Regulation of cysteinyl leukotriene type i receptor

描述(申请人提供)：半胱氨基白三烯激活半胱氨酰白三烯1型受体(CysLT1R)，调节在炎症过程和哮喘等疾病中重要的多种细胞功能。尽管CysLT1R在生理上具有重要意义，但到目前为止，还没有研究检查CysLT1R信号或贩运的调节。我们已经建立了分析重组人CysLT1R的模型系统，并发现CysLT1R的内化和信号转导的调节在GPCR中是独一无二的。观察到野生型(Wt)CysLT1R被LTD4刺激的快速而深刻的内化，而C端截断突变体内化受损但信号强烈，提示氨基酸309-321中的一个区域是内化的关键区域。共表达arrestin2或arrestin3可增加激动剂刺激的wt CysLT1R内化，同时抑制磷脂酰肌醇(PI)的产生。然而，显性负阻滞素的共表达对细胞内化的影响很小，wt CysLT1R内化于同时缺乏抑制素2和3的小鼠胚胎成纤维细胞中，提示阻滞素不是CysLT1R的主要生理调节因子。相反，对PKC的药理抑制深刻地抑制了CysLT1R的内化，同时显著增加了LTD4产生的PI，而对H1组胺受体内化或信号转导几乎没有影响。此外，CysLT1R C-Tail(CysLT1RS(313-316)A)中可能的PKC磷酸化位点的突变减少了受体的内化，增加了LTD4产生的PI，并显著减弱了PKC抑制的作用。在表达wt CysLT1R但不表达CysLT1RS(313-316)A的细胞中，PKC的异源脱敏作用也被观察到，因为佛波酯预处理引起了PI产生的轻微但显著的减少，并伴随着随后的LTD4攻击。这些发现将CysLT1R描述为迄今为止发现的第一个GPCR，其中PKC是快速激动剂依赖的内化和脱敏的主要调节因子。

DESCRIPTION (provided by applicant): Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and find regulation of internalization and signaling of the CysLT1R to be unique among GPCRs. Rapid and profound LTD4-stimulated internalization was observed for the wild type (wt) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, and suggested a region within amino acids 309-321 as critical to internalization. Co-expression of arrestin2 or arrestin3 increased agonist-stimulated internalization of wt CysLT1R while inhibiting phosphoinositide (PI) production. However, co-expression of dominant negative arrestins minimally affected internalization, and wt CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin2 and arrestin3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacological inhibition of PKC profoundly inhibited CysLT1R internalization while greatly increasing PI production by LTD4, yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization and increased PI production by LTD4, and significantly attenuated the effects of PKC inhibition. Heterologous desensitization by PKC was also observed, as pretreatment with phorbol ester caused a small but significant reduction in PI production with subsequent LTD4 challenge in cells expressing wt CysLT1R but not CysLT1RS(313-316)A. These findings characterize the CysLT1R as the first GPCR identified to date in which PKC is the principal regulator of rapid agonist-dependent internalization and desensitization.