Regulation of cysteinyl leukotriene type i receptor

I 型半胱氨酰白三烯受体的调节

• 批准号: 6871340
• 负责人: RAYMOND B. PENN
• 金额: $ 32.29万
• 依托单位: WAKE FOREST UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-15 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6871340
• 关键词: arrestins; calcium flux; cell surface receptors; cysteine; enzyme linked immunosorbent assay; enzyme mechanism; gene mutation; genetic regulation; genetically modified animals; immunofluorescence technique; inflammation; laboratory mouse; leukotrienes; phosphatidylinositols; phosphorylation; protein kinase C; protein structure function; receptor expression; receptor mediated endocytosis; smooth muscle

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.

描述（由申请人提供）：白细胞三烯激活白细胞三烯1型受体（CYSLT1R），以调节在炎症过程和疾病（例如哮喘）中重要的许多细胞功能。尽管具有生理意义，但迄今为止尚未研究CYSLT1R信号传导或贩运的调节。我们已经建立了用于分析重组人Cyslt1R的模型系统，并发现对CYSLT1R的内在化和信号的调控是GPCR中唯一的。对于野生型（WT）Cyslt1r观察到了快速而深刻的LTD4刺激的内在化，而C末端截断突变体的内在化表现出了较强的内在化受损，并建议氨基酸309-321中的区域至关重要。抑制素或抑制素3的共表达增加了激动剂刺激的WT Cyslt1R的内在化，同时抑制磷酸肌醇（PI）的产生。然而，在鼠类胚胎成纤维细胞中均缺乏抑制蛋白2和抑制素3的鼠类胚胎成纤维细胞中的wt cyslt1r，对主要影响的内在化的共同表达，这表明抑制蛋白不是cyslt1rs的主要生理调节剂。取而代之的是，PKC的药理抑制深刻抑制了CYSLT1R的内在化，而LTD4大大增加了PI的产生，但几乎对H1组胺受体的内在化或信号传导几乎没有影响。此外，在Cyslt1r C尾（Cyslt1rs（313-316）a）降低受体内在化并增加了LTD4的PI产生，并显着减弱了PKC抑制的作用。还观察到了PKC异源脱敏，因为用凤凰酯进行了预处理，导致PI产生的较小但显着降低，随后在表达WT Cyslt1R但未表达CYSLT1R的细胞中的LTD4挑战（313-316）a。这些发现将CYSLT1R的特征是迄今为止确定的第一个GPCR，其中PKC是快速激动剂依赖性内在化和脱敏的主要调节剂。