RESUMO
G-protein-coupled receptors enable cells to recognize numerous external stimuli and to transmit corresponding signals across the plasma membrane to trigger appropriate cellular responses. Crystal structures of a number of these receptors have now been determined in inactive and active conformations bound to chemically and functionally distinct ligands. These crystal structures illustrate overall receptor organization and atomic details of ligand-receptor interactions.
Assuntos
Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidoresRESUMO
[This corrects the article DOI: 10.1155/2020/8844963.].
RESUMO
Beta-arrestins (ßarrs) critically mediate desensitization, endocytosis and signalling of G protein-coupled receptors (GPCRs), and they scaffold a large number of interaction partners. However, allosteric modulation of their scaffolding abilities and direct targeting of their interaction interfaces to modulate GPCR functions selectively have not been fully explored yet. Here we identified a series of synthetic antibody fragments (Fabs) against different conformations of ßarrs from phage display libraries. Several of these Fabs allosterically and selectively modulated the interaction of ßarrs with clathrin and ERK MAP kinase. Interestingly, one of these Fabs selectively disrupted ßarr-clathrin interaction, and when expressed as an intrabody, it robustly inhibited agonist-induced endocytosis of a broad set of GPCRs without affecting ERK MAP kinase activation. Our data therefore demonstrate the feasibility of selectively targeting ßarr interactions using intrabodies and provide a novel framework for fine-tuning GPCR functions with potential therapeutic implications.
Assuntos
Endocitose/efeitos dos fármacos , Fragmentos Fab das Imunoglobulinas , Biblioteca de Peptídeos , Receptores Acoplados a Proteínas G/metabolismo , Anticorpos de Cadeia Única , Clatrina/metabolismo , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/farmacologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacologiaRESUMO
G protein-coupled receptors (GPCRs) are the primary targets of drugs prescribed for many human pathophysiological conditions such as hypertension, allergies, schizophrenia, asthma, and various types of cancer. High-resolution structure determination of GPCRs has been a key focus area in GPCR biology to understand the basic mechanism of their activation and signaling and to materialize the long-standing dream of structure-based drug design on these versatile receptors. There has been tremendous effort at this front in the past two decades and it has culminated into crystal structures of 27 different receptors so far. The recent progress in crystallization and structure determination of GPCRs has been driven by innovation and cutting-edge developments at every step involved in the process of crystallization. Here, we present a step-by-step description of various steps involved in GPCR crystallization starting from recombinant expression to obtaining diffracting crystals. We also discuss the next frontiers in GPCR biology that are likely to be a primary focus for crystallography efforts in the next decade or so.