Histamine H2 receptor trafficking: Role of arrestin, dynamin, and clathrin in histamine H2 receptor internalization
Histamine H2 receptor trafficking: Role of arrestin, dynamin, and clathrin in histamine H2 receptor internalization
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DOI：
10.1124/mol.108.045336
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发表时间：
2008-10-01
影响因子：
3.6
通讯作者：
Shayo, Carina
中科院分区：
文献类型：
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作者：
Fernandez, Natalia;Monczor, Federico;Shayo, Carina
激动剂诱导的G蛋白偶联受体（gpcr）内化与受体脱敏、再敏和下调有关。在本研究中，我们试图确定组胺H2受体（H2r）激动剂氨胺除了促进受体脱敏外，是否诱导H2r内化。我们进一步研究了相关机制及其在受体再敏化中的潜在作用。在COS7转染的细胞中，氨胺诱导H2r时间依赖性内化，暴露于氨胺60分钟后显示70%的受体内吞。激动剂的去除导致重敏受体迅速恢复到细胞表面。在环己亚胺（一种蛋白质合成抑制剂）存在的情况下也得到了类似的结果。用蛋白磷酸酶2A （PP2A）家族磷酸酶抑制剂冈田酸治疗，降低了H2r膜位点的恢复和cAMP反应。阻滞蛋白3而非阻滞蛋白2的过表达降低了H2r膜位点和H2r诱发的cAMP反应。受体共转染捕获蛋白、动力蛋白、Eps15（网状蛋白介导的内吞机制的一个组成部分）的显性阴性突变体，或RNA干扰捕获蛋白3，均可消除H2r内化和再敏化。在内源性表达H2r的U937细胞中也得到了类似的结果。我们的研究结果表明，胺胺诱导的H2r内化对H2r再敏化至关重要，这一过程独立于H2r的重新合成，但依赖于pp2a介导的去磷酸化。虽然我们没有提供H2r与β -抑制蛋白、动力蛋白和/或网格蛋白相互作用的直接证据，但我们的研究结果支持它们参与H2r内吞作用。受体在细胞表面的快速循环和特异性参与受体内化进一步表明H2r属于A类gpcr。
Agonist-induced internalization of G protein-coupled receptors (GPCRs) has been implicated in receptor desensitization, resensitization, and down-regulation. In the present study, we sought to establish whether the histamine H2 receptor (H2r) agonist amthamine, besides promoting receptor desensitization, induced H2r internalization. We further studied the mechanisms involved and its potential role in receptor resensitization. In COS7 transfected cells, amthamine induced H2r time-dependent internalization, showing 70% of receptor endocytosis after 60-min exposure to amthamine. Agonist removal led to the rapid recovery of resensitized receptors to the cell surface. Similar results were obtained in the presence of cycloheximide, an inhibitor of protein synthesis. Treatment with okadaic acid, an inhibitor of the protein phosphatase 2A (PP2A) family of phosphatases, reduced the recovery of both H2r membrane sites and cAMP response. Arrestin 3 but not arrestin 2 overexpression reduced both H2r membrane sites and H2r-evoked cAMP response. Receptor cotransfection with dominant-negative mutants for arrestin, dynamin, Eps15 ( a component of the clathrin-mediated endocytosis machinery), or RNA interference against arrestin 3 abolished both H2r internalization and resensitization. Similar results were obtained in U937 cells endogenously expressing H2r. Our findings suggest that amthamine-induced H2r internalization is crucial for H2r resensitization, processes independent of H2r de novo synthesis but dependent on PP2A-mediated dephosphorylation. Although we do not provide direct evidence for H2r interaction with beta-arrestin, dynamin, and/or clathrin, our results support their involvement in H2r endocytosis. The rapid receptor recycling to the cell surface and the specific involvement of arrestin 3 in receptor internalization further suggest that the H2r belongs to class A GPCRs.