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Class II major histocompatibility complex peptide (MHC-IIp) multimers are precisely engineered reagents used to detect T cells specific for antigens from pathogens, tumors, and self-proteins. While the related Class I MHC/peptide (MHC-Ip) multimers are usually produced from subunits expressed in E. coli, most Class II MHC alleles cannot be produced in bacteria, and this has contributed to the perception that MHC-IIp reagents are harder to produce. Herein, we present a robust constitutive expression system for soluble biotinylated MHC-IIp proteins that uses stable lentiviral vector-transduced derivatives of HEK-293T cells. The expression design includes allele-specific peptide ligands tethered to the amino-terminus of the MHC-II β chain via a protease-cleavable linker. Following cleavage of the linker, HLA-DM is used to catalyze efficient peptide exchange, enabling high-throughput production of many distinct MHC-IIp complexes from a single production cell line. Peptide exchange is monitored using either of two label-free methods, native isoelectric focusing gel electrophoresis or matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of eluted peptides. Together, these methods produce MHC-IIp complexes that are highly homogeneous and that form the basis for excellent MHC-IIp multimer reagents. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lentivirus production and expression line creation Support Protocol 1: Six-well assay for estimation of production cell line yield Support Protocol 2: Universal ELISA for quantifying proteins with fused leucine zippers and His-tags Basic Protocol 2: Cultures for production of Class II MHC proteins Basic Protocol 3: Purification of Class II MHC proteins by anti-leucine zipper affinity chromatography Alternate Protocol 1: IMAC purification of His-tagged Class II MHC Support Protocol 3: Protein concentration measurements and adjustments Support Protocol 4: Polishing purification by anion-exchange chromatography Support Protocol 5: Estimating biotinylation percentage by streptavidin precipitation Basic Protocol 4: Peptide exchange Basic Protocol 5: Analysis of peptide exchange by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry Alternate Protocol 2: Native isoelectric focusing to validate MHC-II peptide loading Basic Protocol 6: Multimerization Basic Protocol 7: Staining cells with Class II MHC tetramers., (© 2021 Wiley Periodicals LLC.)

Melatonin is rhythmically secreted by both the pineal gland and retina in a circadian fashion, with its peak synthesis occurring during the night. Once synthesized, melatonin exerts its effects by binding to two specific G-protein coupled receptors-melatonin receptor type 1(MT1) and melatonin receptor type 2(MT2). Recent studies suggest the involvement of MT1 and MT2 in the regulation of glucose homeostasis; however the ability of melatonin signaling to impart timing cues on glucose metabolism remains poorly understood. Here we report that the removal of MT1 or MT2 in mice abolishes the daily rhythm in blood glucose levels. Interestingly, removal of melatonin receptors produced small effects on the rhythmic expression patterns of clock genes within skeletal muscle, liver, and adipose tissue. Taken together, our data suggest that the loss of the daily rhythm in blood glucose observed in MT1(-/-) and MT2(-/-) mice does not occur as a consequence of 'disrupted' clocks within insulin sensitive tissues. Finally our results highlight a diurnal contribution of melatonin receptor signaling in the daily regulation of blood glucose levels.

The formation of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) heteromers enables signaling diversification and holds great promise for improved drug selectivity. Most studies of these oligomerization events have been conducted in heterologous expression systems, and in vivo validation is lacking in most cases, thus questioning the physiological significance of GPCR heteromerization. The melatonin receptors MT1 and MT2 exist as homomers and heteromers when expressed in cultured cells. We showed that melatonin MT1/MT2 heteromers mediated the effect of melatonin on the light sensitivity of rod photoreceptors in mice. This effect of melatonin involved activation of the heteromer-specific phospholipase C and protein kinase C (PLC/PKC) pathway and was abolished in MT1(-/-) or MT2(-/-) mice, as well as in mice overexpressing a nonfunctional MT2 mutant that interfered with the formation of functional MT1/MT2 heteromers in photoreceptor cells. Not only does this study establish an essential role of melatonin receptor heteromers in retinal function, it also provides in vivo support for the physiological importance of GPCR heteromerization. Thus, the MT1/MT2 heteromer complex may provide a specific pharmacological target to improve photoreceptor function.

Complete congenital stationary night blindness (cCSNB) is a clinically and genetically heterogeneous group of retinal disorders characterized by nonprogressive impairment of night vision, absence of the electroretinogram (ERG) b-wave, and variable degrees of involvement of other visual functions. We report here that mutations in GPR179, encoding an orphan G protein receptor, underlie a form of autosomal-recessive cCSNB. The Gpr179(nob5/nob5) mouse model was initially discovered by the absence of the ERG b-wave, a component that reflects depolarizing bipolar cell (DBC) function. We performed genetic mapping, followed by next-generation sequencing of the critical region and detected a large transposon-like DNA insertion in Gpr179. The involvement of GPR179 in DBC function was confirmed in zebrafish and humans. Functional knockdown of gpr179 in zebrafish led to a marked reduction in the amplitude of the ERG b-wave. Candidate gene analysis of GPR179 in DNA extracted from patients with cCSNB identified GPR179-inactivating mutations in two patients. We developed an antibody against mouse GPR179, which robustly labeled DBC dendritic terminals in wild-type mice. This labeling colocalized with the expression of GRM6 and was absent in Gpr179(nob5/nob5) mutant mice. Our results demonstrate that GPR179 plays a critical role in DBC signal transduction and expands our understanding of the mechanisms that mediate normal rod vision., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Retinal melatonin is involved in the modulation of many important retinal functions. Our previous studies have shown that the viability of photoreceptors and ganglion cells is reduced during aging in mice that lack melatonin receptor type 1. This demonstrates that melatonin signaling is important for the survival of retinal neurons. In the present study, we investigate the effects of aging on photoreceptor physiology and retinal organization in CH3-f+/+ mice, a melatonin proficient mouse strain. Our data indicate that the amplitude of the a and b waves of the scotopic and photopic electroretinogram decreases with age. Moreover, the daily rhythm in the amplitude of the a- and b-waves is lost during the aging process. Similarly, the scotopic threshold response is significantly affected by aging, but only when it is measured during the night. Interestingly, the changes observed in the ERGs are not paralleled by relevant changes in retinal morphological features, and administration of exogenous melatonin does not affect the ERGs in C3H-f(+/+) at 12 months of age. This suggests that the responsiveness of the photoreceptors to exogenous melatonin is reduced during aging.

Purpose: The retinal pigment epithelium (RPE) plays an important role in the maintenance of the health and function of photoreceptors. Previous studies have shown that the RPE is also involved in the regulation of disc shedding, a process that is vital for photoreceptor health. This process has been shown to be under circadian control, although the mechanisms that control it are poorly understood. The aim of the present study was to investigate Period 2 (Per2) mRNA levels in the mouse RPE in vivo, and to determine whether the cultured RPE-choroid from PERIOD 2::LUCIFERASE (PER2::LUC) knockin mice expresses a circadian rhythm in bioluminescence., Methods: Per2 mRNA levels were measured using real-time quantitative RT-PCR, and bioluminescence was measured in PER2::LUC knockin mice using a Lumicycle®., Results: Per2 mRNA levels in the RPE-choroid show a clear circadian rhythm in vivo. A circadian rhythm in PER2::LUC bioluminescence was recorded from cultured RPE-choroid explants. Light exposure during the subjective night did not cause a circadian rhythm phase-shift of PER2::LUC bioluminescence. Finally, removal of the suprachiasmatic nuclei of the hypothalamus did not affect the bioluminescence circadian rhythm in the RPE-choroid., Conclusions: Our results demonstrate that the RPE-choroid contains a circadian clock, and the regulation of this circadian rhythm resides within the eye. These new data indicate that it may be useful to design studies with the aim of elucidating the molecular mechanisms responsible for the regulation of the rhythmic event in the RPE.

The incidence of obesity, insulin resistance, and type 2 diabetes (T2D) is increasing at an alarming rate worldwide. Emerging experimental evidence suggests that the hormone melatonin plays an important role in the regulation of glucose metabolisms. In this study, we report that removal of melatonin receptor type 1 (MT1) significantly impairs the ability of mice to metabolize glucose and such inability is probably due to an increased insulin resistance in these mice. Our data suggest that MT1 receptors are implicated in the pathogenesis of T2D and open the door for a detailed exploration on the mechanisms by which MT1 receptors signaling may affect glucose metabolism.

A clear demonstration of the role of melatonin and its receptors in specific retinal functions is lacking. The present study investigated the distribution of MT1 receptors within the retina, and the scotopic and photopic electroretinograms (ERG) and retinal morphology in wild-type (WT) and MT1 receptor-deficient mice. MT1 receptor transcripts were localized in photoreceptor cells and in some inner retinal neurons. A diurnal rhythm in the dark-adapted ERG responses was observed in WT mice, with higher a- and b-wave amplitudes at night, but this rhythm was absent in mice lacking MT1 receptors. Injection of melatonin during the day decreased the scotopic response threshold and the amplitude of the a- and b-waves in the WT mice, but not in the MT1(-/-) mice. The effects of MT1 receptor deficiency on retinal morphology was investigated at three different ages (3, 12, and 18 months). No differences between MT1(-/-) and WT mice were observed at 3 months of age, whereas at 12 months MT1(-/-) mice have a significant reduction in the number of photoreceptor nuclei in the outer nuclear layer compared with WT controls. No differences were observed in the number of cells in inner nuclear layer or in ganglion cells at 12 months of age. At 18 months, the loss of photoreceptor nuclei in the outer nuclear layer was further accentuated and the number of ganglion cells was also significantly lower than that of controls. These data demonstrate the functional significance of melatonin and MT1 receptors in the mammalian retina and create the basis for future studies on the therapeutic use of melatonin in retinal degeneration.

The receptor for avian sarcoma/leukosis virus subtype A (ASLV-A), Tva, is the simplest member of the low density lipoprotein receptor family containing a single ligand-binding repeat (LBR). Most LBRs contain a central Trp (Trp33 in Tva) that is important for ligand binding and, for the low density lipoprotein receptor, is associated with familial hypercholesterolemia. The Tva ligand-binding module contains a second Trp (Trp48) that is part of a DEW motif present in a subset of LBRs. Trp48 is important for ASLV-A infectivity. A soluble Tva (sTva) ligand-binding module is sufficient for ASLV-A infectivity. Tva interacts with the viral glycoprotein, and a soluble receptor-binding domain (SUA) binds sTva with picomolar affinity. We investigated whether Tva, a retroviral receptor, could behave as a classic LBR by assessing sTva interactions with the universal receptor-associated protein (RAP) and comparing these interactions with those between sTva and its viral ligand (SUA). To address the role of the two Trp residues in Tva function, we prepared sTva harboring mutations of Trp33, Trp48, or both and determined the binding kinetics with RAP and SUA. We found that sTva behaved as a "normal" receptor toward RAP, requiring both calcium and Trp33 for binding. However, sTva binding to SUA required neither calcium nor Trp33. Furthermore, sTva could bind both RAP and SUA simultaneously. These results show that the single LBR of Tva has two ligand-binding sites, raising the possibility that other LBRs may also.