GLP1R and GIPR expression and signaling in pancreatic alpha cells, beta cells and delta cells.

Glucagon-like peptide-1 receptor (GLP1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR) are transmembrane receptors involved in insulin, glucagon and somatostatin secretion from the pancreatic islet. Therapeutic targeting of GLP1R and GIPR restores blood glucose levels in part by influencing beta cell, alpha cell and delta cell function. Despite the importance of the incretin-mimetics for diabetes therapy, our understanding of GLP1R and GIPR expression patterns and signaling within the islet remain incomplete. Here, we present the evidence for GLP1R and GIPR expression in the major islet cell types, before addressing signaling pathway(s) engaged, as well as their influence on cell survival and function. While GLP1R is largely a beta cell-specific marker within the islet, GIPR is expressed in alpha cells, beta cells, and (possibly) delta cells. GLP1R and GIPR engage Gs-coupled pathways in most settings, although the exact outcome on hormone release depends on paracrine communication and promiscuous signaling. Biased agonism away from beta-arrestin is an emerging concept for improving therapeutic efficacy, and is also relevant for GLP1R/GIPR dual agonism. Lastly, dual agonists exert multiple effects on islet function through GIPR > GLP1R imbalance, increased GLP1R surface expression and cAMP signaling, as well as beneficial alpha cell-beta cell-delta cell crosstalk.

Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging.

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.

Projection-Targeted Photopharmacology Reveals Distinct Anxiolytic Roles for Presynaptic mGluR2 in Prefrontal- and Insula-Amygdala Synapses.

Dissecting how membrane receptors regulate neural circuit function is critical for deciphering basic principles of neuromodulation and mechanisms of therapeutic drug action. Classical pharmacological and genetic approaches are not well-equipped to untangle the roles of specific receptor populations, especially in long-range projections which coordinate communication between brain regions. Here we use viral tracing, electrophysiological, optogenetic, and photopharmacological approaches to determine how presynaptic metabotropic glutamate receptor 2 (mGluR2) activation in the basolateral amygdala (BLA) alters anxiety-related behavior. We find that mGluR2-expressing neurons from the ventromedial prefrontal cortex (vmPFC) and posterior insular cortex (pIC) preferentially target distinct cell types and subregions of the BLA to regulate different forms of avoidant behavior. Using projection-specific photopharmacological activation, we find that mGluR2-mediated presynaptic inhibition of vmPFC-BLA, but not pIC-BLA, connections can produce long-lasting decreases in spatial avoidance. In contrast, presynaptic inhibition of pIC-BLA connections decreased social avoidance, novelty-induced hypophagia, and increased exploratory behavior without impairing working memory, establishing this projection as a novel target for the treatment of anxiety disorders. Overall, this work reveals new aspects of BLA neuromodulation with therapeutic implications while establishing a powerful approach for optical mapping of drug action via photopharmacology.

Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers.

The metabotropic glutamate receptors (mGluRs) are family C, dimeric G protein-coupled receptors (GPCRs), which play critical roles in synaptic transmission. Despite an increasing appreciation of the molecular diversity of this family, how distinct mGluR subtypes are regulated remains poorly understood. We reveal that different group II/III mGluR subtypes show markedly different beta-arrestin (ß-arr) coupling and endocytic trafficking. While mGluR2 is resistant to internalization and mGluR3 shows transient ß-arr coupling, which enables endocytosis and recycling, mGluR8 and ß-arr form stable complexes, which leads to efficient lysosomal targeting and degradation. Using chimeras and mutagenesis, we pinpoint carboxyl-terminal domain regions that control ß-arr coupling and trafficking, including the identification of an mGluR8 splice variant with impaired internalization. We then use a battery of high-resolution fluorescence assays to find that heterodimerization further expands the diversity of mGluR regulation. Together, this work provides insight into the relationship between GPCR/ß-arr complex formation and trafficking while revealing diversity and intricacy in the regulation of mGluRs.

Fine-tuned photochromic sulfonylureas for optical control of beta cell Ca2+ fluxes.

We previously developed, synthesized and tested light-activated sulfonylureas for optical control of KATP channels and pancreatic beta cell activity in vitro and in vivo. Such technology relies on installation of azobenzene photoswitches onto the sulfonylurea backbone, affording light-dependent isomerization, alteration in ligand affinity for SUR1 and hence KATP channel conductance. Inspired by molecular dynamics simulations and to further improve photoswitching characteristics, we set out to develop a novel push-pull closed ring azobenzene unit, before installing this on the sulfonylurea glimepiride as a small molecule recipient. Three fine-tuned, light-activated sulfonylureas were synthesized, encompassing azetidine, pyrrolidine and piperidine closed rings. Azetidine-, pyrrolidine- and piperidine-based sulfonylureas all increased beta cell Ca2+ -spiking activity upon continuous blue light illumination, similarly to first generation JB253. Notably, the pyrrolidine-based sulfonylurea showed superior switch OFF performance to JB253. As such, third generation sulfonylureas afford more precise optical control over primary pancreatic beta cells, and showcase the potential of pyrrolidine-azobenzenes as chemical photoswitches across drug classes.

Dense 4D nanoscale reconstruction of living brain tissue.

Three-dimensional (3D) reconstruction of living brain tissue down to an individual synapse level would create opportunities for decoding the dynamics and structure-function relationships of the brain's complex and dense information processing network; however, this has been hindered by insufficient 3D resolution, inadequate signal-to-noise ratio and prohibitive light burden in optical imaging, whereas electron microscopy is inherently static. Here we solved these challenges by developing an integrated optical/machine-learning technology, LIONESS (live information-optimized nanoscopy enabling saturated segmentation). This leverages optical modifications to stimulated emission depletion microscopy in comprehensively, extracellularly labeled tissue and previous information on sample structure via machine learning to simultaneously achieve isotropic super-resolution, high signal-to-noise ratio and compatibility with living tissue. This allows dense deep-learning-based instance segmentation and 3D reconstruction at a synapse level, incorporating molecular, activity and morphodynamic information. LIONESS opens up avenues for studying the dynamic functional (nano-)architecture of living brain tissue.

Hypothalamic and brainstem glucose-dependent insulinotropic polypeptide receptor neurons employ distinct mechanisms to affect feeding.

Central glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) signaling is critical in GIP-based therapeutics' ability to lower body weight, but pathways leveraged by GIPR pharmacology in the brain remain incompletely understood. We explored the role of Gipr neurons in the hypothalamus and dorsal vagal complex (DVC) - brain regions critical to the control of energy balance. Hypothalamic Gipr expression was not necessary for the synergistic effect of GIPR/GLP-1R coagonism on body weight. While chemogenetic stimulation of both hypothalamic and DVC Gipr neurons suppressed food intake, activation of DVC Gipr neurons reduced ambulatory activity and induced conditioned taste avoidance, while there was no effect of a short-acting GIPR agonist (GIPRA). Within the DVC, Gipr neurons of the nucleus tractus solitarius (NTS), but not the area postrema (AP), projected to distal brain regions and were transcriptomically distinct. Peripherally dosed fluorescent GIPRAs revealed that access was restricted to circumventricular organs in the CNS. These data demonstrate that Gipr neurons in the hypothalamus, AP, and NTS differ in their connectivity, transcriptomic profile, peripheral accessibility, and appetite-controlling mechanisms. These results highlight the heterogeneity of the central GIPR signaling axis and suggest that studies into the effects of GIP pharmacology on feeding behavior should consider the interplay of multiple regulatory pathways.

Real-Time Library Search Increases Cross-Link Identification Depth across All Levels of Sample Complexity.

Cross-linking mass spectrometry (XL-MS) is a universal tool for probing structural dynamics and protein-protein interactions in vitro and in vivo. Although cross-linked peptides are naturally less abundant than their unlinked counterparts, recent experimental advances improved cross-link identification by enriching the cross-linker-modified peptides chemically with the use of enrichable cross-linkers. However, mono-links (i.e., peptides modified with a hydrolyzed cross-linker) still hinder efficient cross-link identification since a large proportion of measurement time is spent on their MS2 acquisition. Currently, cross-links and mono-links cannot be separated by sample preparation techniques or chromatography because they are chemically almost identical. Here, we found that based on the intensity ratios of four diagnostic peaks when using PhoX/tBu-PhoX cross-linkers, cross-links and mono-links can be partially distinguished. Harnessing their characteristic intensity ratios for real-time library search (RTLS)-based triggering of high-resolution MS2 scans increased the number of cross-link identifications from both single protein samples and intact E. coli cells. Specifically, RTLS improves cross-link identification from unenriched samples and short gradients, emphasizing its advantages in high-throughput approaches and when instrument time or sample amount is limited.

Enhanced Endosomal Signaling and Desensitization of GLP-1R vs GIPR in Pancreatic Beta Cells.

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favored as a therapeutic target due to blunted GIPR responses in T2D patients and conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology, following the realization that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R/GIPR agonists with superior capacity for controlling blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here, we have directly compared surface expression, trafficking, and signaling characteristics of both incretin receptors in pancreatic beta cells to identify potential differences that might underlie distinct pharmacological responses associated with each receptor. Our results indicate increased cell surface levels, internalization, degradation, and endosomal vs plasma membrane activity for the GLP-1R, while the GIPR is instead associated with increased plasma membrane recycling, reduced desensitization, and enhanced downstream signal amplification. These differences might have potential implications for the capacity of each incretin receptor to control beta cell function.

A versatile Halo- and SNAP-tagged BMP/TGFß receptor library for quantification of cell surface ligand binding.

TGFßs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFß receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFß/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFß1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways.

Revealing the tissue-level complexity of endogenous glucagon-like peptide-1 receptor expression and signaling.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in glucose homeostasis and food intake. GLP1R agonists (GLP1RA) are widely used in the treatment of diabetes and obesity, yet visualizing the endogenous localization, organization and dynamics of a GPCR has so far remained out of reach. In the present study, we generate mice harboring an enzyme self-label genome-edited into the endogenous Glp1r locus. We also rationally design and test various fluorescent dyes, spanning cyan to far-red wavelengths, for labeling performance in tissue. By combining these technologies, we show that endogenous GLP1R can be specifically and sensitively detected in primary tissue using multiple colors. Longitudinal analysis of GLP1R dynamics reveals heterogeneous recruitment of neighboring cell subpopulations into signaling and trafficking, with differences observed between GLP1RA classes and dual agonists. At the nanoscopic level, GLP1Rs are found to possess higher organization, undergoing GLP1RA-dependent membrane diffusion. Together, these results show the utility of enzyme self-labels for visualization and interrogation of endogenous proteins, and provide insight into the biology of a class B GPCR in primary cells and tissue.

Enzyme self-label-bound ATTO700 in single-molecule and super-resolution microscopy.

Herein, we evaluate near-infrared ATTO700 as an acceptor in SNAP- and Halo-tag protein labelling for Förster Resonance Energy Transfer (FRET) by ensemble and single molecule measurements. Microscopy of cell surface proteins in live cells is perfomed including super-resolution stimulated emission by depletion (STED) nanoscopy.

Control of Gαq signaling dynamics and GPCR cross-talk by GRKs.

Numerous processes contribute to the regulation of G protein-coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gαq both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gαq-dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gαq-dependent GPCR regulation with wide-ranging pharmacological and physiological implications.

N-Methyl deuterated rhodamines for protein labelling in sensitive fluorescence microscopy.

Rhodamine fluorophores are setting benchmarks in fluorescence microscopy. Herein, we report the deuterium (d12) congeners of tetramethyl(silicon)rhodamine, obtained by isotopic labelling of the four methyl groups, show improved photophysical parameters (i.e. brightness, lifetimes) and reduced chemical bleaching. We explore this finding for SNAP- and Halo-tag labelling in live cells, and highlight enhanced properties in several applications, such as fluorescence activated cell sorting, fluorescence lifetime microscopy, stimulated emission depletion nanoscopy and single-molecule Förster-resonance energy transfer. We finally extend this idea to other dye families and envision deuteration as a generalizable concept to improve existing and to develop new chemical biology probes.

DFT-Guided Discovery of Ethynyl-Triazolyl-Phosphinates as Modular Electrophiles for Chemoselective Cysteine Bioconjugation and Profiling.

We report the density functional theory (DFT) guided discovery of ethynyl-triazolyl-phosphinates (ETPs) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using CuI -catalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer, we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynyl-phosphinate. ETP-reagents were used to obtain fluorescent peptide-conjugates for receptor labelling on live cells and a stable and a biologically active antibody-drug-conjugate. Moreover, we were able to incorporate ETP-electrophiles into an azide-containing ubiquitin under native conditions and demonstrate their potential in protein-protein conjugation. Finally, we showcase the excellent cysteine-selectivity of this new class of electrophile in mass spectrometry based, proteome-wide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules.

Photopharmacology for vision restoration.

Blinding diseases that are caused by degeneration of rod and cone photoreceptor cells often spare the rest of the retinal circuit, from bipolar cells, which are directly innervated by photoreceptor cells, to the output ganglion cells that project axons to the brain. A strategy for restoring vision is to introduce light sensitivity to the surviving cells of the retina. One approach is optogenetics, in which surviving cells are virally transfected with a gene encoding a signaling protein that becomes sensitive to light by binding to the biologically available chromophore retinal, the same chromophore that is used by the opsin photo-detectors of rods and cones. A second approach uses photopharmacology, in which a synthetic photoswitch associates with a native or engineered ion channel or receptor. We review these approaches and look ahead to the next generation of advances that could reconstitute core aspects of natural vision.

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs.

Expanded LUXendin Color Palette for GLP1R Detection and Visualization In Vitro and In Vivo.

The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissues. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615, and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as a chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows noninvasive imaging, highlighting differentially accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using wide-field microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insights into GLP1R biology.

14-3-3ζ Constrains insulin secretion by regulating mitochondrial function in pancreatic ß cells.

While critical for neurotransmitter synthesis, 14-3-3 proteins are often assumed to have redundant functions due to their ubiquitous expression, but despite this assumption, various 14-3-3 isoforms have been implicated in regulating metabolism. We previously reported contributions of 14-3-3ζ in ß cell function, but these studies were performed in tumor-derived MIN6 cells and systemic KO mice. To further characterize the regulatory roles of 14-3-3ζ in ß cell function, we generated ß cell-specific 14-3-3ζ-KO mice. Although no effects on ß cell mass were detected, potentiated glucose-stimulated insulin secretion (GSIS), mitochondrial function, and ATP synthesis were observed. Deletion of 14-3-3ζ also altered the ß cell transcriptome, as genes associated with mitochondrial respiration and oxidative phosphorylation were upregulated. Acute 14-3-3 protein inhibition in mouse and human islets recapitulated the enhancements in GSIS and mitochondrial function, suggesting that 14-3-3ζ is the critical isoform in ß cells. In dysfunctional db/db islets and human islets from type 2 diabetic donors, expression of Ywhaz/YWHAZ, the gene encoding 14-3-3ζ, was inversely associated with insulin secretion, and pan-14-3-3 protein inhibition led to enhanced GSIS and mitochondrial function. Taken together, this study demonstrates important regulatory functions of 14-3-3ζ in the regulation of ß cell function and provides a deeper understanding of how insulin secretion is controlled in ß cells.