ABSTRACT
The glucocorticoid receptor (GR) is a ligand-activated transcription factor that binds DNA and assembles co-regulator complexes to regulate gene transcription. GR agonists are widely prescribed to people with inflammatory and autoimmune diseases. Here we present high-resolution, multidomain structures of GR in complex with ligand, DNA and co-regulator peptide. The structures reveal how the receptor forms an asymmetric dimer on the DNA and provide a detailed view of the domain interactions within and across the two monomers. Hydrogen-deuterium exchange and DNA-binding experiments demonstrate that ligand-dependent structural changes are communicated across the different domains in the full-length receptor. This study demonstrates how GR forms a distinct architecture on DNA and how signal transmission can be modulated by the ligand pharmacophore, provides a platform to build a new level of understanding of how receptor modifications can drive disease progression and offers key insight for future drug design.
Subject(s)
Receptors, Glucocorticoid , Transcription Factors , Humans , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Ligands , Transcription Factors/metabolism , Gene Expression Regulation , DNA/metabolismABSTRACT
Proinsulin C-peptide has previously been proposed to interact with a G-protein coupled receptor (GPCR), specifically the orphan receptor GPR146. To investigate the potential of C-peptide in treating complications of diabetes, such as kidney damage, it is necessary to understand its mode of action. We used CHO-K1 cells expressing human GPR146 to study human and murine C-peptide in dynamic mass redistribution and GPCR ß-arrestin assays, as well as with fluorescence confocal microscopy. Neither assay revealed any significant intracellular response to C-peptide at concentrations of up to 33 µM. We observed no internalisation of C-peptide by fluorescence microscopy. Our results do not support GPR146 as the receptor for C-peptide, but suggest that further investigations of the mode of action of C-peptide should be undertaken.
Subject(s)
C-Peptide/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , CHO Cells , Cricetulus , HEK293 Cells , Humans , Mice , Protein BindingABSTRACT
The provision of high-quality eukaryotic cells through robust cell banking processes is essential for the progression of drug discovery projects throughout the pharmaceutical research process. Numerous models exist to meet this aim, and this review describes many of the underlying principles, challenges and opportunities as well as detailing how these have been addressed within AstraZeneca. Crucial aspects discussed include cell line acquisition, cell bank generation, cryopreservation, storage, tracking and distribution. Because quality assurance underpins much of the process, quality control (QC) testing including mycoplasma screening and cell line authentication are also discussed in detail. Furthermore, because many of the underlying principles of cell banking are applicable in non-pharmaceutical settings, it is hoped that this review will prove a useful resource across the wider scientific community.
