ENDOPEPTIDASES AFFECT G-PROTEIN COUPLED RECEPTOR SIGNALING AND RESENSITIZATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 研究中心，而研究中心不一定是研究者所在的机构。 神经肽对G蛋白偶联受体（GPCR）的激活可通过细胞表面肽酶活性或受体脱敏来终止，从而防止可能导致功能障碍和疾病的不受控制的信号传导。中性内肽酶（NEP）等细胞表面肽酶降解细胞外神经肽并终止其生物学作用。 例如，NEP降解P物质（SP）并通过限制神经激肽-1受体（NK 1 R）的激活来终止炎症。 或者，活化的GPCR信号传导通过脱敏终止，脱敏是一种需要异源三聚体G蛋白解偶联和受体-激动剂复合物内吞到内体中的过程，其中内体的酸性环境促进受体和激动剂的解离。 虽然受体通过知之甚少的机制被分类为再循环或降解途径，但内吞作用后激动剂的最终命运尚不清楚。 内皮素转化酶-1（ECE-1）是内肽酶M-13家族的成员，也包括NEP。 虽然NEP局限于质膜，但ECE-1具有广泛的亚细胞分布，包括质膜和内体。在质膜上表达的ECE-1的主要天然底物是大内皮素（big-ET）。 大ET在质膜上被ECE-1水解形成内皮素-1（ET），一种有效的血管收缩剂。 虽然ECE-1在体外实验中水解多种神经肽，如P物质和缓激肽，但这种活性的生物学意义尚不清楚。 此外，还没有探索内体中所含ECE-1的作用。 本项目的目的是研究ECE-1在GPCR信号转导中的作用。 GPCR的活化导致受体和激动剂内吞到酸化的内体中。 我们提出内肽酶如ECE-1也存在于这些酸化的内体中，在那里它们降解神经肽激动剂，从而控制受体再循环。 质谱法将允许表征内体ECE-1活性并确定其底物特异性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. G-protein coupled receptor (GPCR) activation by neuropeptides is terminated by either cell-surface peptidase activity or receptor desensitization, and thereby prevents uncontrolled signaling that may cause dysfunction and disease. Cell-surface peptidases such as neutral endopeptidase (NEP) degrade extra-cellular neuropeptides and terminate their biological actions. For example, NEP degrades substance P (SP) and terminates inflammation by limiting the activation of the neurokinin-1 receptor (NK1R). Alternatively, activated GPCR signaling is terminated by desensitization, a process that entails uncoupling of heterotrimeric G-proteins and endocytosis of the receptor-agonist complex into endosomes where the acidic environment of the endosomes facilitates dissociation of the receptor and agonist. While the receptor is sorted to either recycling or degradative pathways by poorly understood mechanisms, the ultimate fate of the agonist after endocytosis is unknown. Endothelin-converting enzyeme-1 (ECE-1) is a member of the M-13 family of endopeptidases that also includes NEP. While NEP is confined to the plasma membrane, ECE-1 has a broad subcellular distribution that includes both the plasma membrane and endosomes. The primary natural substrate of ECE-1 expressed at the plasma membrane is big endothelin (big-ET). Big-ET is hydrolyzed by ECE-1 at the plasma membrane to form endothelin-1 (ET), a potent vasoconstrictor. While ECE-1 hydrolyzes a variety of neuropeptides such as substance-P and bradykinin in in-vitro experiments, the biological significance of this activity is unknown. Furthermore, the role of ECE-1 contained in endosomes has not been explored. The aim of this project is to study the role of ECE-1 in GPCR signaling. Activation of GPCRs results in endocytosis of the receptor and agonist into acidified endosomes. We propose that endopeptidases such as ECE-1 are also present in these acidified endosomes where they degrade the neuropeptide agonists and thereby control receptor recycling. Mass spectrometry will allow for characterization of endosomal ECE-1 activity and determine its substrate specificity.