Cellular Signalling

Considered a significant public health issue, the growing resistance to conventional antimalarials necessitates the identification of new targets for drug development. Given that G protein-coupled receptors (GPCRs) are readily druggable targets, we explored the cellular role and potential structure of a GPCR-like protein identified in the P. falciparum genome, serpentine receptor 12 (SR12). Alphafold structure analysis, coupled with molecular dynamics simulations of SR12, revealed structural similarities to the Golgi dynamics domain (GOLD)-seven-transmembrane helix protein family (GOST proteins). This family of proteins, which includes TMEM87A and the orphan GPCRs GPR180, GPR107, and GPR108, is involved in subcellular trafficking. Consistent with such a trafficking role, SR12 is mainly present in the secretory pathway when expressed in mammalian cells. Co-expression of SR12 with GPCRs PAR1 and M3R led to increased plasma membrane targeting of these receptors. SR12 expression in HEK293 cells conferred Gq-dependent calcium signaling in response to the protease activated receptor 1 (PAR1) agonist thrombin. This response was completely abrogated in cells genetically devoid of PARs (PAR KO cells), consistent with its functions as a chaperone-like protein, promoting receptor trafficking to the plasma membrane. Taken together, the data show that the Plasmodium falciparum SR12 promotes GPCR trafficking when expressed in mammalian cells. Although the physiological consequences of such activity remain to be determined, the finding revealed the presence of a GOST protein in the parasite genome.

1.G-protein coupled receptor 27 (GPR27) is part of the "Super Conserved Receptors Expressed in Brain" (SREB) family, alongside GPR85 and GPR173. While the endogenous ligands and functions of SREB receptors are still unknown, GPR27 has been implicated in insulin secretion and tumorigenesis. Here, we show that substituting GPR27s C-terminus domain with that of the {beta}1 adrenergic receptor ({beta}1AR) yields a chimera with {beta}1AR-like ligand selectivity and cellular functions. Interestingly, adrenergic ligands stimulation of GPR27 inhibited EGF-induced serum-responsive element (SRE) activation, independently of G-proteins and {beta}-arrestins, through dephosphorylation of c-Src and EGFR proteins. This unique response was exclusive to GPR27, as GPR85 and GPR173 showed no similar effects. These findings suggest that GPR27 is a receptor responding to adrenergic ligands to transinhibit EGFR through an atypical signaling mechanism.

Several vasoprotective functions of high-density lipoproteins (HDL) on the endothelium have been shown to depend on the presence of sphingosine-1-phosphate (S1P) receptors (S1PRs) as well as scavenger receptor class B type 1 (SR-B1). Interference with the presence of S1P or the activity of S1PR1 or S1PR3 mimics many effects seen by the interference with SR-B1. This raises the question on interactions between S1P receptors and SR-B1. We investigated the influence of S1PRs on SR-B1 expression in human aortic endothelial cells. Silencing or pharmacological inhibition of S1PR1 or S1PR3 down-regulated SCARB1 mRNA expression as well as SR-B1 protein abundance. RNA interference with S1PR1 or S1PR3 also decreased cellular association of 125I-HDL with HAECs. Further mechanistic studies showed that knockdown of S1PR1 or S1PR3 reduced SR-B1 protein by inducing its degradation through deceasing Akt activity. Moreover, silencing of S1PR1 or S1PR3 suppressed SCARB1 mRNA expression by decreasing cellular cAMP levels. In conclusion, we provide evidence for an as yet unappreciated interaction, namely the regulation of SR-B1 abundance by S1PRs on both transcriptional and post-translational levels, suggesting that interactions of S1PRs and SR-B1 regulate signaling functions of HDL as well as uptake of lipoproteins in endothelial cells.

Recently we reported that in human coronary artery endothelial cells, activation of the P2Y2 receptor (P2Y2R) induces up-regulation of tissue factor (TF), a vital initiator of the coagulation cascade. However, others have shown that monocyte TF is more critical than endothelial TF in provoking a pro-thrombotic state. Thus, we aimed to study whether monocytes express the P2Y2R, its role in controlling TF expression, and its relevance in vivo. RT-PCR and receptor activity assays revealed that among the eight P2Y nucleotide receptors, the P2Y2 subtype was selectively and functionally expressed in human monocytic THP-1 cells and primary monocytes. Stimulation of the cells by ATP or UTP dramatically increased TF protein expression, which was abolished by AR-C118925, a selective P2Y2R antagonist, or by siRNA silencing the P2Y2R. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA preceded with an increased TF pre-mRNA, indicating enhanced TF gene transcription. In addition, stimulation of the monocyte P2Y2R significantly activated ERK1/2, JNK, p38, and Akt, along with their downstream transcription factors including c-Jun, c-Fos, and ATF-2, whereas blocking these pathways respectively, all significantly suppressed P2Y2R-mediated TF expression. Furthermore, we found that LPS triggered ATP release and TF expression, the latter of which was suppressed by apyrase or P2Y2R blockage. Importantly, P2Y2R-null mice were more resistant than wild-type mice in response to a lethal dose of LPS, accompanied by much less TF expression in bone marrow cells. These findings demonstrate for the first time that the P2Y2R mediates TF expression in human monocytes through mechanisms involving ERK1/2, JNK, p38, and AKT, and that P2Y2R deletion protects the mice from endotoxemia-induced TF expression and death, highlighting monocyte P2Y2R may be a new drug target for the prevention and/or treatment of relevant thrombotic disease.

HRas is an important node that controls cellular signaling, proliferation, and differentiation. Mutants of HRas (e.g., the constitutively active HRas V12) can be oncogenic, and can also inhibit myoblast differentiation. The C-terminal cysteines of HRas (Cys181 and Cys184) serve as substrates for intra-cellular reversible palmitoylation and de-palmitoylation reactions, which control its subcellular distribution. The relationship between the C-terminal cysteines of HRas, its intracellular distribution, and its cellular activity has remained unclear. Understanding this relationship has important implications for targeting HRas in pathogenic states where it is activated. In this study, we show that a mutation in the C-terminal of HRas, C181S, is sufficient to cause increased levels of HRas V12 in the Golgi, decreased HRas V12-driven Akt and Erk signaling and reverse the ability of HRas V12 to inhibit myoblast differentiation. This demonstrates the importance of C-terminal cysteines in controlling HRas V12. It has been previously shown that Cys184 can also be irreversibly modified by an electrophilic prostaglandin lipid 15d-PGJ2. This lipid is released by senescent cells as a part of senescence-associated secretory phenotype (SASP). In this study, we show that 15d-PGJ2 is secreted by senescent myoblasts formed by treatment with Doxorubicin. We also show that 15d-PGJ2 causes decreased levels of HRas within Golgi, activates Erk signaling (but not Akt signaling), and inhibits differentiation of C2C12 myoblasts in an HRas Cys184-dependent fashion. Chemotherapeutics such as Doxorubicin drive senescence and loss of skeletal muscle homeostasis in cancer patients. This study suggests that targeting the senescence-derived synthesis of 15-PGJ2 might be a target to promote muscle homeostasis after chemotherapy.

Positive allosteric modulators for free fatty acid receptor 2 (FFA2R/GPR43), that affect receptor function through binding to two distinct allosteric binding sites, were used to determine the correlation between the responses induced in neutrophils by two distinct activation modes; FFA2R was activated either by the orthosteric agonist propionate or by a receptor transactivation mechanism that activated FFA2R from the cytosolic side of the neutrophil plasma membrane by signals generated by the neutrophil PAFR (receptor for platelet activating factor), P2Y2R (receptor for ATP), FPR1 (receptor for fMLF) and FPR2 (receptor for WKYMVM). We show that the transactivation signals that activate FFA2R in the absence of any orthosteric agonist were generated downstream of the signaling G protein that couple to PAFR and P2Y2R. This transactivation of allosterically modulated FFA2Rs, by signals generated by PAFR/P2Y2R, represents a novel mechanism by which a G protein coupled receptor can be activated. Weak correlations were obtained when the FFA2R activity was induced by the transactivation signals generated by PAFRs and P2Y2Rs were compared with the FFA2R activity induced by the orthosteric agonist propionate. Comparison of the responses for each allosteric modulator revealed that the ratio values, calculated from the peak values of the ATP and propionate responses, varied from 0.2 to 1. Depending on the allosteric modulator, the response induced by the two different mechanisms (orthosteric activation and receptor transactivation, respectively), was equal or the propionate response was more pronounced. Importantly, we conclude that FFA2R activation from outside (orthosteric activation) and inside (receptor cross-talk/transactivation) can be selectively affected by an allosteric FFA2R modulator. O_LIThe allosterically modulated FFA2R is transactivated by signals generated by other GPCRs. C_LIO_LIThe PAF and ATP receptors transactivate FFA2R from the cytosolic side of the membrane. C_LIO_LIThe mechanisms that regulates activation of FFA2R from outside and inside differ. C_LI

lymphangiogenesis as a process is colorectal cancer first metastasis via lymphatic vessels to proximal lymph nodes. The fuel metabolism in mitochondrial and support proliferation of lymphatic endothelial cells (LECs) remain elusive during lymphangiogenesis in tumor hypoxic microenvironment. Recent studies report that loss of SEMA3F critically contributes to lymphangiogenesis of the CRCs. Here, we silenced SEMA3F expression of CRCs and co-culture with hLECs, the tubulogenesis capacity and hLECs migration were escalated in the hypoxia, the hLECs mainly relied on fatty acid metabolism not aerobic glycolysis during lymphangiogenesis. SEMA3F-deficient CRCs up-regulated PMAKP expression and phosphorylation of hLECs, and activated its peroxisome proliferator-activated receptor (PPARs) and Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1a) facilitated their switched toward fatty acids (FA) catabolism. Furthermore, we observed that activation of the PGCI-PPAR lipid oxidation signaling pathway in hLECs was caused by the secretion of interleukin-6 by tumor cells.Taken together, this study indicates that CRCs with SEMA3F expression depletion significantly promotes lymphangiogenesis in hypoxia and faciliates the secretion of IL-6 in tumor cell, and activates mitochondria fatty acids oxidation (FAO) reaction in the hLECs by PGCI-PPAR signaling pathways to support its growth.

BackgroundIn order to explore the molecular mechanism of cardiomyocyte-dependent myocardial gene expression and cardiomyocyte differentiation in cardiac hypertrophy, and to provide new insights for cardiac hypertrophy. MethodsCardiac myocytes were isolated from day 1-3 Sprague-Dawley rat pups. Real time quantitative PCR, western blot and immunocytochemistry Assay were used to detect the expression and localization of related genes. CO-IP was used to detect direct protein interactions between Myocardin and STAT3. Luciferase reporter assay and chromatin immunoprecipitation were used to detect the binding of Myocardin to the promoter of a downstream target gene. Microinjection of zebrafish embryos was used to examine the effects of STAT3 and Myocardin interactions on cardiac development in vivo ResultsThe N-terminus of STAT3 directly binds to the basic domain of myocardin and inhibits the transcriptional activity of Myocardin-mediated cardiac-specific genes ANF and -actinin, thereby inhibiting their expression, and further inhibit myocardin-mediated cardiac hypertrophy in vivo. ConclusionsIn summary, our report states that signal transduction and transcriptional activation factor 3 (STAT3) are inhibitors of the major cardiac hypertrophic transcription factor Myocardiin, which is required for cardiomyocyte differentiation. The STAT3-cardiacin interaction identified nuclear hormone receptor-mediated and cardiac-specific gene-regulated convergence sites and suggested a possible mechanism for cardioprotective effects.

The TRPV4 cation channel, is expressed in a broad range of tissues where it participates in generation of Ca2+ signal and/or depolarization of membrane potential. Here, we identified post synaptic density protein 95 (PSD95) as an interacting protein of this epithelial Ca2+ channel using confocal microscopy analysis and immunological assay. Using co-immunoprecipitation assays, we demonstrated that PSD95 was part of the TRPV4 protein complex. PSD95 protein was specifically associated with the C-terminal tail of TRPV4 to form a complex. A TRPV4 tail deletion mutant ({Delta}DAPL871: 4d) exhibited a diminished capacity to bind PSD95. Confocal microscopy analysis suggested that apical localization of TRPV4 required PSD95-TRPV4 interaction. Our data clearly suggest that formation of a complex between TRPV4 and PSD95 can regulate TRPV4 membrane localization. Both TRPV4 Ca2+ channel and its autophagy activity of 4d were reduced by more than 80% compared to those of the TRPV4 wild type. Our observation suggests that PSD95-TRPV4 complex plays crucial roles in routing TRPV4 to the apical plasma membrane and maintaining its authentic Ca2+ channel and biological function. CapsuleO_ST_ABSBackgroundC_ST_ABSTRPV4 contain putative PDZ tail motif (DAPL871). ResultsDeletion of TRPV4 tail PDZ motif fails to interact with PSD95 PDZ III domain. ConclusionTRPV4 tail is an authentic PDZ motif to interact with PSD95. SignificanceInteraction between TRPV4 and PSD95 requires for its proper biological functions.

Proteinase activated receptors (PARs) are G protein-coupled receptors (GPCRs) activated by limited N-terminal proteolysis. A variety of proteolytic enzymes derived from the coagulation cascade and inflammatory milieu activate PARs, however specific activators in different physiological and pathophysiological contexts remain poorly defined. PARs are highly expressed in many cancer cells and regulate various aspects of tumor growth and metastasis. Endogenous proteinases that regulate PARs in the setting of various tumors however remains unresolved. Prostate cancer (PCa) remains a major cause of mortality in men despite advances in early detection and clinical intervention. PAR expression has been reported in PCa, however, their role here remains poorly defined. In androgen independent PC3 cells, we find functional expression of PAR1 and PAR2 but not PAR4. Using genetically encoded PAR cleavage biosensors, we find that PCa cells secrete proteolytic enzymes that cleave PARs and trigger autocrine signaling. Deletion of PAR1 and PAR2 using CRISPR/Cas9 combined with microarray analysis revealed genes that are differentially regulated by this autocrine signalling mechanism. Interestingly, several genes that are known PCa prognostic factors or biomarker were differentially expressed in PAR1-KO and PAR2-KO PC3 cells. We also examined PAR1 and PAR2 regulation of PCa cell proliferation and migration using PAR1 and PAR2-KO PC3 cells, as well as PAR1 and PAR2 specific agonists and antagonists. We find that PAR1 and PAR2 have opposite effects on PC3 cell proliferation and migration. In summary, we have identified an autocrine signaling mechanism through PARs as a regulator of PCa cell function.

Cardiac myosin binding protein C (cMyBP-C) is a phosphorylation-dependent force regulator and plays an important role in controlling myosin and actin dynamic interaction. Point-mutations of cMyBP-C that interfere with cMyBP-C threonine/serine phosphorylation resulted in hypertrophic cardiomyopathy and cardiac failure. However, it remains largely unknown how cMyBP-C tyrosine phosphorylation is regulated during cardiac hypertrophy and heart failure. Integrins are receptors of extracellular matrix and are the sensors of cardiac mechanical stretch. Focal adhesion kinase (FAK) plays an essential role in integrin-initiated signal transduction and regulates multiple cellular functions in various types of cells including cardiomyocytes. To identify the regulatory mechanism of cMyBP-C tyrosine phosphorylation during cardiac hypertrophy, we examined the effect of FAK on phosphorylation of cMyBP-C. Immunoprecipitation analysis showed that FAK and cMyBP-C are associated within the intact mouse heart. Results from our mutagenesis experiments demonstrated that the FAK kinase domain was required for FAK to associate with cMyBP-C. Our data also documented that the FAK Y397 site is required for FAK and cMyBP-C association. Importantly, overexpression dominant active Src Y527F with FAK significantly enhanced cMyBP-C phosphorylation. Interestingly, overexpression of cMyBP-C inhibited FAK phosphorylation. Taken together, cMyBP-C is one of effectors of Src/FAK complex in cardiomyocyte.

Complement C3 is a key factor in complement system. Our recently animal study found that C3 may regulate myocardial apoptosis through the intrinsic apoptosis pathway. The current work investigated if C3 regulation of apoptosis occurred in human cardiomyocytes. Our results showed that incubation of exogenous C3 reduced apoptosis in a cell culture system of human cardiomyocytes which did not inherently express C3. In addition, C3 inhibited intrinsic apoptosis pathway in a cell-free apoptosis system. Furthermore, pro-C3 was found to bind with an apoptotic factor, pro-caspase 3, in a cell-free system. Thus, we presented firsthand evidence that exogenous C3 is readily reduce apoptosis in human cardiomyocytes via interaction with the intrinsic apoptotic pathway.

ShcD was previously found to promote cell motility in melanoma cells. Screening of a yeast two hybrid mouse embryo cDNA library identified Nischarin, a negative regulator to cell motility, as an interacting partner to the ShcD-CH2 domain. Therefore, we aimed to investigate the interaction between Nischarin and ShcD in mammalian cells and to determine their functional impact on cell migration. The Nischarin/ShcD interaction was confirmed by transfection and co-immunoprecipitation assays using full-length constructs in HEK293, MCF7 and MM253 cell lines. Deletion of the first 93 amino acids of ShcD abrogated the interaction indicating the importance of these residues for Nischarin binding. Co-expression of Nischarin and ShcD demonstrated an inhibitory effect on the levels of phospho-ERK and phospho-LIMK. In support of this, Nischarin was found to block the migratory activities of ShcD. A brief in silico analysis of publicly available breast cancer patient data was performed to elucidate the effect of Nischarin/ShcD co-expression on the patients overall survival. Patients with high expression of both proteins had better survival than those with only ShcD overexpression. Our results reveal that the novel protein Nischarin is an interacting partner to ShcD. In addition, we report that the tumour suppressive abilities of Nischarin can overcome ShcD-mediated cell migration when both proteins are concomitantly expressed. *This abstract was presented in the National Cancer Research Institute (NCRI)-2019

Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated CCN2 signaling in LECs and its effects on lymphangiogenesis. In primary cultured LECs, gene expressions of lymphatic endothelial markers lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), Podoplanin and prospero homeobox 1 (Prox1) and lymphangiogenic factors vascular endothelial cell growth factor c (Vegfc), vascular endothelial cell growth factor d (Vegfd) and fms-related tyrosine kinase 4 (Flt4, also known as Vegfr3) were upregulated by CCN2. Subsequently, we found that CCN2 induced phospho-ERK and that was decreased by suppression of integrin v. CCN2 slightly decreased the growth of LECs due to enhancement of the interaction of ERK and dual specific protein phosphatase 6 (DUSP6), and knockdown of DUSP6 increased CCN2-induced phospho-ERK levels. In in vivo Matrigel plug assays, the number of Podoplanin-positive vessels was increased by exogenous CCN2, and phospho-ERK-positive LEC and DUSP6-positive LEC were detected in CCN2 plugs. These results suggest that CCN2-related lymphangiogenesis is regulated by DUSP6, which enables negative modulation of ERK-signaling.

We investigated whether the endolysosomal two-pore channel TPC2 is a mediator of adipocyte differentiation. We show that Tpcn2 mRNA is expressed transiently during induction of C3H10T1/2 mesenchymal stem cells to differentiate into adipocytes, and that this expression is triggered by cAMP. This is the first demonstration of a cell signaling pathway that can regulate TPC gene expression. We also identified an important functional role for TPC2 in adipocyte differentiation. First, ectopic TPC2 expression in C3H10T1/2 cells partially rescued the block to adipocyte differentiation caused by cAMP absence. Second, inhibition of endogenous TPC2 expression in primary preadipocytes substantially reduced their ability to differentiate into adipocytes. Finally, genetic variation at the Tpcn2 locus is associated with increased upper-body fat distribution in women concomitant with reduced Tpcn2 expression in abdominal adipose tissue. Our findings implicate TPC2 as an important mediator of adipogenesis and may aid identification of new drug targets for treatment of obesity.

The complement derived neutrophil chemoattractant C5a, is a potent activator of the neutrophil superoxide anion generating NADPH oxidase. An allosteric modulator specific for the free fatty acid 2 receptor increases the activating potency but not the efficacy of C5a. The allosteric modulator also decreases the inhibitory effect of the C5a receptor antagonist avacopan, suggesting that the NADPH oxidase is activated by two different signaling pathways downstream of the receptor for C5a. While the allosteric modulator affected the C5a-mediated activation of the NADPH oxidase, the C5a-induced rise in the intracellular concentration of free calcium ions was unaffected. The C5a receptor and the free fatty acid receptor belong to the family of G protein-coupled receptors family. Our results show that the activated C5a receptors generate signals that directly activate the NADPH oxidase and allosterically modulated free fatty acid receptors which secondarily generate signals that elicit NADPH oxidase activity. This is in line with an earlier described receptor transactivation model, by which the fatty acid receptor is activated by receptor downstream signals generated by several different neutrophil receptors to which we now add the receptor for C5a. In addition, the fatty acid receptor was higher ranked than the receptor for C5a, in the neutrophil receptor hierarchy. The dual receptor trans-regulatory effects, by which the receptor for C5a activates the fatty acid receptor and by which this receptor reduces the C5a response, represent new regulatory mechanisms of importance for the NADPH oxidase activity in neutrophils.

Heteromeric TASK1/3 channels play a fundamental role in oxygen-sensing by carotid body type 1 cells, where hypoxia-induced inhibition of TASK3 and/or TASK1/3 potassium currents leads to depolarisation, voltage-gated calcium entry, exocytotic transmitter release and increases in carotid body afferent input responses that initiate corrective changes in breathing patterns. However, the mechanism by which hypoxia leads to TASK-1/3 channel inhibition is still debated. It had been proposed that the AMP-activated protein kinase (AMPK) might directly phosphorylate and inhibit TASK channels, in particular TASK-3, although subsequent studies on rat type I cells argued against this view. Here we report on the effects of novel, highly selective AMPK activators on recombinant human TASK-3 potassium channels. Sequence alignment identified an AMPK recognition motif in TASK-3, but not TASK-1, with Ser55 representing a potential site for AMPK-dependent phosphorylation in TASK-3. However, neither of the AMPK activators, AICAR or MK-8722, caused a significant reduction of human TASK-3 current amplitude. By contrast, high concentrations of the AMPK activator A-769662 (100-500 {micro}M) inhibited human TASK-3 currents in a concentration-dependent manner. Importantly, A-769662 (300 {micro}M) also inhibited human TASK-3 channels in HEK293 cells that stably over-expressed an AMPK-{beta}1 subunit mutant (S108A) that renders AMPK insensitive to activators binding the Allosteric Drug and Metabolite (ADaM) site, such as A-769662. We therefore identify A-769662 as a novel human TASK-3 channel inhibitor and provide conclusive evidence that AMPK does not regulate TASK-3 channel currents.

The 8th-helix (H8) and carboxyl terminal tail (CT) of GPCRs are crucial for interactions with intracellular signaling molecules and regulatory proteins. We investigated how H8 and CT residues influence signaling in PAR4, a tethered-ligand activated GPCR. Our analysis revealed that PAR4 activation by thrombin or AYPGKF-NH2 stimulates {beta}-arrestin-1/-2 recruitment and activates multiple G proteins (Gq, G11, Gz, G15, G12, G13, Gi1-3, GoA/B). Mutation of the H8 sequence (R352AGLFQRS359) significantly reduced G protein activation and {beta}-arrestin recruitment, with residues Leu355-Glu357 being particularly important. Mutations of specific lysine residues in H8 and CT also impaired signaling. Additionally, we identified a crucial TM7-H8 interaction and found that CT phosphorylation sites regulate {beta}-arrestin recruitment. Finally, using AlphaFold3 we predicted interactions between PAR4, {beta}-arrestins, and G proteins, revealing novel receptor regulatory sites in the intracellular loops, transmembrane domains, H8 and CT.

Unfolded protein response is a dynamic signalling pathway, which is involved in the maintenance of proteostasis and cellular homeostasis. IRE1, a transmembrane signalling protein represents the start point of a highly conserved UPR signalling cascade. IRE1 is endowed with kinase and endoribonuclease activities. The activation of the kinase domain of IRE1 by trans-autophosphorylation leads to the activation of its RNAse domain. RNAse domain performs atypical splicing of Xbp1 mRNA and degradation of mRNAs by an effector function known as Regulated IRE1 Dependent Decay (RIDD). The regulation of the distinctive nature of the IRE1 ribonuclease function is potentially mediated by a dynamic protein structure UPRosome that is an assembly of a huge number of proteins on IRE1. Here, we reported that Bid is a novel recruit to UPRosome, which directly interacts with the cytoplasmic domain of IRE1. Bid controls the auto-phosphorylation of IRE1 in a negative manner where Bid overexpression conditions displayed reduced phosphorylation levels of IRE1 and Bid knockdown cells showed slightly enhanced IRE1 phosphorylation. This effect was reciprocated with JNK, a downstream target of IRE1. Our Insilico analysis revealed that Bid binding to IRE1 dimer averts its structural flexibility and thereby preventing its trans-autophosphorylation activity. We found that the effect of Bid is specific to the IRE1 branch of UPR signalling and competitive in nature. The highlighting observation of the study was that Bid stimulated a differential activity of the IRE1 RNAse domain towards Xbp1 splicing and RIDD. These results together establish that Bid is a part of the UPRosome and modulates IRE1 in a way to differentially regulate its RNAse outputs.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are a common drug target. They can be stabilised in different conformational states by ligands to activate multiple transducers and effectors leading to a variety of cellular responses. The potential of agonists to activate select pathways has important implications for drug discovery. Thus, there is a clear need to profile the initial GPCR signal transduction event, activation of G proteins, to enhance understanding of receptor coupling and guide drug design. The BRET-based biosensor suite, TRUPATH, was recently developed to enable quantification of the activation profiles of all non-visual G proteins (excluding Golf and G14) and has since been utilised in numerous studies. However, it fails to detect Gq/11 activation for a number of GPCRs previously reported to display promiscuous secondary coupling to Gq/11. Here we report modifications to the Gq and G11 biosensors in the switch I region that prevent intrinsic GTPase activity (R183C/Q). Except for the PAC1R, substitution with cancer-associated mutations, Cys or Gln, significantly increased sensitivity to allow detection of robust, reliable, and representative Gq/11 responses to Class B1 GPCRs. We also demonstrate the utility of these modified biosensors for promiscuously coupled class A GPCR that have primary Gs-coupling. Thus, we propose that modification to Gq/11 may also be necessary in other biosensor systems to enable detection of Gq/11 activation.