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: 7593344
• 负责人: John K Northup
• 金额: $ 50.65万
• 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/7593344
• 关键词: Alanine; Amino Acids; Calcium; Calcium-Sensing Receptors; Cell physiology; Cells; Complement; Data; Development; Disruption; Extracellular Domain; Family; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glutamate Receptor; Glutamates; Glycine; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Helix (Snails); Human; Investigation; Ligand Binding; Ligands; Link; Mammalian Cell; Metabotropic Glutamate Receptors; Molecular; Molecular Chaperones; Mutagenesis; Mutation; Neurotransmitters; Pathway interactions; Peptide Signal Sequences; Pharmacologic Substance; Play; Positioning Attribute; Property; Regulation; Research Project Grants; Role; Role playing therapy; Rotation; Scanning; Signal Transduction; Sorting - Cell Movement; Standards of Weights and Measures; Stretching; Structure; Taste Perception; Transmembrane Domain; Transport Vesicles; Work; design; ear helix; expression vector; extracellular; human GPR3 protein; novel; receptor; sweet taste perception; vector

We continued our investigations of the family 3 GPCRs with a study of the role of the linker sequences of the human calcium sensing receptor (hCaR) and the metabotropic glutamate receptor 1 (mGluR1). These studies were designed to reveal the role(s) played by this region of the family3 receptors in transmitting the ligand binding signal from the autonomously folding extracellular domain (ECD) to the transmembrane helix bundle (7TM) for G-protein regulation. Our findings are that disruption of this part of the hCaR structure in most cases leads to misfolded GPCR. Constructs in which a nine amino acid stretch of sequence within the 14 AA linker region was exchanged between the hCaR and mGluR1 yielded nearly wild-type function for both receptor types, highlighting the significant conservation of function of this region. Further, addition of glycine-rich, random coiled sequence, or replacement of this 9 AA with the random coil sequence resulted in misfolded receptors. However, a scanning alanine mutagenesis of the highly conserved residues within the linker sequence revealed 3 positions in which the mutations were tolerated and moderately activating of the hCaR and a fourth in which mutations produced significant misfolding and uncoupling of the ligand regulation through the ECD. These data complement our previous work suggesting essential contacts between the ECD, linker and the extracellular loops 2 and 3 of the 7TM domain. They also indicate that the ECD regulation of the 7TM domain involves changes in linker sequence contact(s) with the 7TM extracellular loop sequence(s) rather than a rotation about the 1st transmembrane domain transmitted by the linker. To complement our 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 initiated an examination of the cellular processing of family c GPCRs in mammalian cells. Our initial findings indicate that the 7TM cores and the ECD constructs 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, and we are currently examining the molecular component(s) linking the ECD structure to Sar1/Rab1 recognition.

我们继续研究了3 GPCR，研究了人钙传感受体（HCAR）和代谢型谷氨酸受体1（MGLUR1）的连接器序列的作用。这些研究的设计旨在揭示该区域的该区域的作用在从自主折叠的细胞外域（ECD）传输到跨膜螺旋螺旋束（7TM）中的配体结合信号中扮演的作用。我们的发现是在大多数情况下，HCAR结构的这一部分的破坏导致GPCR错误折叠。在HCAR和MGLUR1之间交换了14个AA接头区域内的九个氨基酸序列的构建体，两种受体类型都几乎野生型功能，突显了该区域的显着保存。 此外，用随机线圈序列添加了富含甘氨酸的富含甘氨酸，随机盘绕序列或更换该9AA，导致受体错误折叠。 然而，接头序列中高度保守的残基的扫描丙氨酸诱变揭示了3个位置，其中突变被耐受性且中等激活HCAR，而突变则通过ECD对配体调节产生明显的错误折叠和解耦合。这些数据补充了我们先前的工作，表明了7TM域的ECD，接头和细胞外环2和3的基本接触。 他们还表明，7TM域的ECD调节涉及连接器序列接触的变化，而不是7TM细胞外环序列（S），而不是围绕连接器传输的第一个跨膜结构域的旋转。 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检查，我们为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，并且已经开始研究哺乳动物细胞中C gpcr的细胞加工。 我们的初步发现表明，7TM核心和ECD构建体利用HEK293细胞中的替代排序途径，可以使用SAR1和RAB1 GTPases从ER到Golgi的Copii囊泡转运来区分。 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-出口信号序列似乎在此排序中没有作用，并且我们目前正在研究将ECD结构与SAR1/RAB1识别联系起来的分子成分。