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+>-动员受体与G蛋白的Gq家族偶联的脱敏/内化机制，如组胺H_1受体（H_1,Rs）的研究很少，特别是关于通过Ca^<2+>信号传导对脱敏/内化过程的反馈调节。我们发现，在人U373 MG星形细胞瘤细胞中，激动剂诱导的、网状蛋白介导的H_1Rs内化被Ca^<2+>/钙调蛋白（CaM）短暂抑制，其中Ca^<2+>/钙调蛋白依赖的酶，如CaM激酶II和钙调磷酸酶（PP2B），也不涉及蛋白激酶C （PKCs）。由于已知Ca^<2+>/CaM可抑制GRKs对受体的磷酸化，这可能是Ca^<2+>/CaM介导的受体内化抑制的原因。因此，在激动剂刺激的早期阶段，H_1Rs仍停留在细胞膜表面。与受体内化相反，在受体内化开始之前，组胺对H、Rs的亲和力受到脱敏CaM激酶II和重敏PP2B的双重调节。即使在激动剂存在的情况下，细胞内Ca^<2+>浓度的降低也可能使H_1Rs被内化，内化的受体对组胺的亲和力降低。因此，Ca^<2+>/CaM通过调节CaM激酶II、PP2B和GRKs的活性，在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 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.