Biology subjects

Mathiesen, J. M.

Publications and source records attributed to Mathiesen, J. M..

7 recordsLinked to original sources

bioRxiv2024

Discovery and pharmacological characterization of nanobodies acting as potent positive allosteric modulators of the calcium-sensing receptor

The calcium-sensing receptor (CaSR) is responsible for sustaining a stable blood calcium concentration. Consequently, genetic and acquired changes in this G protein-coupled receptor can give rise to various calcium homeostasis disorders. Synthetic positive allosteric modulators targeting CaSR are currently used to treat hypercalcemia, but their usage is highly limited due to the high risk of severe hypocalcemia and gastrointestinal intolerance. In this study, we aimed to generate pharmacologically active CaSR-specific nanobodies that could be employed as a new generation of pharmacological tools to investigate the receptor function and potentially serve as a new drug modality for effective treatment of CaSR-related disorders. Nanobodies were generated by immunization of a llama with CHO cells recombinantly overexpressing a myc-epitope-tagged human CaSR. Following construction of a phage display library representing the repertoire of nanobody genes, nanobodies binding to the CaSR were isolated by FACS of whole HEK293 cells recombinantly overexpressing HA-epitope-tagged human CaSR. Based on sequence comparison, 37 nanobodies from 25 different sequence families were purified and subsequent characterized in vitro for modulation of CaSR signaling. The nanobodies were screened for agonist, as well as positive and negative allosteric modulators activity in in vitro cellular assays downstream of CaSR activation. We identified eight pharmacologically active nanobodies acting as positive allosteric modulators that could be divided into five main families based on their sequence identity. The most potent nanobody (Nb4) binding to the extracellular domain of CaSR was slightly more potent than the reference small molecule PAM NPS R-568. This study describes the discovery and pharmacological characterization of nanobodies acting as potent CaSR positive allosteric modulators. These nanobodies are a new class of pharmacological research tools for the CaSR, which potentially can be developed into new therapeutics in the treatment of CaSR-related disorders.

biochemistry↗

bioRxiv2023

Negative allosteric modulation of the μ-opioid receptor

The {micro}-opioid receptor ({micro}OR) is a well-established target for analgesia, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression, which have led to the present opioid overdose epidemic. {micro}OR negative allosteric modulators (NAMs) may serve as powerful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. We screened a large DNA-encoded chemical library against inactive {micro}OR, counter-screening with active, G-protein and agonist bound receptor to "steer" selections toward functional negative allosteric modulators. We discovered a NAM compound with high and selective enrichment to inactive {micro}OR; the molecule potently blocks the activity of orthosteric agonists and enhances the affinity of the key opioid overdose reversal molecule, naloxone. It accomplishes this by binding to a site on the extracellular vestibule proximal to naloxone, stabilizing a unique inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM perturbs orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone in vivo to inhibit morphine-induced antinociception, respiratory depression and conditioned place preference while minimizing withdrawal behaviors. Our results provide detailed structural insights into the mechanism of a negative allosteric modulator for the {micro}OR and demonstrate how it can be exploited in vivo.

biochemistry↗

bioRxiv2022

Negative allosteric modulation of the glucagon receptor by RAMP2

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The glucagon receptor (GCGR), a Family B GPCR responsible for maintenance of proper blood sugar levels, interacts with RAMP2, though the purpose and consequence of this interaction is poorly understood. Using a series of biochemical and cell-based assays, we show that RAMP2 interacts with and broadly inhibits GCGR-induced downstream signaling. Hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) demonstrates that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) as well as at a key region in the 6th transmembrane helix, while single-molecule fluorescence resonance energy transfer (smFRET) experiments show that this RAMP2-induced ECD disorder results in inhibition of active and intermediate states of the intracellular face of the receptor. Using cryo-electron microscopy (cryoEM), we determined the structure of the GCGR-Gs complex at 2.9 [A] resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner, yet the ECD of GCGR is indeed largely disordered in the presence of RAMP2. This disorder is accompanied by rearrangements of several key areas of the receptor, resulting in the formation of a likely unproductive complex. Together, our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

biochemistry↗

bioRxiv2022

Suitable use of FRET-based Biosensors for Quantitative Detection of GPCR Activation

Cyclic adenosine 3,5-monophosphate is an important second messenger molecule that regulates many downstream signaling pathways in cells. Detection of cAMP levels relies on screenings of cell lysates or the use of genetically encoded biosensors for detection in living cells. Genetically encoded biosensors are, however, primarily used for bioimaging and rarely in high-throughput screenings of potential drug candidates. Here, we describe a quantitative fluorescence-based imaging method based on measurements of single living cells. We used a genetically encoded Epac149 biosensor to investigate cAMP production in living cells following ligand stimulation. The study revealed a dependence of the measured cAMP levels on the expression level of the biosensor in transiently transfected cells. While the biosensor maintained linearity of the signal at high expression levels, the linearity of the biosensor was lost at lower expression levels due to a deficit of the biosensor compared to the maximum possible production of cAMP in the cells. This problem was circumvented by establishment of a stable cell line with constitutive expression of the biosensor. We established dose response curves by stimulation with the {beta}1-adrenergic receptor partial agonist denopamine and observed up to 1.48-fold difference in the cellular response as well as up to 4.27-fold difference in LogEC50 values between cells with insufficient and sufficient biosensor expression. Careful characterization and control of the biosensor expression is therefore important in order to conduct quantitative analysis of the cAMP production and it allows the use of genetically encoded biosensor to be applied in high-throughput screenings.

biophysics↗

bioRxiv2020

Structures of metabotropic GABAB receptor

GABA ({gamma}-aminobutyric acid) stimulation of the metabotropic GABAB receptor results in prolonged inhibition of neurotransmission that is central to brain physiology. GABAB belongs to the Family C of G protein-coupled receptors (GPCRs), which operate as dimers to relay synaptic neurotransmitter signals into a cellular response through the binding and activation of heterotrimeric G proteins. GABAB, however, is unique in its function as an obligate heterodimer in which agonist binding and G protein activation take place on distinct subunits. Here we show structures of heterodimeric and homodimeric full-length GABAB receptors. Complemented by cellular signaling assays and atomistic simulations, the structures reveal an essential role for the GABAB extracellular loop 2 (ECL2) in relaying structural transitions by ordering the linker connecting the extracellular ligand-binding domain to the transmembrane region. Furthermore, the ECL2 of both GABAB subunits caps and interacts with the hydrophilic head of a phospholipid occupying the extracellular half of the transmembrane domain, thereby providing a potentially crucial link between ligand binding and the receptor core that engages G protein. These results provide a starting framework to decipher mechanistic modes of signal transduction mediated by GABAB dimers and have important implications for rational drug design targeting these receptors.

neuroscience↗

bioRxiv2020

Dissecting the roles of GRK2 and GRK3 in μ-opioid receptor internalization and β-arrestin2 recruitment using CRISPR/Cas9-edited HEK293 cells

Most G protein-coupled receptors (GPCRs) recruit {beta}-arrestins and internalize upon agonist stimulation. For the -opioid receptor (-OR), this process has been linked to development of opioid tolerance. GPCR kinases (GRKs), particularly GRK2 and GRK3, have been shown to be important for -OR recruitment of {beta}-arrestin and internalization. However, the contribution of GRK2 and GRK3 to {beta}-arrestin recruitment and receptor internalization, remain to be determined in their complete absence. Using CRISPR/Cas9-mediated genome editing we established HEK293 cells with knockout of GRK2, GRK3 or both to dissect their individual contributions in {beta}-arrestin2 recruitment and -OR internalization upon stimulation with four different agonists. We showed that GRK2/3 removal reduced agonist-induced -OR internalization and {beta}-arrestin2 recruitment substantially and we found GRK2 to be more important for these processes than GRK3. Furthermore, we observed a sustained and GRK2/3 independent component of {beta}-arrestin2 recruitment to the plasma membrane upon -OR activation. Rescue expression experiments restored GRK2/3 functions. Inhibition of GRK2/3 using the small molecule inhibitor CMPD101 showed a high similarity between the genetic and pharmacological approaches, cross-validating the specificity of both. However, off-target effects were observed at high CMPD101 concentrations. These GRK2/3 KO cell lines should prove useful for a wide range of studies on GPCR function.

pharmacology and toxicology↗

bioRxiv2019

Structural insights into ligand efficacy and activation of the glucagon receptor

The glucagon receptor family comprises Class B G protein-coupled receptors (GPCRs) that play a crucial role in regulating blood sugar levels. Receptors of this family represent important therapeutic targets for the treatment of diabetes and obesity. Despite intensive structural studies, we only have a poor understanding of the mechanism of peptide hormone-induced Class B receptor activation. This process involves the formation of a sharp kink in transmembrane helix 6 that moves out to allow formation of the nucleotide-free G protein complex. Here, we present the cryo-EM structure of the glucagon receptor (GCGR), a prototypical Class B GPCR, in complex with an engineered soluble glucagon derivative and the heterotrimeric G-protein, Gs. Comparison with the previously determined crystal structures of GCGR bound to a partial agonist reveals a structural framework to explain the molecular basis of ligand efficacy that is further supported by mutagenesis data.

biophysics↗