Regulation of cysteinyl leukotriene type i receptor

描述（由申请人提供）：半胱氨酰白三烯激活1型半胱氨酰白三烯受体（CysLT 1 R），以调节炎症过程和疾病（如哮喘）中重要的多种细胞功能。尽管它的生理重要性，没有研究到目前为止已经检查了CysLT 1 R信号或贩运的调节。我们已经建立了分析重组人CysLT 1 R的模型系统，并发现CysLT 1 R的内化和信号传导的调节在GPCR中是独特的。观察到野生型（wt）CysLT 1 R的快速和深刻的LTD 4刺激的内化，而C-末端截短突变体表现出受损的内化，但信号强劲，并建议氨基酸309-321内的区域作为内化的关键。arrestin 2或arrestin 3的共表达增加了激动剂刺激的wt CysLT 1 R的内化，同时抑制磷酸肌醇（PI）的产生。然而，显性负性抑制蛋白的共表达对内化的影响最小，而野生型CysLT 1 R在缺乏抑制蛋白2和抑制蛋白3的鼠胚胎成纤维细胞中内化，表明抑制蛋白不是CysLT 1 R的主要生理调节因子。相反，PKC的药理学抑制深刻地抑制CysLT 1 R的内化，同时大大增加PI的生产LTD 4，但几乎没有影响H1组胺受体的内化或信号。此外，CysLT 1 R C-尾（CysLT 1 RS（313-316）A）内推定的PKC磷酸化位点的突变减少了受体内化，增加了LTD 4的PI产生，并显著减弱了PKC抑制作用。还观察到PKC引起的异源性脱敏，因为在表达wt CysLT 1 R但不表达CysLT 1 RS（313-316）A的细胞中，用佛波酯预处理引起PI产生的小但显著的减少，随后进行LTD 4攻击。这些研究结果的特点是CysLT 1 R作为第一个GPCR确定的日期，其中PKC是快速激动剂依赖性内化和脱敏的主要调节。

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.