The WNT signaling pathway regulates cell proliferation and stem cell maintenance. Its sustained and inappropriate activation results in excessive cell division and cancer. WNT signaling is activated upon interaction of t...The WNT signaling pathway regulates cell proliferation and stem cell maintenance. Its sustained and inappropriate activation results in excessive cell division and cancer. WNT signaling is activated upon interaction of the soluble WNT ligand with the Frizzled (FZD) receptor. The RING E3 ubiquitin ligases RNF43 and ZNRF3 promote the ubiquitylation and internalization of FZD, thereby turning off WNT signaling, and their inactivation causes cancer. Here, we identified the determinants of ubiquitin transfer by ZNRF3 and RNF43 and report the structure of the RING domain from ZNRF3. We found that the RING domain was monomeric and that RING dimerization was not required for its ubiquitin ligase activity. However, the ectodomain of ZNRF3 dimerizes, and our data supported a model in which the cytoplasmic domains are in close proximity in cells and interact, even though RING dimerization was not required for ubiquitin transfer. Our studies provide a framework for understanding how the E3 ubiquitin ligase activity of ZNRF3 and RNF43 is regulated.
Individual G protein-coupled receptors (GPCRs) are present on many cell types and regulate multiple physiological responses, making it necessary to design ligands that are selective for the targeted GPCR in a tissue-sele...Individual G protein-coupled receptors (GPCRs) are present on many cell types and regulate multiple physiological responses, making it necessary to design ligands that are selective for the targeted GPCR in a tissue-selective manner. Here, we found ligand-selective effects of the GPCR free fatty acid receptor 2 (FFAR2) mediated through differential activation of G and G family proteins in various tissues. These effects were elucidated with ago-allosteric regulators of FFAR2 that bind to overlapping sites (compound 187 and AZ1729) and to a distinct site (4-CMTB) in tissues from mice expressing a hemagglutinin-tagged human FFAR2 DREADD (designer receptor exclusively activated by designer drugs). The G-activating ligand compound 187 stimulated the release of the gut hormones GLP-1 and PYY from colonic crypts and the secretion of insulin from pancreatic islets, effects that were prevented by the G-biased ligand AZ1729. Both compound 187 and AZ1729 promoted G-mediated neutrophil migration in bone marrow and antilipolytic effects in adipose tissue synergistically with an orthosteric DREADD agonist, whereas 4-CMTB was ineffective in both of these tissues. In contrast, 4-CMTB, alone or with the orthosteric agonist, produced G signaling-dependent effects in colonic crypts and pancreatic islets. These insights into the functional selectivity of allosteric ligands acting on the same GPCR may enable the development of physiologically specific therapeutics.
Fernández-Barrecheguren A, Fernández-Rego A, Fuentes-Cantos L
… +10 more, Pons T, Estrada BS, Iborra-Pernichi M, de la Esperanza MV, Ebrahimi T, Sagrega-Aparisi A, Cogliati S, Martínez-Martín N, Jiménez-Saiz R, Carrasco YR
To mount a robust T cell-dependent immune response, antigen-specific B lymphocytes require the stimulation of the transmembrane receptor CD40 through immune synapse formation with CD4 T follicular helper cells. CD40 stim...To mount a robust T cell-dependent immune response, antigen-specific B lymphocytes require the stimulation of the transmembrane receptor CD40 through immune synapse formation with CD4 T follicular helper cells. CD40 stimulates the activation of mammalian target of rapamycin complex 1 (mTORC1) and remodels mitochondria to meet the increased bioenergetic and anabolic demands of activated B cells. Here, we found that diacylglycerol kinase ζ (DGKζ) supported mTORC1 activation downstream of CD40 stimulation in mouse B cells. We showed that DGKζ was required for organellar translocation to the CD40-mediated immune synapse and for the recruitment of mTORC1 to lysosomes, the latter of which was necessary for mTORC1 activation and function. The production of phosphatidic acid by DGKζ was crucial for these processes. DGKζ B cells exhibited defects in protein biosynthesis, metabolite transporter expression, and cell cycle progression, together with dysregulation of the transcriptional network that determines B cell fate. To sustain their bioenergetic and metabolic demands, DGKζ B cells enhanced their mitochondrial function. Together, these effects of DGKζ loss led to decreases in germinal center responses and in the generation of long-lived plasma cells and memory B cells in mice. Thus, our data identify DGKζ as an essential mediator of CD40 functions in the B cell immune response.
The E3 ubiquitin ligase APC/C with its coactivator Cdh1 is generally considered to be a tumor suppressor, regulating genome stability and the G/S transition of the cell cycle. Cdh1 enhances the stability of immune-cell c...The E3 ubiquitin ligase APC/C with its coactivator Cdh1 is generally considered to be a tumor suppressor, regulating genome stability and the G/S transition of the cell cycle. Cdh1 enhances the stability of immune-cell checkpoint ligand PD-L1 to modulate adaptive immunity. Here, we explored its role in innate immune regulation and identified a noncanonical, degradation-independent function of Cdh1 in clear cell renal cell carcinoma (ccRCC) through its stabilization of the cytosolic double-stranded DNA (dsDNA) sensor STING. Protein levels of Cdh1 and STING were concurrently increased in ccRCC samples from patients, and Cdh1 depletion reduced the abundance and half-life of STING protein in ccRCC cell lines. Mechanistically, Cdh1 bound to the destruction-box degron motif of STING, which sterically prevented the binding of the E3 ubiquitin ligase SPOP, thereby protecting STING from degradation. Upon stimulation of STING with an agonist or dsDNA, Cdh1 bound to STING at the Golgi and increased its abundance and signaling activity. Pharmacologically inhibiting kinases that phosphorylate Cdh1 increased STING abundance and STING-mediated type 1 interferon signaling in ccRCC cells, presumably by promoting the formation of APC/C-Cdh1-STING complexes. These findings reveal a Cdh1-STING axis in ccRCC that might be therapeutically exploited to potentiate antitumor innate immunity.
Oxytocin-induced lipolysis in adipocytes in the lactating mammary gland ensures a high lipid content in milk.Oxytocin-induced lipolysis in adipocytes in the lactating mammary gland ensures a high lipid content in milk.
One-third of all nonodorant G protein-coupled receptors (GPCRs) in humans are orphans with no known ligands. Deorphanization of these GPCRs is hampered by the difficulty of producing purified receptors in their native co...One-third of all nonodorant G protein-coupled receptors (GPCRs) in humans are orphans with no known ligands. Deorphanization of these GPCRs is hampered by the difficulty of producing purified receptors in their native conformations and the lack of a proteome-wide, high-throughput screening approach. Here, we validated a generalizable strategy to deorphanize multipass membrane receptors in the human genome. We leveraged virion display technology to produce recombinant viruses that displayed the four orphan receptors in the GPRC5 family and used them as probes against a human proteome array to identify potential protein ligands. We focused on GPRC5B, a GPCR previously implicated in obesity and metabolic dysfunction. We found that the glycoprotein myeloid differentiation 1 (MD-1) was a ligand that selectively activated GPRC5B on adipocytes, leading to signaling mediated by Gα. The binding of MD-1 to GPRC5B on differentiated 3T3-L1 adipocytes stimulated lipolysis, an effect that was maximally induced by cell-cell contact with MD-1-expressing RAW 264.7 macrophages. Macrophage infiltration into adipose tissue is a common hallmark of obesity and is associated with dysregulated adipose tissue function, and our findings define a signaling axis that regulates lipolysis in a manner dependent on adipocyte-macrophage interactions.
The cyclin-dependent kinase CDK1 is a master regulator of cell cycle progression and the associated changes in cell shape. The biochemical functions of CDK1 have been primarily studied in cultured cells lacking adhesion...The cyclin-dependent kinase CDK1 is a master regulator of cell cycle progression and the associated changes in cell shape. The biochemical functions of CDK1 have been primarily studied in cultured cells lacking adhesion to their neighbors. Within epithelial layers, cells are tightly connected, and cell cycle-associated shape changes must occur without compromising epithelial barrier function. Here, we showed that a pool of CDK1 localized to cell-cell contacts in cultured epithelial cells and phosphorylated substrates at cell-cell junctions throughout the cell cycle. CDK1 substrates identified by proteomic analysis included various components of adherens junctions, tight junctions, and desmosomes, as well as proteins that link cell-cell adhesion complexes to the actomyosin cytoskeleton. CDK1 activity maintained the linear organization of cell-cell junctions and was essential for preserving the integrity of the epithelial barrier. These findings expand the role of CDK1 to the regulation of cell-cell adhesion, establishing that the machinery that governs the cell cycle also controls epithelial integrity.
Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a key negative regulator of T cell activation, acting with C-terminal Src kinase (Csk) to suppress early T cell receptor (TCR) signaling and maintain immune to...Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a key negative regulator of T cell activation, acting with C-terminal Src kinase (Csk) to suppress early T cell receptor (TCR) signaling and maintain immune tolerance. Given that the autoimmune disease-associated R620W variant alters T cell responses, we investigated the effects of PTPN22 on T cell activation. We identified a role for PTPN22 in modulating cytoskeletal dynamics at the immunological synapse in Jurkat cells through its interaction with proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1), a cytoskeletal adaptor protein that recruits actin nucleation-promoting factors, including WASp, to the TCR. PTPN22 deficiency or inhibition disrupted Arp2/3-dependent actin remodeling, leading to excessive central F-actin foci, PSTPIP1 mislocalization, and enhanced Ca signaling, especially under low-affinity stimulation of the TCR. Super-resolution DNA-PAINT analysis revealed that loss of PTPN22 promoted aberrant PSTPIP1-TCR nanoscale colocalization and increased TCR clustering. These findings uncover a PTPN22-PSTPIP1 signaling axis that is critical for regulating cytoskeletal remodeling and receptor organization, providing insights into T cell hyperactivation that may be relevant to autoimmune disease.
Osteoarthritis (OA) is a common chronic joint disease with limited treatment options. Cartilage degeneration in OA is driven by increased matrix catabolism and decreased matrix production and is characterized by chondroc...Osteoarthritis (OA) is a common chronic joint disease with limited treatment options. Cartilage degeneration in OA is driven by increased matrix catabolism and decreased matrix production and is characterized by chondrocyte hypertrophy. Aberrant activation of both β-catenin-dependent and β-catenin-independent Wnt signaling is implicated in OA progression and represents a potential therapeutic target. Here, we investigated the role of Tiki2, a membrane-tethered proteolytic inhibitor of Wnt ligands, in OA. Expression of was reduced in a mouse model of OA induced by joint destabilization, and expression of was reduced across multiple independent human OA transcriptomic datasets. haploinsufficiency in mice led to spontaneous cartilage degeneration, and loss of accelerated the progression of instability-induced OA. Tiki2 suppressed Wnt3a-induced β-catenin signaling and Wnt5a-induced β-catenin-independent signaling in chondrocytes and promoted anabolic gene expression to preserve cartilage integrity. Intra-articular overexpression of human TIKI2 attenuated OA-associated cartilage degeneration in mice. In vitro, Tiki2 inhibited hypertrophic differentiation in primary mouse chondrocytes and ATDC5 cells, and overexpression of TIKI2 promoted chondrogenic differentiation in primary mouse and human chondrocytes. Together, these results identify Tiki2 as a regulator of articular cartilage homeostasis and OA susceptibility and suggest that enhancing Tiki2-mediated Wnt inhibition may be a potential disease-modifying strategy for OA.
Mucosal-associated invariant T (MAIT) cells are a population of innate-like, unconventional T cells characterized by the expression of a semi-invariant, non-MHC-restricted T cell receptor (TCR), which play an important r...Mucosal-associated invariant T (MAIT) cells are a population of innate-like, unconventional T cells characterized by the expression of a semi-invariant, non-MHC-restricted T cell receptor (TCR), which play an important role in mediating innate immune responses to bacterial and viral pathogens. Emerging research continues to describe the many environmental signals that influence the types of responses elicited downstream of MAIT cell activation. In this Review, we highlight five key factors that determine the metabolic and functional responses of MAIT cells, including TCR engagement, costimulation, chemokine signaling, cytokine stimulation, and nutrient availability. We further define the importance of optimal nutrient availability as "signal 5" in promoting MAIT cell fitness and in governing their capacity to respond appropriately to challenge. In understanding the ways in which MAIT cells are influenced by their microenvironment, we can continue to identify the potential factors that drive dysregulated responses in hostile circumstances, enabling restoration of their protective nature in the context of infection or in the tumor microenvironment.
In PDAC, an intrinsic balance of Wnt-secreting and Wnt-receiving cells supports tumor growth.In PDAC, an intrinsic balance of Wnt-secreting and Wnt-receiving cells supports tumor growth.
Hashiuchi E, Inaba Y, Sugimoto H
… +20 more, Kimura K, Watanabe H, Kajino M, Asahara SI, Kobayashi M, Kikuchi O, Hayashi Y, Horike SI, Daikoku T, Mieda M, Sakurai T, Sakai M, Matsumoto M, Kitamura T, Sato M, Ravnskjaer K, Kasuga M, Tanida M, Kuroda S, Inoue H
The vagus nerve connects the brain and pancreas and enhances postprandial endocrine secretion from the pancreas through cholinergic signaling, such as increasing insulin release immediately after food intake in the cepha...The vagus nerve connects the brain and pancreas and enhances postprandial endocrine secretion from the pancreas through cholinergic signaling, such as increasing insulin release immediately after food intake in the cephalic-phase insulin response (CPIR). Here, we investigated how obesity affects vagal regulation of pancreatic endocrine function using designer receptors exclusively activated by designer drugs (DREADDs) to manipulate vagal activity. As expected, the plasma concentration of insulin was increased by vagal activation in mice expressing the excitatory DREADD hM3Dq (M3 mice) and decreased by vagal inactivation in mice expressing inhibitory DREADD hM4Di (M4 mice). However, vagal activation in M3 mice with diet-induced obesity did not elicit an early increase in insulin and instead produced a delayed insulin decrease. Mathematical modeling showed that plasma insulin dynamics in these mice were best explained by a model incorporating both the insulin-increasing and insulin-decreasing effects of the vagus nerve. Furthermore, the insulin-decreasing effect was mediated by nitric oxide (NO)-dependent, noncholinergic signaling and was enhanced in obesity. In obese M3 mice, vagal deficiency of neuronal NO synthase (nNOS) abolished the insulin-decreasing effect and restored insulin release after vagal activation. Vagal nNOS deficiency also enhanced insulin release after voluntary feeding, consistent with the CPIR. These findings suggest that vagal NO action inhibits postprandial insulin release, particularly in obesity.
Neurofibromatosis 1 (NF1) is an inherited tumor-predisposition syndrome in which mutations in the neurofibromin gene cause various dysfunctions in the nervous system, including pain that substantially diminishes quality...Neurofibromatosis 1 (NF1) is an inherited tumor-predisposition syndrome in which mutations in the neurofibromin gene cause various dysfunctions in the nervous system, including pain that substantially diminishes quality of life. Pain can be both tumor-dependent and tumor-independent and is challenging to manage therapeutically. Mice with homozygous loss of in Schwann cells exhibit pain before tumors form, and this pain is associated with increased gene expression of glial cell line-derived neurotrophic factor (GDNF). Here, we investigated the cause of increased GDNF production and its downstream target cells in a mouse model of NF1. We found that Schwann cells were the dominant source of GDNF in NF1 mice. Schwann cell-derived GDNF activated the receptor GFRα1 on high-threshold mechanoreceptors and polymodal C-fibers, which mediated mechanical hypersensitivity in mice. Treating the mice with pharmacological inhibitors of mitogen-activated protein kinase (MAPK) signaling reduced pain-like behaviors and the expression of GDNF at the mRNA and protein levels in Schwann cells. The findings provide insight into the signaling pathways that underlie tumor-independent pain in NF1 and identify a targetable pathway for therapeutic intervention.
Receptor tyrosine kinase (RTK) signaling drives cancer and is a validated therapeutic target. Modulators of RTK signaling can reveal mechanisms of oncogenesis and offer new therapeutic targets. Golgi phosphoprotein 3 (GO...Receptor tyrosine kinase (RTK) signaling drives cancer and is a validated therapeutic target. Modulators of RTK signaling can reveal mechanisms of oncogenesis and offer new therapeutic targets. Golgi phosphoprotein 3 (GOLPH3) is a Golgi-localized oncoprotein that promotes signaling downstream of mTOR. Here, examination of RTK signaling indicated that GOLPH3 acted at the level of the RTK and increased all downstream signaling. We found that GOLPH3 enhanced the delivery of RTKs to the plasma membrane. This role was shared with its binding partner myosin 18A (MYO18A) and depended on the interaction of GOLPH3 with MYO18A. The GOLPH3-MYO18A complex at the Golgi apparatus was required and rate-limiting for RTK signaling across the cell types and receptors assessed. Our findings provide insight into the relationship between the function of GOLPH3 at the Golgi and its role as a cancer driver, highlighting its potential as a therapeutic target in cancer.
An uncontrolled innate immune response to pathogens in the lung causes acute lung injury, in which the cytosolic double-stranded DNA (dsDNA) sensor, cyclic GMP-AMP synthase (cGAS), and its induction of interferon-β (IFN-...An uncontrolled innate immune response to pathogens in the lung causes acute lung injury, in which the cytosolic double-stranded DNA (dsDNA) sensor, cyclic GMP-AMP synthase (cGAS), and its induction of interferon-β (IFN-β) play a crucial role. Given that the vascular endothelium is a point of host contact for both environmental and blood-borne pathogens, we investigated the response of cGAS to dsDNA in endothelial cells in regulating innate immunity in the lung. We found that the phosphorylation of Tyr of cGAS facilitated an interaction between cGAS and activated myosin light chain 6 (MYL6), enabling cGAS condensation and activation and leading to mRNA expression. Furthermore, cGAS binding to dsDNA rapidly increased intracellular Ca in a manner requiring the ER Ca sensor STIM1 to activate MYL6 for cGAS condensation. In cGAS-deficient mice, expression of wild-type cGAS or a phosphomimetic cGAS mutant in the lung endothelium was sufficient to enhance signaling downstream of cGAS, induce neutrophil recruitment, and exacerbate lung damage in response to infection. Our findings suggest a role for phosphorylation of Tyr in cGAS in the endothelium as critical for inducing innate immune responses to pathogens and contributing to acute lung injury.
Insights into G protein-coupled regulation of brain circuitry may reveal new, better antidepressants.Insights into G protein-coupled regulation of brain circuitry may reveal new, better antidepressants.
In neurons, many membrane proteins that are synthesized in the cell body must be efficiently delivered to axons to regulate neuronal connectivity. Transcytosis is an atypical transport mode in which membrane proteins int...In neurons, many membrane proteins that are synthesized in the cell body must be efficiently delivered to axons to regulate neuronal connectivity. Transcytosis is an atypical transport mode in which membrane proteins internalized from soma membranes are transported to axons in an anterograde fashion. Here, we characterized the trafficking dynamics and mechanism of transcytosis of the receptor TrkA from the soma in response to nerve growth factor (NFG) signaling at the axon in mouse sympathetic neurons. Live imaging and electron microscopy of compartmentalized cultures revealed that soma surface-derived TrkA proteins underwent dynamic transport in axons, with changes in speed, direction, and the vesicular organelles that carried them as they moved from proximal to distal axon compartments. In mice, soma surface-labeled TrkA proteins were observed in sympathetic nerve terminals, demonstrating that transcytosis occurs in vivo. Transcytosed TrkA proteins were enriched at presynaptic varicosities, bouton-like structures that store and release neurotransmitters. Disrupting its transcytosis by introducing a point mutation into TrkA reduced the number and size of presynaptic sites and decreased synaptic transmission in vivo and in culture. These findings provide mechanistic insight into an atypical mode of receptor trafficking and demonstrate its physiological relevance in sympathetic neuron connectivity in mice.
Tumor necrosis factor (TNF) is a pivotal inflammatory cytokine, and it predominantly signals by binding to TNF receptor 1 (TNFR1), a type I single-spanning transmembrane protein that is thought to exist primarily as mono...Tumor necrosis factor (TNF) is a pivotal inflammatory cytokine, and it predominantly signals by binding to TNF receptor 1 (TNFR1), a type I single-spanning transmembrane protein that is thought to exist primarily as monomers and dimers. The binding of trimeric ligands induces the formation of signaling-competent trimers and higher-order oligomers that lead to full activation of downstream NF-κB and MAPK signaling pathways. Several TNFR superfamily members, including TNFR1, can form trimeric structures through their transmembrane domains (TMDs). For Fas and DR5, these structures support ligand-induced activation, and ectodomain (ECD) interactions prevent ligand-independent signaling. To explore the structures' role in TNFR1 activation, we replaced the native TNFR1 TMDs with natural and de novo designed versions that formed stable and specific structures of defined monomeric or oligomeric states. We found that none of these TMD variant receptors displayed defects in TNF-induced signaling in mouse fibroblasts, but some showed increased autoactivation in the absence of ligand, particularly where the engineered TMD sequence prevented self-association. Autoactivation depended on intracellular death domain interactions and was exacerbated by a disease-associated mutation but was unaffected by mutation in the preligand assembly domain. Our results demonstrate that, unlike for other TNFR family members, no specific oligomeric TMD structure is required for normal, ligand-induced activation of TNFR1, but self-association through the native TMD may instead act together with ECD interactions to help suppress ligand-free autoactivation.