期刊
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
卷 143, 期 42, 页码 17566-17576出版社
AMER CHEMICAL SOC
DOI: 10.1021/jacs.1c07369
关键词
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资金
- National Key R&D Program of China [2017YFA0505200]
- National Natural Science Foundation of China [22022703, 22177108, 91753205, 81621002, 21621003]
- Science and Technological Fund of Anhui Province for Outstanding Youth [1808085J04]
- Beijing Advanced Innovation Center for Structural Biology
- Beijing Frontier Research Center for Biological Structure
- China Postdoctoral Science Foundation [2015M581075]
This study presents a practical synthesis method for differentially modified, full-length beta(2)AR receptors. The success in preparing a range of homogeneous samples with distinct modification patterns, including a full-length beta(2)AR bearing both monoubiquitination and octaphosphorylation modifications, provides valuable insights into the structure and interactions of beta(2)AR complexes.
The beta(2)-adrenergic receptor (beta(2)AR) is a G-proteincoupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. beta(2)AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length beta(2)AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length beta(2)AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length beta(2)ARs with distinct modification patterns, including a full-length beta(2)AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length beta(2)AR receptors, we found that different phosphorylation patterns mediate different interactions with beta-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between beta(2)AR and beta-arrestin1. Access to full-length beta(2)AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of beta(2)AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.
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