Biology subjects

Gorvin, C. M.

Publications and source records attributed to Gorvin, C. M..

10 recordsLinked to original sources

bioRxiv2026

Loss of phosphorylation sites required for β-arrestin recruitment in the calcium-sensing receptor cytoplasmic region may contribute to hypocalcemia

The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) with an important role in calcium homeostasis, activating mutations of which cause hypocalcemia. Despite the receptors clinical importance and evidence suggesting {beta}-arrestin-1/2 can modulate CaSR signaling, the residues within CaSR required for {beta}-arrestin recruitment and the role of {beta}-arrestin in desensitization, trafficking and signaling are ill defined. We confirmed that {beta}-arrestin-1 and {beta}-arrestin-2 are recruited to CaSR upon receptor activation. Deletion of the distal cytoplasmic region of CaSR, which replicates several mutations identified in individuals with hypocalcemia, reduced {beta}-arrestin-1/2 recruitment and enhanced receptor signaling. Examination of the receptor cytoplasmic region identified three regions of serine and threonine residues that resemble phosphorylation codes identified in other GPCRs. Mutation of each of these residues to alanine demonstrated one region between amino acids 1003-1011 is important for both {beta}-arrestin-1 and {beta}-arrestin-2 recruitment, while disruption of a second region between residues 976-981 impaired {beta}-arrestin-2 recruitment. Alanine mutagenesis of these residues also reduced CaSR signaling and impaired receptor internalization suggesting an important role in receptor desensitization. Three variants in the ClinVar dataset reported in disorders of calcium homeostasis were identified and two, T1006M and T1008P, were shown to reduce {beta}-arrestin recruitment and enhance CaSR signaling. Further analysis of the T1006M variant showed it reduced CaSR internalization. Thus, we have identified two regions within the CaSR cytoplasmic region that are important for {beta}-arrestin recruitment, receptor desensitization and internalization. Mutation of residues within these regions may represent another mechanism for the pathogenesis of hypocalcemia.

molecular biology↗

bioRxiv2025

MRAP2 potentiates GPCR signaling by conserved mechanisms that are disrupted by obesity-associated genetic variants

Accessory proteins such as members of the melanocortin-2 receptor accessory protein family (MRAP) have been described to interact with and regulate the signaling of diverse G protein-coupled receptors (GPCRs), however, surprisingly little is known about the mechanisms by which they mediate these effects. MRAP2 modifies signaling of three distinct GPCRs, melanocortin receptor 4 (MC4R), MC3R and the ghrelin receptor (GHSR), which each play essential roles in appetite regulation. Human mutations in MRAP2 cause obesity with hyperglycaemia and hypertension, suggesting that its regulation of GPCRs is critical for maintaining metabolic homeostasis. However, the nature of MRAP2/GPCR complexes and whether there are shared mechanisms for complex assembly, critical structural regions or consistent effects on receptor signaling and trafficking remains unknown. Here we showed all three GPCRs preferentially interact with MRAP2 as 1:1 complexes and that MRAP2 binding disrupts GPCR homodimerization. MRAP2 interacts with the same receptor transmembrane regions to promote GPCR signaling, and the accessory protein impairs {beta}-arrestin-2 recruitment to prolong signaling and delay internalization. Deletion of the cytoplasmic region of MRAP2 impairs GPCR signaling by modulating receptor constitutive activity. Genetic variants in MRAP2 associated with overweight or obesity modulate the constitutive activity of all three GPCRs. Thus, MRAP2 regulates GPCR function using shared molecular mechanisms and these studies provide further evidence of the importance of GHSR constitutive activity.

molecular biology↗

bioRxiv2025

G protein-coupled receptor 35 (GPR35) stimulation reduces osteoclast activity in primary human bone cells

G protein-coupled receptor 35 (GPR35) is an orphan receptor that is widely expressed in tissues including human osteoblasts and osteoclasts. Expression of the GPR35 gene and protein are downregulated in osteoporosis patients and in mouse models of the disease. Gpr35-knockout mice have reduced bone mass, while GPR35 agonism rescues bone loss in rodent osteoporosis models indicating that GPR35 has an important role in bone. Our previous studies demonstrated GPR35 is expressed in human osteoclasts, and we sought to determine the receptors function in these cells. We differentiated human peripheral blood mononuclear cells to mature osteoclasts and assessed effects of the GPR35 synthetic agonists, TCG1001 and Zaprinast on osteoclast activity and differentiation. Both agonists stimulated significant reductions in osteoclast bone resorption and TRAP activity, and downregulated expression of MMP9, a gene that regulates osteoclast bone resorption. These effects were prevented by pre-incubation of cells with a GPR35-specific antagonist. To understand GPR35 signaling pathways, we measured the phosphorylation of secondary messengers known to have important roles in osteoclast activity using AlphaLISA assays. Upon GPR35 stimulation, we observed reduced phosphorylation of cSrc, which stimulates actin ring formation necessary for bone resorption, and decreased phosphorylation of Akt, CREB and NF{kappa}B that drive transcription of genes required for bone resorption. Additionally, we used chemical inhibitors and siRNA knockdown to show that GPR35 couples to Gi/o and G12/13 to stimulate these signaling pathways. Finally, we compared the ability of GPR35 agonists to suppress osteoclast activity to that of current osteoporosis drugs, denosumab and alendronic acid, and showed TRAP activity was similar suppressed under all conditions. Our findings demonstrate that GPR35 has an important inhibitory role in human osteoclast activity and have defined the signaling pathways that drive these processes. GPR35 represents a promising novel target to reduce osteoclast activity that could be exploited for osteoporosis treatments. Lay summaryExpression of G protein-coupled receptor 35 (GPR35) is reduced in osteoporosis and Gpr35-knockout mice have reduced bone mass. Here we showed stimulation of GPR35 activates Gi/o and G12/13 signaling pathways to reduce bone resorption in human osteoclast. The anti-resorptive activity of GPR35 agonists was comparable to current osteoporosis drugs, denosumab and alendronic acid. Our findings demonstrate that GPR35 has an important inhibitory role in human osteoclast activity and have defined the signaling pathways that drive these processes. GPR35 represents a promising novel target to reduce osteoclast activity that could be exploited for osteoporosis treatments.

pharmacology and toxicology↗

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↗

bioRxiv2024

Dual agonism of sodium iodide symporter function in vivo

New approaches are urgently needed to enhance the radioiodide (RAI) ablation of aggressive and metastatic thyroid cancer. We recently discovered that valosin-containing protein inhibitors (VCPi) such as clotrimazole and disulfiram transiently block sodium iodide symporter (NIS) proteasomal degradation, hence promoting RAI uptake. However, poor bioavailability diminishes their potential impact in vivo. Following 3D modelling and iterative drug design we appraised 26 novel analogues of clotrimazole, as well as albumin nano-encapsulated copper-diethyldithiocarbamate [Cu(DDC)2-alb] - a stabilised reformulation of a disulfiram metabolite. While several clotrimazole analogues specifically increased RAI uptake, the greatest impact was observed with Cu(DDC)2-alb in thyroid cancer cells as well as human primary thyrocytes from patients with thyroid hyperplasia. NanoBRET assays revealed that Cu(DDC)2 enhanced the plasma membrane accumulation of NIS in living cells. In BALB/c mice, both intraperitoneal and intravenous administration of Cu(DDC)2-alb significantly enhanced thyroidal 99mTc-uptake. RNA-Seq revealed the surprising observation that Cu(DDC)2-alb induced key thyroid transcription factors. Accordingly, expression of PAX8 and NKX2.1 was upregulated in thyroid glands from drug treated mice, with NIS levels correlating closely to 99mTc-uptake. As Cu(DDC)2 inhibits the VCP cofactor NPL4, with VCP being critical to the proteostatic processing of NIS protein, we identify a new dual agonist of RAI uptake in vivo, with the potential to directly impact RAI therapy for patients with aggressive thyroid cancer.

cancer biology↗

bioRxiv2023

Dissecting endocytic mechanisms reveals new molecular targets to enhance sodium iodide symporter activity with clinical relevance to radioiodide therapy

The sodium/iodide symporter (NIS) frequently shows diminished plasma membrane (PM) targeting in differentiated thyroid cancer (DTC), resulting in suboptimal radioiodide (RAI) treatment and poor prognosis. The mechanisms which govern the endocytosis of NIS away from the PM are ill-defined. Here, we challenged the hypothesis that new mechanistic understanding of NIS endocytosis would facilitate prediction of patient outcomes and enable specific drug modulation of RAI uptake in vivo. Through mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake and NanoBRET, we identify an acidic dipeptide within the NIS C-terminus which mediates binding to the {sigma}2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, chloroquine treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. We propose that NIS internalisation is orchestrated by the interaction of a C-terminal diacidic motif with AP2{sigma}2, together with the proto-oncogene PBF acting via AP22. Given that NIS internalisation was specifically druggable in vivo, our data provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. SummaryWe delineate the role of endocytic genes in regulating NIS activity at the plasma membrane and highlight the potential for systemic targeting of endocytosis to enhance radioiodine effectiveness in radioiodine-refractory cancer cells.

cancer 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↗

bioRxiv2020

Ap2s1 mutation in mice causes familial hypocalciuric hypercalcemia type 3

Mutations of the adaptor protein-2 sigma subunit (AP2S1) gene which encodes AP2{sigma}2, a component of the ubiquitous AP2 heterotetrameric complex involved in endosomal trafficking of the calcium-sensing receptor (CaSR), cause familial hypocalciuric hypercalcemia type 3 (FHH3). FHH3 patients have heterozygous AP2S1 missense Arg15 mutations (p.Arg15Cys, p.Arg15His or p.Arg15Leu) with marked hypercalcemia and occasional hypophosphatemia and osteomalacia. To further characterise the phenotypic spectrum and calcitropic pathophysiology of FHH3, we used CRISPR/Cas9 genome editing to generate mice harboring the AP2S1 p.Arg15Leu mutation, which causes the most severe FHH3 phenotype. Heterozygous (Ap2s1+/L15) mice were viable, and had marked hypercalcemia, hypermagnesemia, hypophosphatemia, and increased plasma concentrations of parathyroid hormone, fibroblast growth factor 23 and alkaline phosphatase activity, but normal pro-collagen type 1 N-terminal pro-peptide and 1,25 dihydroxyvitamin D. Homozygous (Ap2s1L15/L15) mice invariably died perinatally. The AP2S1 p.Arg15Leu mutation impaired protein-protein interactions between AP2{sigma}2 and the other AP2 subunits, and the CaSR. Cinacalcet, a CaSR allosteric activator, ameliorated the hypercalcemia and elevated PTH concentrations, but not the diminished AP2{sigma}2-CaSR interaction. Thus, our studies have established a mouse model with a germline loss-of-function AP2S1 mutation that is representative for FHH3 in humans, and demonstrated that cinacalcet corrects the abnormalities of plasma calcium and PTH.

genetics↗