Regulation of cysteinyl leukotriene type i receptor

描述（由申请人提供）：半胱氨酸白三烯激活半胱氨酸白三烯1型受体（cysllt1r），调节炎症过程和疾病（如哮喘）中重要的许多细胞功能。尽管它具有重要的生理意义，但迄今为止还没有研究检查CysLT1R信号传导或贩运的调节。我们建立了用于分析重组人CysLT1R的模型系统，并发现CysLT1R的内化和信号传导调控在gpcr中是独一无二的。在野生型（wt） CysLT1R中观察到快速而深刻的ltd4刺激内化，而c端截断突变体内化受损，但信号强烈，表明氨基酸309-321内的区域对内化至关重要。arrestin2或arrestin3的共表达增加了激动剂刺激的wt CysLT1R内化，同时抑制磷酸肌苷（PI）的产生。然而，显性阴性抑制因子的共表达对抑制因子内化的影响最小，并且在缺乏抑制因子2和抑制因子3的小鼠胚胎成纤维细胞中，抑制因子wt CysLT1R内化，这表明抑制因子不是CysLT1Rs的主要生理调节因子。相反，PKC的药理抑制深刻地抑制了cysllt1r的内化，同时大大增加了LTD4产生PI，但对H1组胺受体的内化或信号传导几乎没有影响。此外，在CysLT1R C-tail中假定的PKC磷酸化位点（CysLT1RS(313-316)A）的突变减少了LTD4的受体内化，增加了PI的产生，并显著减弱了PKC抑制的作用。PKC的异源脱敏也被观察到，因为在表达wt CysLT1R而不表达CysLT1RS(313-316) a的细胞中，用磷酸酯预处理导致随后LTD4激发的PI产量小幅但显著降低。这些发现表明cysllt1r是迄今为止鉴定的第一个PKC是快速激动剂依赖性内化和脱敏的主要调节因子的GPCR。

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.