β-Arrestin Recruitment and Biased Agonism at Free Fatty Acid Receptor 1

FFAR1/GPR40 is a seven-transmembrane domain receptor (7TMR) expressed in pancreatic β cells and activated by FFAs. Pharmacological activation of GPR40 is a strategy under consideration to increase insulin secretion in type 2 diabetes. GPR40 is known to signal predominantly via the heterotrimeric G proteins G_q/11. However, 7TMRs can also activate functionally distinct G protein-independent signaling via β-arrestins. Further, G protein- and β-arrestin-based signaling can be differentially modulated by different ligands, thus eliciting ligand-specific responses (“biased agonism”). Whether GPR40 engages β-arrestin-dependent mechanisms and is subject to biased agonism is unknown. Using bioluminescence resonance energy transfer-based biosensors for real-time monitoring of cell signaling in living cells, we detected a ligand-induced GPR40-β-arrestin interaction, with the synthetic GPR40 agonist TAK-875 being more effective than palmitate or oleate in recruiting β-arrestins 1 and 2. Conversely, TAK-875 acted as a partial agonist of G_q/11-dependent GPR40 signaling relative to both FFAs. Pharmacological blockade of G_q activity decreased FFA-induced insulin secretion. In contrast, knockdown or genetic ablation of β-arrestin 2 in an insulin-secreting cell line and mouse pancreatic islets, respectively, uniquely attenuated the insulinotropic activity of TAK-875, thus providing functional validation of the biosensor data. Collectively, these data reveal that in addition to coupling to G_q/11, GPR40 is functionally linked to a β-arrestin 2-mediated insulinotropic signaling axis. These observations expose previously unrecognized complexity for GPR40 signal transduction and may guide the development of biased agonists showing improved clinical profile in type 2 diabetes.     

Retinol-Binding Protein 4 and Its Membrane Receptor STRA6 Control Adipogenesis by Regulating Cellular Retinoid Homeostasis and Retinoic Acid Receptor α Activity

Retinoids are vitamin A (retinol) derivatives and complex regulators of adipogenesis by activating specific nuclear receptors, including the retinoic acid receptor (RAR) and retinoid X receptor (RXR). Circulating retinol-binding protein 4 (RBP4) and its membrane receptor STRA6 coordinate cellular retinol uptake. It is unknown whether retinol levels and the activity of RAR and RXR in adipocyte precursors are linked via RBP4/STRA6. Here, we show that STRA6 is expressed in precursor cells and, dictated by the apo- and holo-RBP4 isoforms, mediates bidirectional retinol transport that controls RARα activity and subsequent adipocyte differentiation. Mobilization of retinoid stores in mice by inducing RBP4 secretion from the liver activated RARα signaling in the precursor cell containing the stromal-vascular fraction of adipose tissue. Retinol-loaded holo-RBP4 blocked adipocyte differentiation of cultured precursors by activating RARα. Remarkably, retinol-free apo-RBP4 triggered retinol efflux that reduced cellular retinoids, RARα activity, and target gene expression and enhanced adipogenesis synergistically with ectopic STRA6. Thus, STRA6 in adipocyte precursor cells links nuclear RARα activity to the circulating RBP4 isoforms, whose ratio in obese mice was shifted toward limiting the adipogenic potential of their precursors. This novel cross talk identifies a retinol-dependent metabolic function of RBP4 that may have important implications for the treatment of obesity.     

Analysis of γ-Aminobutyric Acid (GABA) Type A Receptor Subtypes Using Isosteric and Allosteric Ligands

The GABA_A receptors (GABA_ARs) play an important role in inhibitory transmission in the brain. The GABA_ARs could be identified using a medicinal chemistry approach to characterize with a series of chemical structural analogues, some identified in nature, some synthesized, to control the structural conformational rigidity/flexibility so as to define the ‘receptor-specific’ GABA agonist ligand structure. In addition to the isosteric site ligands, these ligand-gated chloride ion channel proteins exhibited modulation by several chemotypes of allosteric ligands, that help define structure and function. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA_ARs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Also in the trans-membrane domain are allosteric modulatory ligand sites, mostly positive, for diverse chemotypes with general anesthetic efficacy, namely, the volatile and intravenous agents: barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are apparent endogenous positive allosteric modulators of GABA_ARs. These binding sites depend on the GABA_AR heteropentameric subunit composition, i.e., subtypes. Two classes of pharmacologically very important allosteric modulatory ligand binding site reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site, and the low-dose ethanol site. The benzodiazepine site is specific for certain subunit combination subtypes, mainly synaptically localized. In contrast, the low-dose (high affinity) ethanol site(s) is found at a modified benzodiazepine site on different, extrasynaptic, subtypes.     

Clinical and Genomic Crosstalk between Glucocorticoid Receptor and Estrogen Receptor α In Endometrial Cancer

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Isolation and Identification of Tubaic Acid and β-Tubaic Acid from Derris Roots

A tobacco callus strain, OMT-53, was selected from many cultures as a desirable strain having high nicotine producing capacity. Several culture conditions were examined, aiming to get higher nicotine production with the callus strain, OMT-53. It was revealed that the nicotine production was remarkably enhanced when the callus tissues were cultured at a limited concentration of α-NAA in culture medium. The optimal concentrations of sucrose and nitrogen in the culture medium were 3 % and 840 mg N/L respectively. Some precursors in nicotine biosynthesis were examined, and only ornithine gave a slightly positive effect at 2x10^-4m concentration. Cultures at 25°C produced the highest yield for nicotine. Considerable amounts of nicotine (ca. 20% of total nicotine) were also recognized in the culture medium. Under the best culture condition mentioned above, nicotine production in tobacco callus tissues has been elevated to 2.14% on D.W, basis at 4 weeks’ culture. This value is near to that of the intact tobacco plants.     

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor �� and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor α (PPARα) activation biomarkers 21-steroid carboxylic acids 11β-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11β,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARα-specific time-dependent increases in HDOPA and 20α-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARα induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARα and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARα activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20α-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARα resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.     

Demonstration of a Direct Interaction between β2-Adrenergic Receptor and Insulin Receptor by BRET and Bioinformatics

Glucose metabolism is under the cooperative regulation of both insulin receptor (IR) and β₂-adrenergic receptor (β₂AR), which represent the receptor tyrosine kinases (RTKs) and seven transmembrane receptors (7TMRs), respectively. Studies demonstrating cross-talk between these two receptors and their endogenous coexpression have suggested their possible interactions. To evaluate the effect of IR and prospective heteromerization on β₂AR properties, we showed that IR coexpression had no effect on the ligand binding properties of β₂AR; however, IR reduced β₂AR surface expression and accelerated its internalization. Additionally, both receptors displayed a similar distribution pattern with a high degree of colocalization. To test the possible direct interaction between β₂AR and IR, we employed quantitative BRET² saturation and competition assays. Saturation assay data suggested constitutive β₂AR and IR homo- and heteromerization. Calculated acceptor/donor (AD₅₀) values as a measure of the relative affinity for homo- and heteromer formation differed among the heteromers that could not be explained by a simple dimer model. In heterologous competition assays, a transient increase in the BRET² signal with a subsequent hyperbolical decrease was observed, suggesting higher-order heteromer formation. To complement the BRET² data, we employed the informational spectrum method (ISM), a virtual spectroscopy method to investigate protein-protein interactions. Computational peptide scanning of β₂AR and IR identified intracellular domains encompassing residues at the end of the 7^th TM domain and C-terminal tail of β₂AR and a cytoplasmic part of the IR β chain as prospective interaction domains. ISM further suggested a high probability of heteromer formation and homodimers as basic units engaged in heteromerization. In summary, our data suggest direct interaction and higher-order β₂AR:IR oligomer formation, likely comprising heteromers of homodimers.     

Opioid Receptor Trafficking and Signaling: What Happens After Opioid Receptor Activation?

Prolonged opioid treatment leads to a comprehensive cellular adaptation mediated by opioid receptors, a basis to understand the development of opioid tolerance and dependence. However, the molecular mechanisms underlying opioid-induced cellular adaptation remain obscure. Recent advances in opioid receptor trafficking and signaling in cells have extensively increased our insight into the network of intracellular signal integration. This review focuses on those important intracellular biochemical processes that play critical roles in the development of opioid tolerance and dependence after opioid receptor activation, and tries to explain what happens after opioid receptor activation, and how the cellular adaptation develops from cell membrane to nucleus. Decades of research have delineated a network on opioid receptor trafficking and signaling, but the challenge remains to explain opioid tolerance and dependence from a single cellular signal network.     

Retinoic Acid-related Orphan Receptor α Regulates Diurnal Rhythm and Fasting Induction of Sterol 12α-Hydroxylase in Bile Acid Synthesis

Sterol 12α-hydroxylase (CYP8B1) is required for cholic acid synthesis and plays a critical role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone, dyslipidemia, and diabetes. In this study we investigated the underlying mechanism of fasting induction and circadian rhythm of CYP8B1 by a cholesterol-activated nuclear receptor and core clock gene retinoic acid-related orphan receptor α (RORα). Fasting stimulated, whereas restricted-feeding reduced expression of CYP8B1 mRNA and protein. However, fasting and feeding had little effect on the diurnal rhythm of RORα mRNA expression, but fasting increased RORα protein levels by cAMP-activated protein kinase A-mediated phosphorylation and stabilization of the protein. Adenovirus-mediated gene transduction of RORα to mice strongly induced CYP8B1 expression, and increased liver cholesterol and 12α-hydroxylated bile acids in the bile acid pool and serum. A reporter assay identified a functional RORα response element in the CYP8B1 promoter. RORα recruited cAMP response element-binding protein-binding protein (CBP) to stimulate histone acetylation on the CYP8B1 gene promoter. In conclusion, RORα is a key regulator of diurnal rhythm and fasting induction of CYP8B1, which regulates bile acid composition and serum and liver cholesterol levels. Antagonizing RORα activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease and type 2 diabetes.     

Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function

Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α₁-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α₁-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α₁-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca²⁺ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α₁-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α₁-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure.     

Blocking L-type Voltage-gated Ca2+ Channels with Dihydropyridines Reduces ��-Aminobutyric Acid Type A Receptor Expression and Synaptic Inhibition

γ-Aminobutyric acid type A receptors (GABA_ARs) are the major sites of fast inhibitory neurotransmission in the brain, and the numbers of these receptors at the cell surface can determine the strength of GABAergic neurotransmission. Chronic changes in neuronal activity lead to an adaptive modulation in the efficacy of GABAergic synaptic inhibition, brought about in part by changes in the number of synaptic GABA_ARs, a mechanism known as homeostatic synaptic plasticity. Reduction in the number of GABA_ARs in response to prolonged neuronal activity blockade is dependent on the ubiquitin-proteasome system. The underlying biochemical pathways linking chronic activity blockade to proteasome-dependent degradation of GABA_ARs are unknown. Here, we show that chronic blockade of L-type voltage-gated calcium channels (VGCCs) with nifedipine decreases the number of GABA_ARs at synaptic sites but not the overall number of inhibitory synapses. In parallel, blockade of L-type VGCCs decreases the amplitude but not the frequency of miniature inhibitory postsynaptic currents or expression of the glutamic acid decarboxylase GAD65. We further reveal that the activation of L-type VGCCs regulates the turnover of newly translated GABA_AR subunits in a mechanism dependent upon the activity of the proteasome and thus regulates GABA_AR insertion into the plasma membrane. Together, these observations suggest that activation of L-type VGCCs can regulate the abundance of synaptic GABA_ARs and the efficacy of synaptic inhibition, revealing a potential mechanism underlying the homeostatic adaptation of fast GABAergic inhibition to prolonged changes in activity.     

Retinoic Acid Mediates Regulation of Network Formation by COUP-TFII and VE-Cadherin Expression by TGFβ Receptor Kinase in Breast Cancer Cells

Tumor development, growth, and metastasis depend on the provision of an adequate vascular supply. This can be due to regulated angiogenesis, recruitment of circulating endothelial progenitors, and/or vascular transdifferentiation. Our previous studies showed that retinoic acid (RA) treatment converts a subset of breast cancer cells into cells with significant endothelial genotypic and phenotypic elements including marked induction of VE-cadherin, which was responsible for some but not all morphological changes. The present study demonstrates that of the endothelial-related genes induced by RA treatment, only a few were affected by knockdown of VE-cadherin, ruling it out as a regulator of the RA-induced endothelial genotypic switch. In contrast, knockdown of the RA-induced gene COUP-TFII prevented the formation of networks in Matrigel but had no effect on VE-cadherin induction or cell fusion. Two pan-kinase inhibitors markedly blocked RA-induced VE-cadherin expression and cell fusion. However, RA treatment resulted in a marked and broad reduction in tyrosine kinase activity. Several genes in the TGFβ signaling pathway were induced by RA, and specific inhibition of the TGFβ type I receptor blocked both RA-induced VE-cadherin expression and cell fusion. Together these data indicate a role for the TGFβ pathway and COUP-TFII in mediating the endothelial transdifferentiating properties of RA.     

Isolation and Identification of (−)- Methylcitric Acid and 2-Methyl-cis-aconitic Acid from the Culture of Candida lipolytica

Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc₂ (3.4%), Man₂FucGlcNAc₂ (2.1%), Man₃FucGlcNAc₂ (2.5%), Man₃FucXylGlcNAc₂ (42.5%), two isomers of Man₂FucXylGlcNAc₂ (39.1%), Man₃XylGlcNAc₂ (6.5%), and Man₂XylGlcNAc₂ (3.9%).