Biology subjects

Wyatt, R. A.

Publications and source records attributed to Wyatt, R. A..

4 recordsLinked to original sources

bioRxiv2024

Identification of anti-resorptive GPCRs by high-content imaging in human osteoclasts

Osteoporosis diagnoses are increasing in the ageing population and although several treatments exist, these have several disadvantages, highlighting the need to identify new drug targets. G protein-coupled receptors (GPCRs) are transmembrane proteins whose surface expression and extracellular activation make them desirable drug targets. Our previous studies have identified 144 GPCR genes to be expressed in primary human osteoclasts, which could provide novel drug targets. The development of high-throughput assays to assess osteoclast activity would improve the efficiency at which we could assess the effect of GPCR activation on human bone cells and could be utilised for future compound screening. Here we assessed the utility of a high-content imaging (HCI) assay that measured cytoplasmic-to-nuclear translocation of the nuclear factor of activated T cells-1 (NFATc1), a transcription factor that is essential for osteoclast differentiation and resorptive activity. We first demonstrated that the HCI assay detected changes in NFATc1 nuclear translocation in human primary osteoclasts using GIPR as a positive control, then developed an automated analysis platform to assess NFATc1 in nuclei in an efficient and unbiased manner. We assessed six GPCRs simultaneously and identified four receptors (FFAR2, FFAR4, FPR1, GPR35) that reduced osteoclast activity. Bone resorption assays and measurements of TRAP activity verified that activation of these GPCRs reduced osteoclast activity, and that receptor-specific antagonists prevented these effects. These studies demonstrate that HCI of NFATc1 can accurately assess osteoclast activity in human cells, reducing observer bias and increasing efficiency of target detection for future osteoclast-targeted osteoporosis therapies.

cell biology↗

bioRxiv2024

Obesity-associated MRAP2 variants impair multiple MC4R-mediated signaling pathways

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed at hypothalamic neurons that has an important role in appetite suppression and food intake. Mutations in MC4R are the most common cause of monogenic obesity and can affect multiple signaling pathways including Gs-cAMP, Gq, ERK1/2, {beta}-arrestin recruitment, internalization and cell surface expression. The melanocortin-2 receptor accessory protein 2 (MRAP2), is a single-pass transmembrane protein that interacts with and regulates signaling by MC4R. Variants in MRAP2 have also been identified in overweight and obese individuals. However, functional studies that have only measured the effect of MRAP2 variants on MC4R-mediated cAMP signaling have produced inconsistent findings and most do not reduce MC4R function. Here we investigated the effect of twelve of these previously reported MRAP2 variants and showed that all variants that have been identified in overweight or obese individuals impair MC4R function. When expressed at equal concentrations, seven MRAP2 variants impaired MC4R-mediated cAMP signaling, while nine variants impaired IP3 signaling. Four mutations in the MRAP2 C-terminus affected internalization. MRAP2 variants had no effect on total or cell surface expression of either the MRAP2 or MC4R proteins. Structural models predicted that MRAP2 interacts with MC4R transmembrane helices 5 and 6, and mutations in two MRAP2 residues in putative contact sites impaired the ability of MRAP2 to facilitate MC4R signaling. In summary, our studies demonstrate that human MRAP2 variants associated with obesity impair multiple MC4R signaling pathways and that both Gs-cAMP and Gq-IP3 pathways should be assessed to determine variant pathogenicity.

molecular biology↗

bioRxiv2024

The MRAP2 accessory protein directly interacts with melanocortin-3 receptor to enhance signaling

The central melanocortin system links nutrition to energy expenditure, with melanocortin-4 receptor (MC4R) controlling appetite and food intake, and MC3R regulating timing of sexual maturation, rate of linear growth and lean mass accumulation. Melanocortin-2 receptor accessory protein-2 (MRAP2) is a single transmembrane protein that interacts with MC4R to potentiate its signalling, and human mutations in MRAP2 cause obesity. Previous studies have been unable to consistently show whether MRAP2 affects MC3R activity. Here we used single-molecule pull-down (SiMPull) to confirm that MC3R and MRAP2 interact in HEK293 cells. Analysis of fluorescent photobleaching steps showed that MC3R and MRAP2 readily form heterodimers most commonly with a 1:1 stoichiometry. Human single-nucleus and spatial transcriptomics show MRAP2 is co-expressed with MC3R in hypothalamic neurons with important roles in energy homeostasis and appetite control. Functional analyses showed MRAP2 enhances MC3R cAMP signalling, impairs {beta}-arrestin recruitment, and reduces internalization in HEK293 cells. Structural homology models revealed putative interactions between the two proteins and alanine mutagenesis of five MRAP2 and three MC3R transmembrane residues significantly reduced MRAP2 effects on MC3R signalling. Finally, we showed genetic variants in MRAP2 that have been identified in individuals that are overweight or obese prevent MRAP2s enhancement of MC3R-driven signalling. Thus, these studies reveal MRAP2 as an important regulator of MC3R function and provide further evidence for the crucial role of MRAP2 in energy homeostasis.

molecular biology↗

bioRxiv2022

GIP receptor reduces osteoclast activity and improves osteoblast survival by activating multiple signaling pathways

Bone is a dynamic tissue that is remodeled throughout life by bone resorbing osteoclasts and bone forming osteoblasts, to adapt to physiological or mechanical demands. These processes are impaired in osteoporosis, and understanding how bone remodeling is regulated could improve anti-osteoporotic treatments. Clinical investigations show that short-term treatment with glucose-dependent insulinotropic polypeptide (GIP) acutely decreases serum markers of bone resorption and may increase bone formation. However, evidence for direct effects of GIP intracellular signaling and functions in mature human osteoclasts and osteoblasts have not been investigated. We report that the GIP receptor (GIPR) is robustly expressed in mature human osteoclasts. Exposure of osteoclasts to GIP inhibits osteoclastogenesis, delays bone resorption, and increases osteoclast apoptosis by acting upon multiple signaling pathways (cAMP, Src, Akt, calcium, p38) to impair nuclear translocation of nuclear factor of activated T cells 1 (NFATc1) and nuclear factor-{kappa}B (NF{kappa}B). Human osteoblasts also express GIPR, and GIP improves osteoblast survival via cAMP and Akt-mediated pathways. GIP treatment of co-cultures of osteoclasts and osteoblasts also decreased bone resorption. Antagonizing GIPR with GIP(3-30)NH2 abolished the effects of GIP on osteoclasts and osteoblasts. This study demonstrates that GIP inhibits bone resorption and improves survival of human osteoblasts, which could increase bone mass and strength, supporting clinical investigations of the effect of GIP on bone. Moreover, this study demonstrates that GIPR agonism could be beneficial in the treatment of disorders of bone remodeling, such as osteoporosis. One-sentence SummaryGIP acts directly on bone cells to regulate bone remodeling

cell biology↗