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.

G蛋白偶联受体（GPCR）在细胞表面调节大多数物理过程，并且 重要的药物靶标有〜34％的所有针对性药物。从经典上讲，在激动剂刺激下， GPCR激活异三聚体G蛋白，在整个细胞中引起下游信号传导。为了 终止G蛋白信号传导，细胞设计了一种专门的脱敏机制，其中包括接收器 GPCR激酶的磷酸化以及随后募集β-arrestin（βArrs）到磷酸化的 受体。 GPCR –βarr相互作用均可阻止受体核心的G蛋白结合位点，并促进 受体内吞作用。但是，最近，我们发现某些GPCR与βarr相互作用 通过其磷酸化的C末端尾巴。由于β不会阻止此“尾巴”中的G蛋白结合位点 构象，接收器可以与βArrs和G蛋白相关联，仅形成GPCR – G蛋白 - βarr“ Megaplexes”。这些巨珠的组装允许接收器继续刺激G蛋白 信号传导被βarr内部化为内体。那是核心和尾巴GPCR –βarr的存在 复杂构象表明βars充当G蛋白信号的时空主调节剂：何时 与接收器核心结合，βARR调节G蛋白信号的持续时间，而βARR控制细胞 从与接收器C末端尾部相关联时，从G蛋白中激活G蛋白的位置。作为 促进这两个复杂构象的基本特性仍然难以捉摸，我的研究目标 在接下来的5年中，涉及一般规模确定这些分子驱动力。我们的初步数据 表明位于接收器C末端尾巴内的磷酸化位点簇需要 与尾部构象中的βarr相关。因此，我们计划确定是否存在这些 磷酸化位点簇与GPCR的能力相关，从而从事导致的机制 持续的内体G蛋白信号传导。关于GPCR –βarr核心构象，Fingerloop结构域 βARR的（FLD）通过其疏水尖端和接收器插入大多数GPCR的跨膜核心 特定残差。为了在一般规模上表征这种相互作用，我们将检查这些受体是否是否 特定的βarr-fld残基与不同GPCR的G蛋白亚型偶联相关。最后，βarrrs调制 磷酸二酯酶（PDES）的活性终止GS-cAMP信号传导。但是，我们的初步数据 提出了这种调制的可能性，该调制是由核心构象中的βarr专门发生的。因此，我们会的 采用细胞生物学，生化和蛋白质组学方法的组合来检查是否调节 PDE和其他脱敏机制的特征是由不同的βarr构象专门介导的。我的视野 通过该研究计划，阐明了GPCR信号如何由βarrs的空间调节，这 可能会导致分化的身体反应。这里获取的知识将用于设计新的知识和 专门针对GPCR的创新理论。