INVESTIGATION OF DESENSITIZATION/RESENSITIZATION MECHANISMS FOR Gq PROTEIN-COUPLED RECEPTORS

许多G蛋白偶联受体(GPCRs)在激动剂的刺激下会发生功能和分布的变化，即受体的脱敏/内化。G蛋白偶联受体激酶(GRKs)对激动剂占据的GPCRs的磷酸化是诱导受体脱敏和随后Arrestin介导的内化的关键事件。然而，组胺H_1受体(H_1，Rs)等与G蛋白Gq家族偶联的钙动员受体的脱敏/内化机制研究较少，尤其是关于通过钙信号对脱敏/内化过程的反馈调控。我们在人U373 MG星形细胞瘤细胞中发现，激动剂诱导的、由网状蛋白介导的H_1RS内化被钙调素(CaM)瞬时抑制，而钙调素(CaM)既不涉及CaM依赖的酶，如CaM激酶II和钙调神经磷酸酶(PP2B)，也不涉及蛋白激酶C(PKCs)。由于已知Ca^&lt；2+&gt；/CaM抑制GRKs的受体磷酸化，这可能是Ca^&lt；2+/CaM介导的抑制受体内化的原因。结果表明，在激动剂刺激的早期，H_1RS仍停留在细胞表膜上。与受体内化相反，组胺对H、Rs的亲和力在受体内化开始之前通过脱敏CaM激酶II和重新增敏PP2B而双重调节。即使在激动剂存在的情况下，细胞内钙离子浓度的降低也可能使H_1RS内化，内化的受体对组胺的亲和力降低。因此，提示Ca~(2+)&Gt；/CaM通过调节CaM激酶II、PP2B和GRKs的活性，在决定Gq蛋白偶联受体的功能和分布中起重要作用。

Many G protein-coupled receptors (GPCRs) undergo functional and distributional changes on stimulation with agonist, i.e., desensitization/internalization of receptors. Phosphorylation of agonist-occupied GPCRs by G protein-coupled receptor kinases (GRKs) is a key event for induction of receptor desensitization and the subsequent arrestin-mediated internalization. However, desensitization/internalization mechanisms of Ca^<2+>-mobilizing receptors coupled to the Gq family of G proteins, such as histamine H_1, receptors (H_1,Rs), has been much less studied, in particular, with respect to feedback modulation of the desensitization/internalization process via the Ca^<2+> signaling. We have found in human U373 MG astrocytoma cells that agonist-induced, clathrin-mediated internalization of H_1Rs is transiently inhibited by Ca^<2+>/calmodulin (CaM), where neither Ca^<2+>/CaM-dependent enzymes, such as CaM kinase II and calcineurin (PP2B), nor protein kinase C (PKCs) is involved. Since Ca^<2+>/CaM is known to inhibit receptor Phosphorylation by GRKs, this might be responsible for the Ca^<2+>/CaM-mediated inhibition of receptor internalization. As a result, H_1Rs remain on the cell surface membrane during the early stage of agonist stimulation. In contrast to the receptor internalization, the affinity of histamine for the H,Rs was dually regulated by desensitizing CaM kinase II and resensitizing PP2B before initiation of the receptor internalization. The subsequent decrease in the intracellular Ca^<2+> concentration even in the presence of agonist may allow H_1Rs to be internalized, and the internalized receptors showed a reduced affinity for histamine. Thus, it is suggested that Ca^<2+>/CaM play a crucial role in determining both function and distribution of Gq protein-coupled receptors by regulating activity of CaM kinase II, PP2B and GRKs.