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: 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
• 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

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.)

与甜和氨基酸传感T1R受体相同族的细胞外钙感应受体3，最近发现它参与了Kokumi味道增强的感知。属于此类的代谢性谷氨酸受体也参与了鲜味。 我们希望了解家族3 GPCR的细胞处理，因为家族3 GPCR的异源细胞表达一直是为这些受体开发功能筛选策略的主要瓶颈之一。 使用人类钙传感受体HCAR，我们以前曾关注与将新合成的HCAR从ER转移到细胞表面的细胞分类途径，并且我们已经识别出一种新型的RAB1/SAR1依赖机制，以识别N端外脉冲外载体外部配体结合结构域进行运输。 我们目前的工作集中在HCAR的重新骑行上。 我们检查了已知调节HCAR细胞表达的C末端域中的序列。在HCAR中，残基S895至V1075之间的大型天然羧基末端缺失，导致常染色体显性低钙（ADH）在HEK-293细胞中表现出功能的增强和增加的细胞表面表达。我们的研究表明，在溶酶体降解之前，HCAR，早期（RAB7阳性）和晚期内体（LAMP-1阳性）分选室的积累快速组成型受体内在化。也很明显地将受体回收回到细胞表面。缺失分析定义了靶向溶酶体降解所需的53个残基序列，但不能用于内在化或回收受体。在我们的研究中，未发现单个序列基序，这些基序消除了已知的钙调蛋白结合，蛋白激酶C塑料和靶向蛋白质体降解的赖氨酸。在此间隔中，此间隔包括高比例的酸性和羟基化氨基酸残基，表明与害虫样降解基序相似。结果定义了一个新型的大虫害样序列，该序列参与了内部化的HCAR的分选，该序列将其路由到溶酶体降解途径中，该途径调节受体的细胞表面丰度（Zhuang等人，J。Biol。Chem。Chem。Chem。）的手稿。