在AT(1)受体的CAM N111G中具有反向激动剂活性。该突变体的内化作用远低于WT受体，在ARBs存在的情况下显著增加。结果表明，ARBs诱导了AT(1)受体的CAM N111G的内化，这可能是ARBs在N111G中反向激动剂活性的一个重要特征。
