Spatiotemporal Regulation of GPCR Signaling by Different Beta-Arrestin Conformations

不同 Beta-arrestin 构象对 GPCR 信号传导的时空调节

• 批准号: 10501076
• 负责人: Alex Rojas Bie Thomsen
• 金额: $ 37.09万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10501076
• 关键词: Agonist; Arrestins; Binding Sites; Biochemical; Biological; C-terminal; Cell surface; Cells; Complex; Coupling; Cyclic AMP; Data; Drug Prescriptions; Drug Targeting; Endocytosis; Endosomes; Future; G Protein-Coupled Receptor Signaling; G protein coupled receptor kinase; G-Protein-Coupled Receptors; GTP-Binding Protein Regulators; GTP-Binding Proteins; Heterotrimeric GTP-Binding Proteins; Hydrophobicity; Knowledge; Lead; Location; Mediating; Molecular; Molecular Conformation; Phosphorylation; Phosphorylation Site; Physiological; Physiological Processes; Property; Proteomics; Regulation; Research; Signal Transduction; Signaling Protein; Tail; Therapeutic; Time; Vision; beta-arrestin; desensitization; design; driving force; drug development; innovation; phosphoric diester hydrolase; programs; receptor; receptor internalization; recruit; response; spatiotemporal

G protein-coupled receptors (GPCRs) at the cell surface regulate most physiological processes and are important drug targets with ~34% of all prescribed drugs targeting them. Classically, upon agonist stimulation, GPCRs activate heterotrimeric G proteins, causing downstream signaling throughout the cell. In order to terminate G protein signaling, cells have devised a specialized desensitization mechanism that includes receptor phosphorylation by GPCR kinases and subsequent recruitment of β-arrestins (βarrs) to the phosphorylated receptors. The GPCR–βarrs interaction both blocks the G protein-binding site at the receptor core and promotes receptor endocytosis. Recently, however, we discovered that some GPCRs interact with βarrs exclusively through their phosphorylated C-terminal tails. Since βarrs do not block the G protein-binding site in this `tail' conformation, the receptor can associate with βarrs and G proteins simultaneously to form GPCR–G protein– βarr `megaplexes.' The assembly of these megaplexes allows the receptor to continue to stimulate G protein signaling while being internalized into endosomes by βarrs. Thus, the existence of the core and tail GPCR–βarr complex conformations suggests that βarrs act as spatiotemporal master regulators of G protein signaling: When bound to the receptor core, βarrs regulate the duration of G protein signaling whereas βarrs control the cellular location from where G proteins are activated from when associated with the receptor C-terminal tail. As the underlying properties that promote these two complex conformations remain elusive, my research objectives over the next 5 years involve determining these molecular driving forces on a general scale. Our preliminary data suggest that phosphorylation site clusters located within the receptor C-terminal tail are required for the association with βarrs in the tail conformation. Therefore, we plan to establish whether the presence of these phosphorylation site clusters correlates with the capacity of GPCRs to engage in mechanisms that lead to sustained endosomal G protein signaling. In regards to the GPCR–βarr core conformation, the fingerloop domain (FLD) of βarrs inserts itself into the transmembrane core of most GPCRs via its hydrophobic tip and receptor- specific residues. To characterize this interaction on a general scale, we will examine whether these receptor- specific βarr-FLD residues correlate with G protein subtype coupling of different GPCRs. Finally, βarrs modulate the activity of phosphodiesterases (PDEs), which terminate Gs-cAMP signaling. However, our preliminary data raise the possibility that this modulation occurs specifically by βarrs in the core conformation. Therefore, we will apply a combination of cell biological, biochemical, and proteomics approaches to examine whether modulation of PDEs and other desensitization mechanism is mediated specifically by distinct βarr conformations. My vision with this research program is to elucidate how GPCR signaling is regulated spatiotemporally by βarrs, which may lead to differentiated physiological responses. The knowledge acquired here will be used to design new and innovative therapeutics that specifically target GPCRs in time and space.

细胞表面的G蛋白偶联受体(GPCRs)调节大多数生理过程，并 重要的药物靶点，约占所有处方药的34%。经典地说，在激动剂刺激下， GPCRs激活异源三聚体G蛋白，在整个细胞内产生下行信号。为了 终止G蛋白信号，细胞已经设计出一种特殊的脱敏机制，包括受体 GPCRK的磷酸化和随后的β阻滞素(βARRs)对磷酸化的 感受器。GPCR-βARRS相互作用既阻断受体核心的G蛋白结合部位，又促进 受体内吞作用。然而，最近我们发现，一些GPCR只与βARR相互作用 通过它们磷酸化的C-末端尾巴。由于βARR不阻断这条“尾巴”中的G蛋白结合部位 构象上，该受体可同时与βARRs和G蛋白结合，形成GPCRG蛋白。 β是‘巨型复合体’。这些巨型神经丛的组装使受体能够继续刺激G蛋白 信号被βARRs内化到内体中。因此，核心和尾部GPCRARR的存在 复杂的构象表明βARRs作为G蛋白信号的时空主调节器：何时 结合受体核心，βARRs调节G蛋白信号的持续时间，而βARRs控制细胞 当G蛋白与受体C末端尾巴相关时，G蛋白被激活的位置。作为 促进这两种复杂构象的潜在特性仍然难以捉摸，我的研究目标 在接下来的5年里，需要在一般规模上确定这些分子驱动力。我们的初步数据 提示位于受体C末端尾部内的磷酸化位点簇是 在尾部构象中与βARRs结合。因此，我们计划确定是否存在这些 磷酸化位点簇与GPCRs参与导致 持续的内体G蛋白信号。关于GPCR-βARR核心构象，指环结构域 βARRS的FLD通过其疏水末端和受体插入大多数GPCRs的跨膜核心。 特定残留物。为了在一般规模上描述这种相互作用，我们将检查这些受体是否- 特异性βARR-FLD残基与不同GPCR的G蛋白亚型偶联有关。最后，βARRS调制 磷酸二酯酶(PDE)的活性，终止Gs-cAMP信号。然而，我们的初步数据 提高这种调制特定地由核心构象中的βARR发生的可能性。因此，我们将 应用细胞生物学、生物化学和蛋白质组学方法相结合的方法来检查 PDE和其他脱敏机制是由不同的βARR构象特异性地介导的。我的愿景 这个研究计划是为了阐明βARRs是如何在时空上调节gpr信号的， 可能导致不同的生理反应。在这里获得的知识将用于设计新的和 在时间和空间上专门针对GPCRs的创新疗法。