Subtype selective fluorescent ligands based on ICI 118,551 to study the human β2‐adrenoceptor in CRISPR/Cas9 genome‐edited HEK293T cells at low expression levels

Fluorescent ligand technologies have proved to be powerful tools to improve our understanding of ligand-receptor interactions. Here we have characterized a small focused library of nine fluorescent ligands based on the highly selective β2-adrenoceptor (β2AR) antagonist ICI 118,551. The majority of fluorescent ICI 118,551 analogs had good affinity for the β2AR (pKD >7.0) with good selectivity over the β1AR (pK

The use of fluorescence correlation spectroscopy to monitor cell surface β2‐adrenoceptors at low expression levels in human embryonic stem cell‐derived cardiomyocytes and fibroblasts

The importance of cell phenotype in determining the molecular mechanisms underlying β2- adrenoceptor (β2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged β2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged b2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface b2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of embryonic stem cell technology in combination with FCS allowed us to demonstrate that cell surface b2ARs internalise in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalisation is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting b2AR agonists

Application of BRET to monitor ligand binding to GPCRs

Bioluminescence resonance energy transfer (BRET) is a well-established method for investigating protein-protein interactions. Here we present a BRET approach to monitor ligand binding to G protein–coupled receptors (GPCRs) on the surface of living cells made possible by the use of fluorescent ligands in combination with a bioluminescent protein (NanoLuc) that can be readily expressed on the N terminus of GPCRs

Small molecule fluorescent ligands for the CXCR4 chemokine receptor

The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small molecule-based fluorescent probes were designed and synthesized based on previously reported small molecule antagonists. Here we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (pKD ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor

CRISPR/Cas9-mediated generation and analysis of N terminus polymorphic models of β2AR in isogenic hPSC-derived cardiomyocytes

© 2020 During normal- and patho-physiological situations, the behavior of the beta2-adrenoreceptor (β2AR) is influenced by polymorphic variants. The functional impact of such polymorphisms has been suggested from data derived from genetic association studies, in vitro experiments with primary cells, and transgenic overexpression models. However, heterogeneous genetic background and non-physiological transgene expression levels confound interpretation, leading to conflicting mechanistic conclusions. To overcome these limitations, we used CRISPR/Cas9 gene editing technology in human pluripotent stem cells (hPSCs) to create a unique suite of four isogenic homozygous variants at amino acid positions 16(G/R) and 27(G/Q), which reside in the N terminus of the β2AR. By producing cardiomyocytes from these hPSC lines, we determined that at a functional level β2AR signaling dominated over β1AR. Examining changes in beat rates and responses to isoprenaline, Gi coupling, cyclic AMP (cAMP) production, downregulation, and desensitization indicated that responses were often heightened for the GE variant, implying differential dominance of both polymorphic location and amino acid substitution. This finding was corroborated, since GE showed hypersensitivity to doxorubicin-induced cardiotoxicity relative to GQ and RQ variants. Thus, understanding the effect of β2AR polymorphisms on cardiac response to anticancer therapy may provide a route for personalized medicine and facilitate immediate clinical impact. During normal- and patho-physiological situations, the behavior of beta2-adrenoreceptor (β2AR) is determined by its polymorphic variants. A human-based isogenic model system represents a promising tool for systematic analysis of mechanisms of β2AR variant-mediated cellular response under normal and stressed conditions. This allows important subtleties of polymorphisms in β2AR to be unraveled

Plasma membrane preassociation drives β-arrestin coupling to receptors and activation

beta-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-b-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in b-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that b arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover,they indicate that, following receptor interaction, the plasma membrane stabilizes b-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of b-arrestin function at the plasma membrane, revealing a critical role for b-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation