Family 3 G-protein-coupled receptor signaling mechanisms

家族 3 G 蛋白偶联受体信号传导机制

• 批准号: 7733884
• 负责人: John K Northup
• 金额: $ 54.18万
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733884
• 关键词: Amino Acid Sequence; Amino Acids; Calcium; Cell physiology; Cells; Complement; Development; Extracellular Domain; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; Glutamates; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Mammalian Cell; Manuscripts; Molecular Chaperones; Neurotransmitters; Pathway interactions; Peptide Sequence Determination; Peptide Signal Sequences; Pharmacologic Substance; Pheromone; Play; Property; Research Project Grants; Role; Sensory; Signal Transduction; Sorting - Cell Movement; Standards of Weights and Measures; Structure; Taste Perception; Transport Vesicles; Work; design; expression vector; extracellular; link protein; novel; receptor; sweet taste perception; vector

To complement our prior examination of the signaling properties of the 7TM cores of the family 3 GPCRs (hCaR and hT1Rs), we have constructed expression vectors for the ECD portions of the hCaR, mGluR1 and hT1Rs. Attempts to express these in several E.coli strains have uniformly failed, despite co-transformation of the expression hosts with identified bacterial chaperones and thioreductase. We are now constructing baculoviral vectors for this project, and we have examined the cellular processing of family 3 GPCRs in mammalian cells. Our findings indicate that the 7TM cores and the ECD domains utilize alternate sorting pathways within HEK293 cells which can be distinguished by COPII vesicle transport from ER to Golgi using Sar1 and Rab1 GTPases. The wild-type structures for hCaR and mGluR1 appear to utilize both sorting pathways. Surprisingly, the standard ER-export signal sequence does not appear to play a role in this sorting. Further, constructs lacking the carboxyl-terminal sequences of the hCaR or ECD constructs lacking any transmembrane segments utilize a sorting pathway with an as yet unidentified cellular recognition of the extracellular sequence(s) of the proteins linking them to Sar1-Rab1 assembly (Zhuang, Northup & Ray, manuscript submitted).

为了补充我们对家族3 GPCR（HCAR和HT1RS）7TM核心的信号传导特性的补充，我们已经为HCAR，MGLUR1和HT1RS的ECD部分构建了表达向量。 尽管表达宿主与已鉴定的细菌伴侣和硫糖酶共同转化，但在几种大肠杆菌菌株中表达它们的尝试均匀失败。 现在，我们正在为该项目构建细菌病毒载体，并研究了哺乳动物细胞中3个GPCR的细胞加工。 我们的发现表明，7TM核心和ECD结构域利用HEK293细胞中的替代排序途径可以使用SAR1和RAB1 GTPases从ER到Golgi进行copi囊泡传输来区分。 HCAR和MGLUR1的野生型结构似乎利用了这两种排序途径。 出人意料的是，标准的ER-EXPORT信号序列似乎在此分类中没有作用。 此外，缺乏缺乏HCAR或ECD构建体的羧基末端序列，缺乏任何跨膜段的构建体利用了一种分类途径，具有尚未识别的细胞识别蛋白质外细胞序列（S）的蛋白质（s），将其链接到SAR1-RAB1组件（Zhuang，Northup＆Ray，Manuscript，Manscript）。