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

许多G蛋白偶联受体（gpcr）在激动剂刺激下发生功能和分布变化，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gpcr的磷酸化是诱导受体脱敏和随后的抑制素介导的内化的关键事件。然而，Ca^<2+>受体与G蛋白的Gq家族，如组胺H_1受体（H_1Rs）偶联的脱敏/内化机制的研究很少，特别是关于通过Ca^<2+>对脱敏/内化过程的反馈调节。我们研究了Ca~<2+> (CaM)介导的H_1Rs在人U373 MG星形细胞瘤细胞中的调节，发现受体兴奋剂诱导的H_1Rs的发生，Ca^<2+>/CaM的激活延迟了网格蛋白介导的H_1Rs内化，这可能是由于grk介导的内化过程受到抑制。虽然由于Ca^<2+>/CaM介导的受体内化抑制，H_1Rs仍留在细胞膜表面，但Ca^<2+>/CaM通过激活CaM激酶II（脱敏）和蛋白磷酸酶2B（再敏化）来调节激动剂对细胞表面H_1Rs的亲和力。即使在激动剂存在的情况下，细胞内Ca^<2+>浓度的下降也可能使H_1Rs被内化。相比之下，Ca^<2+>离子载体离子霉素对细胞内Ca^<2+>的非受体介导和持续增加未能抑制组胺介导的H_1Rs内化。因此，我们认为受体介导的Ca^<2+>/CaM的激活通过调节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着至关重要的作用。

Many G protein-coupled receptors (GPCRs) undergo functional and distributional changes upon 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+> receptors coupled to the Gq family of G proteins, such as histamine H_1 receptors (H_1Rs), has been much less studied, in particular, with respect to feedback modulation of the desensitization/internalization process via the Ca^<2+> We investigated Ca~<2+> (CaM)-mediated regulation of H_1Rs in human U373 MG astrocytoma cells, and found that the onset of agonist-induced, clathrin-mediated internalization of H_1Rs was delayed by activation of Ca^<2+>/CaM, possibly due to inhibition of GRK-mediated internalization process. While the H_1Rs remained on the cell surface membrane owing to the Ca^<2+>/CaM-mediated inhibition of receptor internalization, the agonist affinity for the cell surface H_1Rs was regulated by Ca^<2+>/CaM via actiyation of CaM kinase II (for desensitization) and protein phosphatase 2B (for resenisitization). The subsequent decrease in the intracellular Ca^<2+> concentration even in the presence of agonist may allow H_1Rs to be internalized. In contrast, a receptor-non-mediated and sustained increase in the intracellular Ca^<2+> by a Ca^<2+> ionophore, ionomycin, failed to inhibit histamine-mediated internalization of H_1Rs. Thus, it is suggested that the receptor-mediated activation of Ca^<2+>/CaM plays a crucial role in determining both function and distribution of Gq protein-coupled receptors by regulating activity of CaM kinase II, PP2B and GRKs.