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 (GPCR) 调节大多数生理过程，并且 传统上，在激动剂刺激下，约 34% 的处方药物都针对这些重要药物靶点。 GPCR 激活异源三聚体 G 蛋白，在整个细胞中引起下游信号传导。 终止 G 蛋白信号传导，细胞设计了一种专门的脱敏机制，其中包括受体 GPCR 激酶进行磷酸化，随后将 β-抑制蛋白 (βarrs) 募集到磷酸化的 GPCR-βarrs 相互作用既阻断受体核心的 G 蛋白结合位点，又促进 然而，最近我们发现一些 GPCR 只与 βarrs 相互作用。 由于 βarrs 不会阻断该“尾部”中的 G 蛋白结合位点。 构象下，受体可以同时与βarrs和G蛋白结合，形成GPCR–G蛋白– βarr ‘megaplexes’。这些megaplexes的组装允许受体继续刺激G蛋白 信号传导同时被 βarrs 内化到核内体中，因此，核心和尾部 GPCR-βarr 的存在。 复杂的构象表明 βarrs 充当 G 蛋白信号传导的时空主调节因子： βarrs 与受体核心结合，调节 G 蛋白信号传导的持续时间，而 βarrs 控制细胞 当与受体 C 末端尾部结合时，G 蛋白被激活的位置。 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 未来 5 年，我们将在总体范围内确定这些分子驱动力。 表明位于受体 C 末端尾部内的磷酸化位点簇是 因此，我们计划确定这些是否存在。 磷酸化位点簇与 GPCR 参与导致磷酸化的机制的能力相关 持续的内体 G 蛋白信号传导关于 GPCR-βarr 核心构象，即指环结构域。 βarrs (FLD) 通过其疏水性尖端和受体将自身插入大多数 GPCR 的跨膜核心中 为了在一般范围内表征这种相互作用，我们将检查这些受体是否- 特定的 βarr-FLD 残基与不同 GPCR 的 G 蛋白亚型偶联相关。最后，βarrs 进行调节。 磷酸二酯酶 (PDE) 的活性，它终止 Gs-cAMP 信号传导。 提出这种调节是由核心构象中的 βarrs 特异性发生的可能性。 应用细胞生物学、生化和蛋白质组学方法的组合来检查调节是否 我的观点是，偏微分方程和其他脱敏机制是由不同的 βarr 构象专门介导的。 这项研究计划的目的是阐明 GPCR 信号传导如何受 βarrs 的时空调节， 可能会导致不同的生理反应，此处获得的知识将用于设计新的和 专门针对时间和空间上的 GPCR 的创新疗法。