G-protein-coupled serotonin receptor type 4 (5-HT(4)R) is a pharmacological target implicated in a variety of gastrointestinal and nervous system disorders. As for many other integral membrane proteins, structural and functional studies of this receptor could be facilitated by its heterologous overexpression in eukaryotic systems that can perform appropriate post-translational modifications (PTMs) on the protein. We previously reported the development of an expression system that employs rhodopsin's biosynthetic machinery in rod cells of the retina to express heterologous G-protein-coupled receptors (GPCRs) in a pharmacologically functional form.
View Article and Find Full Text PDFCrucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions.
View Article and Find Full Text PDFThe ability to combine primary hit identification assays with target profiling would significantly streamline the current drug discovery process. Working towards this end, we report here the development of a microarray-based ligand binding assay that supports multiplexed analysis of G protein-coupled receptor systems in a 96-well microplate format that is compatible with the equipment and infrastructure typical of high-throughput screening laboratories. A prototype microarray was generated by pin-printing seven different receptors within the wells of a specially coated glass-bottom microplate and assaying with a cocktail of fluorescent ligands.
View Article and Find Full Text PDFRecent breakthroughs in the solution of X-ray structures for G protein-coupled receptors (GPCRs) with diffusible ligands have employed extensively mutated or recombined receptor fusion proteins heterologously expressed in conventional in vitro cell-based systems. While these advances now show that crystallization of non-rhodopsin members of this superfamily can be accomplished, the use of radically modified proteins may limit the relevance of the derived structures for precision-guided drug design. To better enable the study of native GPCR structures, we report here efforts to engineer an in vivo expression system that harnesses the photoreceptor system of the retina to express heterologous GPCRs with native human sequences in a biochemically homogeneous and pharmacologically functional conformation.
View Article and Find Full Text PDFTraditional cell-based systems used to express integral membrane receptors have yet to produce protein samples of sufficient quality for structural study. Herein we report an in vivo method that harnesses the photoreceptor system of the retina to heterologously express G protein-coupled receptors in a biochemically homogeneous and pharmacologically functional conformation. As an example we show that the adenosine A1 receptor, when placed under the influence of the mouse opsin promoter and rhodopsin rod outer segment targeting sequence, localized to the photoreceptor cells of transgenic retina.
View Article and Find Full Text PDFBioorg Med Chem Lett
July 2002
The discovery of a new class of sulfonamide NPY Y5 receptor antagonists is described. Optimization of this series led to the identification of compounds with high affinity for the hY5 subtype and excellent selectivity over the other NPY receptor subtypes. The SAR for this series was examined and a model for understanding the ligand-receptor interactions was developed.
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