RESUMEN
G protein-coupled receptors (GPCRs) have been found to trigger G protein-independent signalling. However, the regulation of G protein-independent pathways, especially their desensitization, is poorly characterized. Here, we show that the G protein-independent 5-HT(4) receptor (5-HT(4)R)-operated Src/ERK (extracellular signal-regulated kinase) pathway, but not the G(s) pathway, is inhibited by GPCR kinase 5 (GRK5), physically associated with the proximal region of receptor' C-terminus in both human embryonic kidney (HEK)-293 cells and colliculi neurons. This inhibition required two sequences of events: the association of beta-arrestin1 to a phosphorylated serine/threonine cluster located within the receptor C-t domain and the phosphorylation, by GRK5, of beta-arrestin1 (at Ser(412)) bound to the receptor. Phosphorylated beta-arrestin1 in turn prevented activation of Src constitutively bound to 5-HT(4)Rs, a necessary step in receptor-stimulated ERK signalling. This is the first demonstration that beta-arrestin1 phosphorylation by GRK5 regulates G protein-independent signalling.
Asunto(s)
Arrestinas/biosíntesis , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Receptores de Serotonina 5-HT4/metabolismo , Línea Celular , Proteínas de Unión al GTP/metabolismo , Humanos , Mutación , Neuronas/metabolismo , Péptidos/química , Fosforilación , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Serina/química , beta-Arrestinas , Familia-src Quinasas/metabolismoRESUMEN
Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.
Asunto(s)
Anticuerpos Neutralizantes , Preparaciones Farmacéuticas , Tolerancia a MedicamentosRESUMEN
Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by ß-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of ß-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the µ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs.
Asunto(s)
Complejo 2 de Proteína Adaptadora/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Señales de Clasificación de Proteína , Receptor PAR-1/química , Receptor PAR-1/metabolismo , Ubiquitinación , Proteínas Adaptadoras del Transporte Vesicular/química , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células HEK293 , Células HeLa , Humanos , Datos de Secuencia Molecular , Fosforilación , Transporte de ProteínasRESUMEN
During biotherapeutic drug development, immunogenicity is evaluated by measuring anti-drug antibodies (ADAs). The presence and magnitude of ADA responses is assessed using a multi-tier workflow where samples are screened, confirmed, and titered. Recent reports suggest that the assay signal to noise ratio (S/N) obtained during the screening tier correlates well with titer. To determine whether S/N could more broadly replace titer, anonymized ADA data from a consortium of sponsors was collected and analyzed. Datasets from clinical programs with therapeutics of varying immunogenicity risk levels (low to high), common ADA assay platforms (ELISA and MSD) and formats (bridging, direct, solid-phase extraction with acid dissociation), and titration approaches (endpoint and interpolated) were included in the analysis. A statistically significant correlation between S/N and titer was observed in all datasets, with a strong correlation (Spearman's r > 0.8) in 11 out of 15 assays (73%). For assays with available data, conclusions regarding ADA impact on pharmacokinetics and pharmacodynamics were similar using S/N or titer. Subject ADA kinetic profiles were also comparable using the two measurements. Determination of antibody boosting in patients with pre-existing responses could be accomplished using similar approaches for titer and S/N. Investigation of factors that impacted the accuracy of ADA magnitude measurements revealed advantages and disadvantages to both approaches. In general, S/N had superior precision and ability to detect potentially low affinity/avidity responses compared to titer. This analysis indicates that S/N could serve as an equivalent and in some cases preferable alternative to titer for assessing ADA magnitude and evaluation of impact on clinical responses.
Asunto(s)
Anticuerpos , Ensayo de Inmunoadsorción Enzimática , HumanosRESUMEN
A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.
Asunto(s)
Anticuerpos , Anticuerpos NeutralizantesRESUMEN
Regulation of the magnitude, duration, and localization of G protein-coupled receptor (GPCR) signaling responses is controlled by desensitization, internalization, and downregulation of the activated receptor. Desensitization is initiated by the phosphorylation of the activated receptor by GPCR kinases (GRKs) and the binding of the adaptor protein arrestin. In addition to phosphorylating activated GPCRs, GRKs have been shown to phosphorylate a variety of additional substrates. An in vitro screen for novel GRK substrates revealed Hsp70 interacting protein (Hip) as a substrate. GRK5, but not GRK2, bound to and stoichiometrically phosphorylated Hip in vitro. The primary binding domain of GRK5 was mapped to residues 303-319 on Hip, while the major site of phosphorylation was identified to be Ser-346. GRK5 also bound to and phosphorylated Hip on Ser-346 in cells. While Hip was previously implicated in chemokine receptor trafficking, we found that the phosphorylation of Ser-346 was required for proper agonist-induced internalization of the chemokine receptor CXCR4. Taken together, Hip has been identified as a novel substrate of GRK5 in vitro and in cells, and phosphorylation of Hip by GRK5 plays a role in modulating CXCR4 internalization.
Asunto(s)
Proteínas Portadoras/metabolismo , Endocitosis , Receptores CXCR4/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Bases , Línea Celular , Quinasa 5 del Receptor Acoplado a Proteína-G , Humanos , Inmunoprecipitación , Fosforilación , ARN Interferente Pequeño , Serina/metabolismo , Especificidad por SustratoRESUMEN
G protein-coupled receptor kinases (GRKs) specifically phosphorylate activated G protein-coupled receptors. While the X-ray crystal structures of several GRKs have been determined, the mechanism of interaction of GRK with GPCRs is currently unknown. To further characterize the role of the GRK2 amino terminus in receptor interaction and phosphorylation, we generated a series of point mutations within the first 10 amino acids of GRK2 and tested their ability to phosphorylate receptor and nonreceptor substrates. Although all mutants exhibited some impairment in receptor phosphorylation, three of the mutants, D3K, L4A, and D10A, were the most severely affected. Using the beta2-adrenergic receptor and rhodopsin as receptor substrates and tubulin as a nonreceptor substrate, we demonstrated that the kinase activity toward the receptors was severely decreased in the mutants, while they fully retained their ability to phosphorylate tubulin. Moreover, the amino-terminal mutants were able to bind to the receptor but, in contrast to wild-type GRK2, were not activated by receptor binding. A synthetic peptide containing residues 1-14 of GRK2 served as a noncompetitive inhibitor of receptor phosphorylation by GRK2, while a comparable peptide from GRK5 had no effect on GRK2 activity. Secondary structure prediction and circular dichroism suggest that the GRK2 amino-terminal peptide forms an amphipathic alpha-helix. Taken together, we propose a mechanism whereby the extreme amino terminus of GRK2 forms an intramolecular interaction that selectively enhances the catalytic activity of the kinase toward receptor substrates.