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Parallel Interrogation of β-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay
Published on: March 10, 2020
Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors
X Edward Zhou1, Yuanzheng He2, Parker W de Waal2
1VARI-SIMM Center, Center for Structure and Function of Drug Targets, CAS-Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; Laboratory of Structural Sciences, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, Grand Rapids, MI 49503, USA.
Abstract:
G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular β sheet with the N-terminal β strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to β-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.
Insights
Researchers discovered how phosphorylated G protein-coupled receptors (GPCRs) bind to arrestins. This finding reveals a common mechanism, termed phosphorylation codes, for arrestin recruitment by GPCRs, impacting cellular signaling.
Area of Science:
- Structural biology
- Molecular and cellular signaling
Background:
- G protein-coupled receptors (GPCRs) are crucial cell surface receptors involved in diverse physiological processes.
- Arrestins bind to GPCRs, mediating receptor desensitization, internalization, and G protein-independent signaling pathways.
- High-affinity arrestin binding necessitates prior GPCR phosphorylation, typically on the C-terminal tail.
Purpose of the Study:
- To elucidate the structural basis of the interaction between phosphorylated rhodopsin and arrestin.
- To identify the specific phosphorylation sites and their role in mediating arrestin binding.
- To propose a general mechanism for phosphorylation-dependent arrestin recruitment by GPCRs.
Main Methods:
- X-ray free electron laser (XFEL) crystallography was employed to determine the structure of the rhodopsin-arrestin complex.
- Biochemical analysis was used to identify and validate phosphorylation sites on the rhodopsin C-terminal tail.
- Structural data was analyzed to understand the electrostatic interactions governing the complex formation.
Main Results:
- The crystal structure revealed an extended intermolecular β-sheet formed between the phosphorylated rhodopsin C terminus and arrestin's N-terminal β strands.
- Phosphorylation at threonine 336 (T336) and serine 338 (S338) of rhodopsin was identified as critical for high-affinity binding.
- A network of electrostatic interactions between these phospho-residues and positively charged pockets in arrestin was observed, similar to other GPCR-arrestin interactions.
- A set of 'phosphorylation codes' was derived, explaining the common mechanism of arrestin recruitment.
Conclusions:
- The study provides a high-resolution structural insight into the phosphorylated rhodopsin-arrestin complex.
- Phosphorylation acts as a key determinant for high-affinity arrestin binding to GPCRs.
- The identified 'phosphorylation codes' represent a conserved mechanism for arrestin recruitment across various GPCRs, impacting cellular signaling pathways.
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