Inositol Phosphate Production Research Articles

Orphan GPCR GPRC5C Facilitates Angiotensin II-Induced Smooth Muscle Contraction.

GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.

Relevance of augmented kisspeptin signaling through H364 KISS1R in central precocious puberty

Understanding the pathophysiology of idiopathic central precocious puberty (ICPP) is essential, in view of its consequences on reproductive health and metabolic disorders in later life. Towards this, estimation of circulating levels of the neuropeptides, viz; Kisspeptin (Kp-10), Neurokinin B (NKB) and Neuropeptide Y (NPY), acting upstream to Gonadotropin-Releasing Hormone (GnRH), has shown promise. Insights can also be gained from functional studies on genetic variations implicated in ICPP. This study investigated the pathophysiology of ICPP in a girl by exploring the therapeutic relevance of the circulating levels of Kp-10, NKB, NPY and characterizing the nonsynonymous KISS1R variant, L364H, that she harbours, in a homozygous condition. Plasma levels of Kp-10, NKB and NPY before and after GnRH analog (GnRHa) treatment, were determined by ELISA. It was observed that GnRHa treatment resulted in suppression of circulating levels of Kp-10, NKB and NPY. Further, the H364 variant in KISS1R was generated by site directed mutagenesis. Post transient transfection of either L364 or H364 KISS1R variant in CHO cells, receptor expression was ascertained by western blotting, indirect immunofluorescence and flow cytometry. Kp-10 stimulated signalling response was also determined by phospho-ERK and inositol phosphate production. Structure-function studies revealed that, although the receptor expression in H364 KISS1R was comparable to L364 KISS1R, there was an enhanced signalling response through this variant at high doses of Kp-10. Thus, elevated levels of Kp-10, acting through H364 KISS1R, contributed to the manifestation of ICPP, providing further evidence that dysregulation of Kp-10/KISS1R axis impacts the onset of puberty.

Gonadotropin-Releasing Hormone Receptor (GnRHR) and Hypogonadotropic Hypogonadism

Human sexual and reproductive development is regulated by the hypothalamic-pituitary-gonadal (HPG) axis, which is primarily controlled by the gonadotropin-releasing hormone (GnRH) acting on its receptor (GnRHR). Dysregulation of the axis leads to conditions such as congenital hypogonadotropic hypogonadism (CHH) and delayed puberty. The pathophysiology of GnRHR makes it a potential target for treatments in several reproductive diseases and in congenital adrenal hyperplasia. GnRHR belongs to the G protein-coupled receptor family and its GnRH ligand, when bound, activates several complex and tissue-specific signaling pathways. In the pituitary gonadotrope cells, it triggers the G protein subunit dissociation and initiates a cascade of events that lead to the production and secretion of the luteinizing hormone (LH) and follicle-stimulating hormone (FSH) accompanied with the phospholipase C, inositol phosphate production, and protein kinase C activation. Pharmacologically, GnRHR can be modulated by synthetic analogues. Such analogues include the agonists, antagonists, and the pharmacoperones. The agonists stimulate the gonadotropin release and lead to receptor desensitization with prolonged use while the antagonists directly block the GnRHR and rapidly reduce the sex hormone production. Pharmacoperones include the most recent GnRHR therapeutic approaches that directly correct the misfolded GnRHRs, which are caused by genetic mutations and hold serious promise for CHH treatment. Understanding of the GnRHR’s genomic and protein structure is crucial for the most appropriate assessing of the mutation impact. Such mutations in the GNRHR are linked to normosmic hypogonadotropic hypogonadism and lead to various clinical symptoms, including delayed puberty, infertility, and impaired sexual development. These mutations vary regarding their mode of inheritance and can be found in the homozygous, compound heterozygous, or in the digenic state. GnRHR expression extends beyond the pituitary gland, and is found in reproductive tissues such as ovaries, uterus, and prostate and non-reproductive tissues such as heart, muscles, liver and melanoma cells. This comprehensive review explores GnRHR’s multifaceted role in human reproduction and its clinical implications for reproductive disorders.

Hormone profiling and the root proteome analysis of itpk1 mutant seedlings of barley (Hordeum vulgare) during the red-light induced photomorphogenesis

Inositol phosphates represent crucial primary metabolites, known as phosphate storage molecules or molecules enabling protein interactions. Inositol phosphates and their pyrophosphate forms direct the assembly of the COP9 signalosome and Cullin Ring Ligase complexes. Thus, inositol phosphates participate on protein degradation, which is a means of signal transduction for several plant hormone classes and light signaling cascade. In plants, production of inositol phosphates is maintained by several inositol phosphate kinases including the group of inositol-1,3,4-trisphosphate 5/6 kinases (ITPKs). The members of ITPKs connect plant phosphate status with light perception and hormonal balance. Photomorphogenesis in red light was investigated using allelic variants of barley Hvitpk1 mutants produced by gene editing. Barley ITPK promoter regions, conserved protein motifs, and expression profiles were investigated to characterize their evolutionary diversification and transcriptional regulation. We focused on ITPK1 relation to auxins, cytokinins, gibberellins, jasmonates, abscisic acid, and salicylic acid in red light-induced photomorphogenesis. Global proteome analyses was conducted in the mutant and wild-type (WT) seedlings grown under continuous red light. Our results demonstrate that different allelic variants of itpk1 mutants manifested defects in producing important phytohormones with consequences for root growth. Mutations in itpk1 affected hormone biosynthesis and metabolite, while proteome analysis revealed significant differences in seedling root proteomes between mutant lines. Based on our results and published data, a model of ITPK activity is proposed depicting them as regulators of hormone signal transduction at the level of protein degradation.

Development of a Non-Peptide Angiotensin II Type 1 Receptor Ligand by Structural Modification of Olmesartan as a Biased Agonist.

As a biased agonist, peptide angiotensin II (Ang II) type 1 (AT1) receptor ligand antagonizes Ang II-stimulated G protein signaling but stimulates several kinase pathways. Here, we developed a non-peptide AT1 receptor compound as a biased ligand. We synthesized three non-peptide AT1 receptor ligands (R239470, R781253, and R794847) as candidates of biased ligands. Extracellular signal-regulated kinase (ERK) 1/2 activation and inositol phosphate (IP) production were measured using a cell system that overexpressed AT1 receptors (wild-type, L112A, Q257A, Y292A, and N295A receptors). We also examined the modes of receptor-ligand binding using a competition binding study. The Kd values of R239470, R781253, and R794847 for the AT1 wild-type receptor were 0.8, 21, and 48 nM, respectively, as assessed in a competition binding study. Those of R239470, R781253, and R794847 for the L112A receptor were 37, 23, and 31 nM, respectively. R781253 and R794847 decreased and increased IP production, respectively, whereas R239470 did not change IP production. R781253 and R794847, but not R239470, activated ERK1/2. In conclusion, R239470, R781253, and R794847 act as a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. On the other hand, R781253 and R794847, but not R239470, are agonists toward ERK1/2 activation. Thus, we developed a non-peptide AT1 receptor compound as a biased ligand.

PS-BPB08-2: DEVELOPMENT OF NON-PEPTIDE TYPE OF ANGIOTENSIN II TYPE 1 RECEPTOR LIGAND AS A BIASED AGONIST

Objective: Peptide type of angiotensin II (Ang II) type 1 (AT1) receptor ligand as a biased agonist antagonized Ang II-stimulated G protein signaling, but stimulated several kinase pathways. Here, we developed non-peptide type of AT1 receptor compound as a biased ligand. Design and method: We synthesized three non-peptide type of AT1 receptor ligands (Compound A, B and C) as candidates of biased ligands. These compounds were provided by Daiichi Sankyo Co. LTD. In vitro studies, inositol phosphate (IP) production and extracellular signal-regulated kinase (ERK) 1/2 activation were measured using AT1 receptor overexpressing cell system. We mutated the AT1 receptor (L112A, Q257A, Y292A and N295A receptors) and examined binding modes of receptor-ligand by competition binding study, and subsequently analyzed whether these ligands would induce IP production and ERK1/2 activation. Results: Kd of Compound A, B and C to AT1 wild-type receptor by competition binding study were 0.8, 21 and 48 nM, respectively. Kd of Compound A, B and C to L112A receptor were 37, 23 and 31 nM, respectively. Compound B and C decreased and increased IP production, respectively, whereas Compound A did not change IP production. Compound B and C, but not Compound A, activated ERK1/2. L112A had a key role of IP production. Conclusions: Compound A, B and C were a neutral antagonist, an inverse agonist, and an agonist with regard to IP production, respectively. On the other hand, Compound B and C, but not Compound A, were agonists to ERK1/2 activation. Thus, we developed non-peptide type of AT1 receptor compound as a biased ligand.

Rapeseed meal processing and dietary enzymes modulate excreta inositol phosphate profile, nutrient availability, and production performance of broiler chickens

This study aimed to assess the effect of rapeseed meal (RSM) processing method, where solvent extraction occurred under standard industry conditions (ST) or cold-pressed hexane extraction was employed (MT), and exogenous enzyme supplementation (phytase [PHY] and xylanase [XYL]) alone or in combination on key nutritional factors of broiler chickens. A randomized control experiment was performed using 144 male Ross 308 broilers in a 2 × 2 × 3 factorial arrangement. Three diets including a nutritionally complete wheat-based basal diet (BD), a diet containing 200 g/kg of RSM extracted under ST and another diet containing 200 g/kg of RSM extracted under MT were produced. Each diet was then split into 4 parts and was fed as is, or supplemented with PHY at 1,500 FTU/kg or XYL at 16,000 BXU/kg, alone or in combination, resulting in 12 diets in total. Response criteria: feed intake (FI), weight gain (WG), and feed conversion ratio (FCR), from 7 to 21 d age, AMEn, retention coefficients for dry matter (DMR), nitrogen (NR), fat (FR), and the profile of inositol phosphate esters (IP2-6) and myo-inositol (MI) in excreta. Diets containing MT had higher AMEn compared to ST diets (P < 0.05). There was RSM by PHY interaction for FI, as only birds fed MT diet responded to PHY supplementation with reduced FI and FCR (P < 0.001). Feeding XYL reduced overall FI and FCR (P < 0.05). Feeding PHY reduced IP6 and increased MI in excreta (P < 0.001). Feeding XYL and PHY in combination reduced MI in excreta compared to PHY only (P = 0.05). Compared to BD, birds fed RSM diets had an increased IP6 (P < 0.05) and MI concentration in excreta (P < 0.01). This may be due to IP ester differences in RSM and BD.

A naturally occurring membrane-anchored Gαs variant, XLαs, activates phospholipase Cβ4

Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein–coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cβ (PLCβ), PLCβ4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCβ stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gβγ, suggesting a mechanism of activation that relies on Gβγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCβ4. We also show that PLCβ4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology.

Discovery of the cyclotide caripe 11 as a ligand of the cholecystokinin-2 receptor

The cholecystokinin-2 receptor (CCK2R) is a G protein-coupled receptor (GPCR) that is expressed in peripheral tissues and the central nervous system and constitutes a promising target for drug development in several diseases, such as gastrointestinal cancer. The search for ligands of this receptor over the past years mainly resulted in the discovery of a set of distinct synthetic small molecule chemicals. Here, we carried out a pharmacological screening of cyclotide-containing plant extracts using HEK293 cells transiently-expressing mouse CCK2R, and inositol phosphate (IP1) production as a readout. Our data demonstrated that cyclotide-enriched plant extracts from Oldenlandia affinis, Viola tricolor and Carapichea ipecacuanha activate the CCK2R as measured by the production of IP1. These findings prompted the isolation of a representative cyclotide, namely caripe 11 from C. ipecacuanha for detailed pharmacological analysis. Caripe 11 is a partial agonist of the CCK2R (Emax = 71%) with a moderate potency of 8.5 µM, in comparison to the endogenous full agonist cholecystokinin-8 (CCK-8; EC50 = 11.5 nM). The partial agonism of caripe 11 is further characterized by an increase on basal activity (at low concentrations) and a dextral-shift of the potency of CCK-8 (at higher concentrations) following its co-incubation with the cyclotide. Therefore, cyclotides such as caripe 11 may be explored in the future for the design and development of cyclotide-based ligands or imaging probes targeting the CCK2R and related peptide GPCRs.

PH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis.

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD.

Structure activity relationship of 3-nitro-2-(trifluoromethyl)-2H-chromene derivatives as P2Y6 receptor antagonists

Various 6-alkynyl analogues of a known 3-nitro-2-(trifluoromethyl)-2H-chromene antagonist 3 of the Gq-coupled P2Y6 receptor (P2Y6R) were synthesized using a Sonogashira reaction to replace a 6-iodo group. The analogues were tested in a functional assay consisting of inhibition of calcium mobilization in P2Y6R-expressing astrocytoma cells elicited by native P2Y6R agonist UDP. 6-Ethynyl and 6-cyano groups were installed, and the alkynes were extended through both alkyl and aryl spacers. The most potent antagonists, with IC50 of ~1 µM, were found to be trialkylsilyl-ethynyl 7 and 8 (3–5 fold greater affinity than reference 3), t-butyl prop-2-yn-1-ylcarbamate 14 and p-carboxyphenyl-ethynyl 16 derivatives, and 3 and 8 displayed surmountable antagonism of UDP-induced production of inositol phosphates. Other chain-extended terminal carboxylate derivatives were less potent than the corresponding methyl ester derivatives. Thus, the 6 position in this chromene series is suitable for derivatization with flexibility of substitution, even with sterically extended chains, without losing P2Y6R affinity. However, a 3-carboxylic acid or 3-ester substitution did not serve as a nitro bioisostere, as the affinity was eliminated. These compounds provide additional ligand tools for the underexplored P2Y6R, which is a target for inflammatory, neurodegenerative and metabolic diseases.

Serotonin 2B Receptor by Interacting with NMDA Receptor and CIPP Protein Complex May Control Structural Plasticity at Glutamatergic Synapses.

The serotonin 2B (5-HT2B) receptor coupled to Gq-protein contributes to the control of neuronal excitability and is implicated in various psychiatric disorders. The mechanisms underlying its brain function are not fully described. Using peptide affinity chromatography combined with mass spectrometry, we found that the PDZ binding motif of the 5-HT2B receptor located at its C-terminal end interacts with the scaffolding protein channel interacting PDZ protein (CIPP). We then showed, in COS-7 cells, that the association of the 5-HT2B receptor to CIPP enhanced receptor-operated inositol phosphate (IP) production without affecting its cell surface and intracellular levels. Co-immunoprecipitation experiments revealed that CIPP, the 5-HT2B receptor, and the NR1 subunit of the NMDA receptor form a macromolecular complex. CIPP increased 5-HT2B receptor clustering at the surface of primary cultured hippocampal neurons and prevented receptor dispersion following agonist stimulation, thus potentiating IP production and intracellular calcium mobilization in dendrites. CIPP or 5-HT2B receptor stimulation in turn dispersed NR1 clusters colocalized with 5-HT2B receptors and increased the density and maturation of dendritic spines. Collectively, our results suggest that the 5-HT2B receptor, the NMDA receptor, and CIPP may form a signaling platform by which serotonin can influence structural plasticity of excitatory glutamatergic synapses.

Dissecting Gq/11-Mediated Plasma Membrane Translocation of Sphingosine Kinase-1.

Diverse extracellular signals induce plasma membrane translocation of sphingosine kinase-1 (SphK1), thereby enabling inside-out signaling of sphingosine-1-phosphate. We have shown before that Gq-coupled receptors and constitutively active Gαq/11 specifically induced a rapid and long-lasting SphK1 translocation, independently of canonical Gq/phospholipase C (PLC) signaling. Here, we further characterized Gq/11 regulation of SphK1. SphK1 translocation by the M3 receptor in HEK-293 cells was delayed by expression of catalytically inactive G-protein-coupled receptor kinase-2, p63Rho guanine nucleotide exchange factor (p63RhoGEF), and catalytically inactive PLCβ3, but accelerated by wild-type PLCβ3 and the PLCδ PH domain. Both wild-type SphK1 and catalytically inactive SphK1-G82D reduced M3 receptor-stimulated inositol phosphate production, suggesting competition at Gαq. Embryonic fibroblasts from Gαq/11 double-deficient mice were used to show that amino acids W263 and T257 of Gαq, which interact directly with PLCβ3 and p63RhoGEF, were important for bradykinin B2 receptor-induced SphK1 translocation. Finally, an AIXXPL motif was identified in vertebrate SphK1 (positions 100–105 in human SphK1a), which resembles the Gαq binding motif, ALXXPI, in PLCβ and p63RhoGEF. After M3 receptor stimulation, SphK1-A100E-I101E and SphK1-P104A-L105A translocated in only 25% and 56% of cells, respectively, and translocation efficiency was significantly reduced. The data suggest that both the AIXXPL motif and currently unknown consequences of PLCβ/PLCδ(PH) expression are important for regulation of SphK1 by Gq/11.

The WD40 repeat protein, WDR36, orchestrates sphingosine kinase-1 recruitment and phospholipase C-β activation by Gq-coupled receptors

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that Gq-coupled receptors induce a rapid and long-lasting translocation of SphK1 to the plasma membrane and cross-activation of S1P receptors. Here, we further addressed Gq regulation of SphK1 by analysing the influence of the WD40 repeat protein, WDR36. WDR36 has been described as a scaffold tethering Gαq to phospholipase C (PLC)-β and the thromboxane A2 receptor-β (TPβ receptor). Overexpression of WDR36 in HEK-293 cells enhanced TPβ receptor-induced inositol phosphate production, as reported (Cartier et al. 2011), but significantly attenuated inositol phosphate production induced by muscarinic M3 and bradykinin B2 receptors. In agreement with its effect on PLCβ, WDR36 augmented TPβ receptor-induced [Ca2+]i increases. Surprisingly, WDR36 also augmented M3 receptor-induced [Ca2+]i increases, which was due to increased Ca2+ mobilization while the Ca2+ content of thapsigargin-sensitive stores remained unaltered. Interestingly, overexpression of WDR36 significantly delayed SphK1 translocation by Gq-coupled M3, B2 and H1 receptors in HEK-293 cells, while TPβ receptor-induced SphK1 translocation was generally slow and not altered by WDR36 in these cells. Finally, in C2C12 myoblasts, overexpression of WDR36 delayed SphK1 translocation induced by B2 receptors. It is concluded that WDR36 reduces signalling of Gq-coupled receptors other than TPβ towards PLC and SphK1, most likely by scavenging Gαq and PLCβ. Our results support a role of WDR36 in orchestration of Gq signalling complexes, and might help to functionally unravel its genetic association with asthma and allergy.

Structure-Activity Relationship Study of Psychostimulant Synthetic Cathinones Reveals Nanomolar Antagonist Potency of α-Pyrrolidinohexiophenone at Human Muscarinic M2 Receptors.

Synthetic cathinones (SCs) are designer, psychostimulant drugs of abuse that primarily act on monoamine transporters; little is known about their off-target liability. Abuse of pyrrolidine-containing SCs, such as α-PHP, has been linked to clinical features, including tachycardia and hypertension, and psychiatric events, including delusions and memory impairments-effects mimicking deliriant hallucinogens that are acetylcholine muscarinic receptor (MR) antagonists. α-PHP and nine analogs with modifications in the α-carbon side chain length and/or containing a methylenedioxy moiety were screened for activity at each of the five human MRs. Increasing the length of the α-carbon side chain of 1-phenyl-2-(pyrrolidin-1-yl)ethan-1-one analogs from a methyl (α-PPP) to a propyl (α-PVP) group caused a steep increase in affinity at all MR subtypes, and one extra carbon (α-PHP) further enhanced MR affinity; the presence of a methylenedioxy moiety generally hindered this effect. Highest MR affinity was observed with α-PHP at M2Rs-its M2R affinity (Ki = 251 nM) was 302-fold higher than α-PPP's. M2R-cAMP inhibition and β-arrestin recruitment assays showed that α-PHP is an M2R antagonist (Kb = 120 and 502 nM, respectively). Additional experiments showed α-PHP is also an antagonist of M1R-inositol phosphate production (Kb = 1.4 μM). Human toxicology studies report blood concentrations of pyrrolidine-containing SCs, including α-PHP, that reach micromolar levels during intoxication, indicating α-PHP's MR activity might have physiological relevance. As M2Rs and M1Rs are widely expressed in the autonomic and central nervous systems, α-PHP's anticholinergic activity might be relevant to adverse events associated with α-PHP intoxication.

Frequent and Persistent PLCG1 Mutations in Sézary Cells Directly Enhance PLCγ1 Activity and Stimulate NFκB, AP-1, and NFAT Signaling

Phospholipase C Gamma 1 (PLCG1) is frequently mutated in primary cutaneous T-cell lymphoma (CTCL). This study functionally interrogated nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and the in-frame indel p.VYEEDM1161V) identified in Sézary Syndrome, the leukemic variant of CTCL. The mutations were demonstrated in diagnostic samples and persisted in multiple tumor compartments over time, except in patients who achieved a complete clinical remission. In basal conditions, the majority of the mutations confer PLCγ1 gain-of-function activity through increased inositol phosphate production and the downstream activation of NFκB, AP-1, and NFAT transcriptional activity. Phosphorylation of the p.Y783 residue is essential for the proximal activity of wild-type PLCγ1, but we provide evidence that activating mutations do not require p.Y783 phosphorylation to stimulate downstream NFκB, NFAT, and AP-1 transcriptional activity. Finally, the gain-of-function effects associated with the p.VYEEDM1161V indel suggest that the C2 domain may have a role in regulating PLCγ1 activity. These data provide compelling evidence to support the development of therapeutic strategies targeting mutant PLCγ1.

Changes in the function of angiotensin II type 1 receptor due to cholesterol depletion from cell membrane

Blockers of G-protein coupled receptors (GPCRs), angiotensin II (Ang II) type 1 (AT1) receptor and β1-adrenergic (Ad) receptor, have been shown to improve the prognosis of cardiovascular disease. Cholesterol molecules in the cell membrane are needed to stabilize GPCRs as well as the cell membrane itself. We determined whether the functions of AT1 and β1-Ad receptors were changed by cholesterol depletion from cardiovascular cell membranes. Ang II-induced inositol phosphate production through AT1 receptor was suppressed by cholesterol depletion from cell membranes using rosuvastatin or methyl-β-cyclodextrin (MβCD), whereas isoproterenol-induced cyclic AMP production through β1-Ad receptor did not change after cholesterol depletion. In addition, the binding affinities of Ang II and AT1 receptor blocker after cholesterol depletion were significantly lower than those before depletion. Although AT1 receptor expression levels did not change after cholesterol depletion, the expression levels of AT1 receptor that could bind to Ang II significantly decreased after depletion. The changes in the structure of AT1 receptor due to depletion were confirmed by substituted-cysteine accessibility mapping. In conclusion, Ang II-induced activation of AT1 receptor is reduced without affecting the function of β1-Ad receptor after cholesterol depletion from cardiovascular cell membranes.

N-Terminal Liver-Expressed Antimicrobial Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the Ghrelin Receptor.

The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.

Unique mode of binding between angiotensin II type 1 receptor and its blockers

ABSTRACTBACKGROUND: We hypothesized that the 5-oxo-1,2,4-oxadiazole moiety of azilsartan (AZL), which represents a small difference in the molecular structures of AZL and candesartan (CAN) [angiotensin II type 1 receptor (AT1R) blockers], may be responsible for the molecular effects of AZL. METHODS: We examined the binding affinities of AZL and CAN to AT1R, along with their ability to block receptor activity. A competition binding study, inositol phosphate (IP) production assay and extracellular signal-regulated kinase (ERK) assay were performed using wild-type (WT) and mutants AT1R-transfected cells. RESULTS: The binding affinities of CAN and AZL were reduced by > 5-fold for Y35F, W84F, R167K, K199Q and I288A compared with WT. In addition, AZL showed a > 5-fold reduction in its binding affinity to V108A. CAN and AZL exhibited > 20-fold and > 100-fold reductions in binding affinity to R167K, respectively. The loss of binding affinity of AZL to R167K was greater than that of CAN. CAN-7H exhibited a > 10-fold reduction in binding affinity to R167K compared with CAN. On the other hand, the binding affinity of AZL-7H to R167K was comparable to that of AZL. While 10-6M CAN and CAN-7H partly blocked Ang II-induced IP production in R167K, 10-6M AZL and AZL-7H did not. In addition, 10-6M CAN, but not 10-6M AZL, partly blocked Ang II-induced ERK activation in R167K. CONCLUSIONS: The interaction between 5-oxo-1,2,4-oxadiazole in AZL and Arg167 in the AT1R appears to be more important than the interaction between the tetrazole ring in CAN and Arg167.

Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production.

Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. We previously found that stimulation of G protein-coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. We hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. We measured GPCR activation-dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P2-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P2 from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P2 Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P2 pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.