Family 3 G-protein-coupled receptor signaling mechanisms

家族 3 G 蛋白偶联受体信号传导机制

基本信息

批准号：
8349631
负责人：
John K Northup
金额：
$ 59.96万
依托单位：
NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
依托单位国家：
美国
项目类别：
财政年份：
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/8349631
关键词：
Amino Acids Aminobutyric Acids Anabolism Back Binding Sites C-terminal Calcium Calcium-Sensing Receptors Calmodulin-Binding Proteins Cations Cell Separation Cell physiology Cell surface Cells Degradation Pathway Disulfide Linkage Extracellular Domain Family G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Homo Human Hypocalcemia result LAMP-1 Laboratories Ligand Binding Domain Link Lysine Lysosomes Manuscripts Metabotropic Glutamate Receptors Molecular Molecular Conformation N-terminal Neurotransmitters Pathway interactions Perception Pheromone Receptors Process Protein Kinase C Research Project Grants Rodent Screening procedure Signal Transduction Sorting - Cell Movement Structure T1R receptor Taste Perception Transmembrane Domain Work deletion analysis design dimer extracellular gain of function glycosylation novel protein protein interaction receptor receptor internalization receptor recycling sweet receptor sweet taste perception trafficking

项目摘要

The extracellular calcium-sensing receptor belonging to the same family3 as the sweet and amino acid sensing T1R receptors and it has recently been found to be involved in kokumi taste enhancing perception. A metabotropic glutamate receptor belonging in this class also participates in umami taste. We wished to understand cellular processing of the family 3 GPCRs because heterologous cell expression of family 3 GPCRs has been one of the major bottlenecks in developing functional screening strategies for these receptors. Using the human calcium sensing receptor, hCaR, we had previously focused on the cell-sorting pathway(s) involved in transporting the newly synthesized hCaR from the ER to the cell surface, and we had identified a novel Rab1/Sar1 dependent mechanism recognizing the N-terminal extraceullar ligand binding domain for transport. Our current work has focused on the re-cycling of the hCaR. We examined sequence in the C-terminal domain known to modulate cell-surface expression of the hCaR. A large naturally-occurring carboxyl terminus deletion between residues S895 to V1075 of the hCaR causing autosomal dominant hypocalcemia (ADH) showed gain-of-function and increased cell surface expression in HEK-293 cells. Our studies revealed a rapid constitutive receptor internalization of hCaR, accumulation in early (Rab7 positive) and late endosomal (LAMP-1 positive) sorting compartments, before lysosomal degradation. Recycling of receptors back to the cell surface was also evident. Deletion analysis has defined a 53 residue sequence required for targeting to lysosomes for degradation but not for internalization or recycling of the receptor. No single sequence motif was identified in our studies, which have eliminated known calmodulin-binding, protein kinase C phorphorylation and lysines targeting proteosomal degradation. This interval includes a high proportion of acidic and hydroxylated amino acid residues in this interval suggesting a similarity to a PEST-like degradation motif. The results define a novel large PEST-like sequence that participates in the sorting of internalized hCaR routed to the lysosomal degradation pathway that regulates cell surface abundance of the receptor (Zhuang et al, manuscript in revision for J. Biol. Chem.)

细胞外钙感应受体与甜味和氨基酸感应 T1R 受体属于同一家族3，最近发现它与醇厚味觉增强有关。属于此类的代谢型谷氨酸受体也参与鲜味。我们希望了解家族 3 GPCR 的细胞加工，因为家族 3 GPCR 的异源细胞表达一直是开发这些受体功能筛选策略的主要瓶颈之一。使用人类钙传感受体 hCaR，我们之前重点研究了将新合成的 hCaR 从 ER 转运到细胞表面所涉及的细胞分选途径，并且我们已经确定了一种新的 Rab1/Sar1 依赖性机制，可识别用于运输的 N 端胞外配体结合域。我们目前的工作重点是 hCaR 的回收。我们检查了已知调节 hCaR 细胞表面表达的 C 端结构域中的序列。 hCaR 残基 S895 至 V1075 之间天然存在的大量羧基末端缺失导致常染色体显性低钙血症 (ADH)，在 HEK-293 细胞中显示出功能获得和细胞表面表达增加。我们的研究揭示了 hCaR 的快速组成性受体内化，在溶酶体降解之前在早期（Rab7 阳性）和晚期内体（LAMP-1 阳性）分选区室中积累。受体再循环回到细胞表面也很明显。删除分析确定了靶向溶酶体降解所需的 53 个残基序列，但不用于受体的内化或再循环。我们的研究中没有发现单一序列基序，该基序消除了已知的钙调蛋白结合、蛋白激酶 C 磷酸化和靶向蛋白体降解的赖氨酸。该区间包含高比例的酸性和羟基化氨基酸残基，表明与 PEST 样降解基序相似。结果定义了一个新的大型 PEST 样序列，该序列参与内化 hCaR 的分选，该内化 hCaR 路由至调节受体细胞表面丰度的溶酶体降解途径（Zhuang 等人，J. Biol. Chem. 修订中的手稿）