The thromboxane A2 receptor and peroxisomes

g蛋白偶联受体（gpcr）调节多种生理功能，如嗅觉、味觉、视觉、分泌、神经传递、代谢、炎症和免疫反应。GPCR信号被认为起源于细胞表面，受体与外界刺激的相互作用可以被转导成细胞反应。越来越多的证据表明，许多gpcr也从细胞内部发出信号。过氧化物酶体参与细胞代谢的中心途径，如脂肪酸的ß-氧化和活性氧的解毒。过氧化物酶体具有复杂的生物发生和降解途径，活跃分布于细胞内，并具有自身的靶向和转运机制。它们是一种动态结构，其形态、丰度、内容和功能可以根据不同的信号快速调节。过氧化物酶体在GPCR生物学中一直被忽视。通过我们对与tps2（血栓素A2受体）相互作用和调节的蛋白质的研究项目，我们确定了几种参与过氧化物酶体的蛋白质：A)蛋白质靶向和转运到过氧化物酶体，B)过氧化物酶体的运动/分布，以及C)各种过氧化物酶体代谢途径。在后一组中特别感兴趣的是ACOX1，这种酶参与脂肪酸ß-氧化的第一步，产生H2O2。免疫荧光显微镜检测TPß与过氧化物酶体标记物共定位。通过免疫共沉淀和western blot实验证实了受体与过氧化物酶体蛋白之间的相互作用。据我们所知，这是gpcr在过氧化物酶体中存在的第一个证据。在未来5年，我们将专注于以下3个短期目标：1)在HeLa细胞内源性水平上，通过电子显微镜研究TPß对过氧化物酶体的定位。放射性配体结合实验也将在HeLa细胞纯化过氧化物酶体上进行，以进一步研究TPß的存在。TPß在过氧化物酶体膜中的取向将被确定。将研究将tpβ靶向过氧化物酶体的分子机制。2)刺激、阻断和过氧化物酶体中TPß的存在对该细胞器的数量、运动和分布的影响将被表征。在这些过程中所涉及的分子机制将被探讨。3)研究tpβ与过氧化物酶体酶的相互作用。我们将首先关注与ACOX1的交互作用。我们将确定ACOX1是否调控TPß的功能反应。我们将探讨TPß是否调节ACOX1的活性。据我们所知，gpcr从未在过氧化物酶体中被描述过，它们在过氧化物酶体中的功能仍然未知。此外，我们的计划将为我们对细胞信号响应的过氧化物酶体生物学分子调控的理解提供重大进展。

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological functions, such as smell, taste, vision, secretion, neurotransmission, metabolism, inflammatory and immune response. GPCR signaling is thought to stem from the cell surface where receptor interactions with external stimuli can be transduced into cellular responses. Accumulating evidence indicates that many GPCRs also signal from inside the cell. Peroxisomes participate in central pathways of cellular metabolism such as ß-oxydation of fatty acids and detoxification of reactive oxygen species. Peroxisomes have complex biogenesis and degradation pathways, are actively distributed intracellularly and possess their own targeting and translocation machineries. They are dynamic structures whose morphology, abundance, content and function can be rapidly regulated in response to diverse signals. Peroxisomes have been overlooked so far in GPCR biology. Through our research program on the proteins that interact with and regulate TPß (thromboxane A2 receptor), we identified several peroxisomal proteins involved in: A) targeting and translocation of proteins to peroxisomes, B) motility/distribution of peroxisomes, and C) various peroxisomal metabolic pathways. Of particular interest in the latter group is ACOX1, the enzyme involved in the first step of fatty acid ß-oxydation, producing H2O2. TPß co-localization with peroxisomal markers was detected by immunofluorescence microscopy. Interaction between the receptor and peroxisomal proteins were confirmed by co-immunoprecipitation and western blot experiments. To the best of our knowledge, this is the first evidence of the presence of GPCRs in peroxisomes. We will concentrate on the 3 following short-term objectives for the next 5 years: 1)TPß localization to peroxisomes will be studied by electron microscopy at the endogenous level in HeLa cells. Radioligand binding experiments will also be carried out on purified peroxisomes from HeLa cells to further study the presence of TPß. The orientation of TPß in the peroxisomal membrane will be determined. The molecular mechanisms involved in targeting TPß to peroxisomes will be studied. 2)The effects of the stimulation, the blockade and the presence of TPß in peroxisomes on the number, motility, and distribution of this organelle will be characterized. The molecular mechanisms involved in these processes will be explored.  3)The interaction of TPß and peroxisomal enzymes will be studied. We will first focus on the interaction with ACOX1. We will determine whether ACOX1 regulates the functional responses of TPß. We will address whether TPß modulates the activity of ACOX1. To the best of our knowledge, GPCRs have never been described in peroxisomes and their function in this organelle is still unknown. Furthermore, our program will provide significant advancement in our understanding of the molecular regulation of peroxisome biology in response to cellular signals.