The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

The glucagon-like peptide-1 receptor (GLP-1R), a key pharmacological target in type 2 diabetes (T2D) and obesity, undergoes rapid endocytosis after stimulation by endogenous and therapeutic agonists. We have previously highlighted the relevance of this process in fine-tuning GLP-1R responses in pancreatic beta cells to control insulin secretion. In the present study, we demonstrate an important role for the translocation of active GLP-1Rs into liquid-ordered plasma membrane nanodomains, which act as hotspots for optimal coordination of intracellular signaling and clathrin-mediated endocytosis. This process is dynamically regulated by agonist binding through palmitoylation of the GLP-1R at its carboxyl-terminal tail. Biased GLP-1R agonists and small molecule allosteric modulation both influence GLP-1R palmitoylation, clustering, nanodomain signaling, and internalization. Downstream effects on insulin secretion from pancreatic beta cells indicate that these processes are relevant to GLP-1R physiological actions and might be therapeutically targetable.

μ‐Opioid receptors (μ‐ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ‐ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ‐OR antagonist E‐p‐nitrocinnamoylamino‐dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single‐molecule imaging of μ‐ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ‐ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ‐ORs interact with each other to form short‐lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ‐OR pharmacology at single‐molecule level.

G protein-coupled receptors (GPCRs) regulate many cellular and physiological processes, responding to a diverse range of extracellular stimuli including hormones, neurotransmitters, odorants and light. Decades of biochemical and pharmacological studies have provided fundamental insights into the mechanisms of GPCR signaling. Thanks to recent advances in structural biology, we now possess an atomistic understanding of receptor activation and G protein coupling. However, how GPCRs and G proteins interact in living cells to confer signaling efficiency and specificity remains insufficiently understood. The development of advanced optical methods, including single-molecule microscopy, has provided the means to study receptors and G proteins in living cells with unprecedented spatio-temporal resolution. The results of these studies reveal an unexpected level of complexity, whereby GPCRs undergo transient interactions among themselves as well as with G proteins and structural elements of the plasma membrane to form short-lived signaling nanodomains that likely confer both rapidity and specificity to GPCR signaling. These findings may provide new strategies to pharmaceutically modulate GPCR function, which might eventually pave the way to innovative drugs for common diseases such as diabetes or heart failure.

17β-Estradiol (E2) treatment exerts rapid, nonclassical actions via intracellular signal transduction system in basal forebrain cholinergic (BFC) neurons in vivo. Here we examined the effect of E2 treatment on lesioned BFC neurons in ovariectomized mice and the role of E2-induced nonclassical action in this treatment. Mice given an N-methyl-d-aspartic acid (NMDA) injection into the substantia innominata-nucleus basalis magnocellularis complex (SI-NBM) exhibited cholinergic cell loss in the SI-NBM and ipsilateral cholinergic fiber loss in the cortex. A single injection of E2 after NMDA lesion did not have an effect on cholinergic cell loss in the SI-NBM, but it restored the ipsilateral cholinergic fiber density in the cortex in a time- and dose-dependent manner. The most effective cholinergic fiber restoration was observed with 33 ng/g E2 treatment at 1 h after NMDA lesion. The E2-induced cholinergic fiber restoration was absent in neuron-specific estrogen receptor-α knockout mice in vivo. Selective activation of nonclassical estrogen signaling in vivo by estren induced E2-like restorative actions. Selective blockade of the MAPK or protein kinase A pathway in vivo prevented E2's ability to restore cholinergic fiber loss. Finally, studies in intact female mice revealed an E2-induced restorative effect that was similar to that of E2-treated ovariectomized mice. These observations demonstrate that a single E2 treatment restores the BFC fiber loss in the cortex, regardless of endogenous E2 levels. They also reveal the critical role of nonclassical estrogen signaling via estrogen receptor-α and protein kinase A-MAPK pathways in E2-induced restorative action in the cholinergic system in vivo.

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Pituitary adenylate cyclase-activating polypeptide (PACAP) is neuroprotective in animal models of different brain pathologies and injuries, including cerebral ischemia, Parkinson's disease, and different types of retinal degenerations. We have previously shown that PACAP is protective against monosodium glutamate (MSG)-induced retinal degeneration, where PACAP-treated retinas has more retained structure and PACAP induces anti-apoptotic while it inhibits pro-apoptotic signaling pathways. The aim of the present study was to investigate cell-type specific effects of PACAP in MSG-induced retinal degeneration by means of immunohistochemistry. Rat pups received MSG (2 mg/g b.w.) applied on postnatal days 1, 5, and 9. PACAP (100 pmol in 5 microl saline) was injected into the right vitreous body, while the left eye received only saline. Retinas were processed for immunocytochemistry after 3 weeks. Immunolabeling was determined for vesicular glutamate transporter 1, tyrosine hydroxylase, calretinin, calbindin, parvalbumin, and vesicular gamma-aminobutyric acid (GABA) transporter. In the MSG-treated retinas, the cell bodies and processes in the inner nuclear, inner plexiform, and ganglion cell layers displayed less immunoreactivity for all antisera. Apart from photoreceptors, only one major retinal cell type examined in this study; the calbindin-immunoreactive horizontal cell seemed not to be affected by MSG application. After simultaneous application of MSG and PACAP, staining of retinas was similar to that of normal eyes, with no significant alterations in immunoreactive patterns. These findings further support the neuroprotective function of PACAP in MSG-induced retinal degeneration.

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