Apelin receptor homodimer-oligomers revealed by single-molecule imaging and novel G protein-dependent signaling

The apelin receptor (APJ) belongs to family A of the G protein-coupled receptors (GPCRs) and is a potential pharmacotherapeutic target for heart failure, hypertension, and other cardiovascular diseases. There is evidence APJ heterodimerizes with other GPCRs; however, the existence of APJ homodimers and oligomers remains to be investigated. Here, we measured APJ monomer-homodimer-oligomer interconversion by monitoring APJ dynamically on cells and compared their proportions, spatial arrangement, and mobility using total internal reflection fluorescence microscopy, resonance energy transfer, and proximity biotinylation. In cells with <0.3 receptor particles/μm2, approximately 60% of APJ molecules were present as dimers or oligomers. APJ dimers were present on the cell surface in a dynamic equilibrium with constant formation and dissociation of receptor complexes. Furthermore, we applied interference peptides and MALDI-TOF mass spectrometry to confirm APJ homo-dimer and explore the dimer-interfaces. Peptides corresponding to transmembrane domain (TMD)1, 2, 3, and 4, but not TMD5, 6, and 7, disrupted APJ dimerization. APJ mutants in TMD1 and TMD2 also decreased bioluminescence resonance energy transfer of APJ dimer. APJ dimerization resulted in novel functional characteristics, such as a distinct G-protein binding profile and cell responses after agonist stimulation. Thus, dimerization may serve as a unique mechanism for fine-tuning APJ-mediated functions

Molecular Characterization and Expression Profile Analysis of Heat Shock Transcription Factors in Mungbean

Heat shock transcription factors (Hsfs) are essential elements in plant signal transduction pathways that mediate gene expression in response to various abiotic stresses. Mungbean (Vigna radiata) is an important crop worldwide. The emergence of a genome database now allows for functional analysis of mungbean genes. In this study, we dissect the mungbean Hsfs using genome-wide identification and expression profiles. We characterized a total of 24 VrHsf genes and classified them into three groups (A, B, and C) based on their phylogeny and conserved domain structures. All VrHsf genes exhibit highly conserved exon-intron organization, with two exons and one intron. In addition, all VrHsf proteins contain 16 distinct motifs. Chromosome location analysis revealed that VrHsf genes are located on 8 of the 11 mungbean chromosomes, and that seven duplicated gene pairs had formed among them. Moreover, transcription patterns of VrHsf genes varied in different tissues, indicating their different roles in plant growth and development. We identified multiple stress related cis-elements in VrHsf promoter regions 2 kb upstream of the translation initiation codons, and the expression of most VrHsf genes was altered under different stress conditions, suggesting their potential functions in stress resistance pathways. These molecular characterization and expression profile analyses of VrHsf genes provide essential information for further function investigation

Individual phosphorylation sites at the C-terminus of the apelin receptor play different roles in signal transduction

The apelin and Elabela proteins constitute a spatiotemporal double-ligand system that controls apelin receptor (APJ) signal transduction. Phosphorylation of multiple sites within the C-terminus of APJ is essential for the recruitment of β-arrestins. We sought to determine the precise mechanisms by which apelin and Elabela promote APJ phosphorylation, and to elucidate the influence of β-arrestin phosphorylation on G-protein-coupled receptor (GPCR)/β-arrestin-dependent signaling. We used techniques including mass spectrometry (MS), mutation analysis, and bioluminescence resonance energy transfer (BRET) to evaluate the role of phosphorylation sites in APJ-mediated G-protein-dependent and β-dependent signaling. Phosphorylation of APJ occurred at five serine residues in the C-terminal region (Ser335, Ser339, Ser345, Ser348 and Ser369). We also identified two phosphorylation sites in β-arrestin1 and three in β-arrestin2, including three previously identified residues (Ser412, Ser361, and Thr383) and two new sites, Tyr47 in β-arrestin1 and Tyr48 in β-arrestin2. APJ mutations did not affect the phosphorylation of β-arrestins, but it affects the β-arrestin signaling pathway, specifically Ser335 and Ser339. Mutation of Ser335 decreased the ability of the receptor to interact with β-arrestin1/2 and AP2, indicating that APJ affects the β-arrestin signaling pathway by stimulating Elabela. Mutation of Ser339 abolished the capability of the receptor to interact with GRK2 and β-arrestin1/2 upon stimulation with apelin-36, and disrupted receptor internalization and β-arrestin-dependent ERK1/2 activation. Five peptides act on distinct phosphorylation sites at the APJ C-terminus, differentially regulating APJ signal transduction and causing different biological effects. These findings may facilitate screening for drugs to treat cardiovascular and metabolic diseases

A method for identifying G protein-coupled receptor dimers and their interfaces

The G protein-coupled receptor (GPCR) dimer interface plays an important role in the formation and stabilization of the dimer. Therefore, identifying the potential receptor-receptor interface is an important part of studying GPCRs. Various strategies have been employed to study the GPCR dimer interface and explore its functional significance, but experimental methods lack robustness and calculations are laborious. Herein, we report a combined optimized experimental and calculation approach for identifying and structurally characterizing GPCR dimer interfaces, and constructing atomic resolution models. Using a transmembrane domain (TM) peptide containing a human immunodeficiency virus trans-acting transcriptional activator (HIV-TAT) protein transduction motif, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDITOF-MS), and bioluminescence resonance energy transfer (BRET), we successfully identified Apelin receptor (APJ)/Nociceptin receptor 1 (ORL1) and APJ/Vasopressin receptor 2 (V2R) heterodimer interfaces, and two key sites mediating dimerization. This method can identify dimer interfaces of GPCR homodimers and heterodimers

Detection Methods and Clinical Applications of Circulating Tumor Cells in Breast Cancer

Circulating Tumor Cells (CTCs) are cancer cells that split away from the primary tumor and appear in the circulatory system as singular units or clusters, which was first reported by Dr. Thomas Ashworth in 1869. CTCs migrate and implantation occurs at a new site, in a process commonly known as tumor metastasis. In the case of breast cancer, the tumor cells often migrate into locations such as the lungs, brain, and bones, even during the early stages, and this is a notable characteristic of breast cancer. Survival rates have increased significantly over the past few decades because of progress made in radiology and tissue biopsy, making early detection and diagnosis of breast cancer possible. However, liquid biopsy, particularly that involving the collection of CTCs, is a non-invasive method to detect tumor cells in the circulatory system, which can be easily isolated from human plasma, serum, and other body fluids. Compared to traditional tissue biopsies, fluid sample collection has the advantages of being readily available and more acceptable to the patient. It can also detect tumor cells in blood earlier and in smaller numbers, possibly allowing for diagnosis prior to any tumor detection using imaging methods. Because of the scarcity of CTCs circulating in blood vessels (only a few CTCs among billions of erythrocytes and leukocytes), thorough but accurate detection methods are particularly important for further clinical applications

A novel phosphorylation site on orexin receptor 1 regulating orexinA-induced GRK2-biased signaling

Drug discovery efforts targeting G protein–coupled receptors (GPCRs) have succeeded in developing multiple medications for treating various human diseases including cancer, metabolic disorders, and inflammatory disorders. These medications are broadly classified as either agonists or antagonists that respectively promote or inhibit receptor activation by endogenous stimuli. However, there has been a growing appreciation that GPCR biased signaling between G protein- and β-arrestin-dependent signaling in particular is a promising method for improving drug efficacy and therapy. Orexin receptor 1 (OX1R), a member of the GPCRs, is an important drug target in the central nervous system. In this study, we identified a novel regulatory phosphorylation site (Ser-262) on OX1R that abolished its capability to interact with GRK2, but did not affect its interaction with G proteins, GRK5, or β-arrestin1/2 activation, indicating that Ser-262 is a key amino acid for OX1R internalization that contributes to induction of GRK2-dependent biased signaling via orexin A. Our findings could potentially lead to the development of new drug targets for the prevention and treatment of insomnia, narcolepsy, and substance abuse, with fewer side effects than existing therapies

Time-restricted feeding improves metabolic and endocrine profiles in mice with polycystic ovary syndrome

ObjectivesPolycystic ovary syndrome (PCOS) is one of the most common endocrinopathy disorders in premenopausal women, which is characterized by hyperandrogenemia, anovulation, and polycystic ovarian morphology (PCOM). Time-restricted feeding (TRF) is a new intermittent restriction dietary pattern, which has been shown to have positive benefits on obesity and glycolipid metabolism disorders. We aimed to explore the effect of the feeding regimen (ad libitum vs. TRF) on the glycolipid metabolism and reproductive endocrine disorders in a PCOS mouse model.MethodsPCOS mouse model was induced by continuous subcutaneous administration of dihydrotestosterone for 21 days. Mice were fed a high-fat diet (HFD) for 8 weeks on an ad libitum or time- restricted diet (from 10:30 p.m. to 6:30 a.m.).ResultsCompared to control mice, PCOS mice that received TRF treatment had significantly lower body weight, reduced adiposity, lower area under the curve (AUC) of glucose response in the oral glucose tolerance test (OGTT), and lower AUC in the insulin tolerance test (ITT). TRF also ameliorated lipid metabolism, as shown by a reduction in plasma lipid profiles (triglycerides and cholesterol) and the triglyceride content in the liver of PCOS mice. In terms of reproduction, the plasma androgen level, plasma estrogen (E2) level, and luteinizing hormone (LH)/follicle stimulating hormone (FSH) ratio in PCOS mice were significantly reduced after 8 weeks of TRF treatment. In addition, ovarian histology showed that TRF inhibits cyst formation and promotes corpus luteum formation.ConclusionIn conclusion, TRF improved metabolic and endocrine profiles in mice with PCOS

5-HT recruits distinct neurocircuits to inhibit hunger-driven and non-hunger-driven feeding

Funding Information: The investigators were supported by grants from the NIH (R01DK114279, R01DK109934, and R21NS108091 to QT; R01ES027544 and R01DK111436 to ZS; R00DK107008 to PX; R01DK109194 and R56DK109194 to QW; P01DK113954, R01DK115761, R01DK117281, and R01DK125480 to YX; R01DK120858 to QT and YX; K01DK119471 to CW; and P20GM135002 to YH), USDA/CRIS (51000-064-01 S to YX and QW), American Diabetes Association (1-17-PDF-138 to YH, 7-13-JF-61 to QW, and 1-15-BS-184 to QT), American Heart Association awards (16POST27260254 to CW), the Pew Charitable Trust awards to QW (0026188), Baylor Collaborative Faculty Research Investment Program grants to QW, the Faculty Start-up grants from USDA/ ARS to QW, the Biotechnology and Biological Sciences Research Council (BB/ K001418/1 and BB/NO17838/1 to LKH), and the Medical Research Council (MC/PC/ 15077 to LKH). QW is the Pew Scholar of Biomedical Sciences and the Kavli Scholar. The anxiety tests (e.g., open-field test, light–dark test, and elevated plus maze test) were performed in the Mouse Neurobehavior Core, Baylor College of Medicine, which was supported by National Institutes of Health Grant No. P30HD024064. The Ad-iN/ WED virus was kindly provided by Dr. Martin Myers (University of Michigan). The AAV9-CBA-DIO-WGA-zsGreen virus was kindly provided by Dr. Richard Palmiter (University of Washington).Peer reviewedPublisher PD