Putative Membrane Receptor Research Articles

Guanine inhibits the growth of human glioma and melanoma cell lines by interacting with GPR23.

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint effector of GBP antiproliferative effects. Accordingly, cells stably expressing GPR23 showed increased sensitivity to GUA. Furthermore, while GPR23 expression was low in a hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-mutated melanoma cell line showing poor sensitivity to GBPs, and in HGPRT-silenced glioma cells, GPR23-induced expression in both cell types rescued GUA-mediated cell growth inhibition. Finally, binding experiments using [3H]-GUA and U87 cell membranes revealed the existence of a selective GUA binding (KD = 29.44 ± 4.07 nM; Bmax 1.007 ± 0.035 pmol/mg prot) likely to GPR23. Overall, these data suggest GPR23 involvement in modulating responses to GUA in tumor cell lines, although further research needs to verify whether this receptor mediates other GUA effects.

Dormant spores sense amino acids through the B subunits of their germination receptors

Bacteria from the orders Bacillales and Clostridiales differentiate into stress-resistant spores that can remain dormant for years, yet rapidly germinate upon nutrient sensing. How spores monitor nutrients is poorly understood but in most cases requires putative membrane receptors. The prototypical receptor from Bacillus subtilis consists of three proteins (GerAA, GerAB, GerAC) required for germination in response to L-alanine. GerAB belongs to the Amino Acid-Polyamine-Organocation superfamily of transporters. Using evolutionary co-variation analysis, we provide evidence that GerAB adopts a structure similar to an L-alanine transporter from this superfamily. We show that mutations in gerAB predicted to disrupt the ligand-binding pocket impair germination, while mutations predicted to function in L-alanine recognition enable spores to respond to L-leucine or L-serine. Finally, substitutions of bulkier residues at these positions cause constitutive germination. These data suggest that GerAB is the L-alanine sensor and that B subunits in this broadly conserved family function in nutrient detection.

Classic and Novel Sex Hormone Binding Globulin Effects on the Cardiovascular System in Men.

In men, 70% of circulating testosterone binds with high affinity to plasma sex hormone binding globulin (SHBG), which determines its bioavailability in their target cells. In recent years, a growing body of evidence has shown that circulating SHBG not only is a passive carrier for steroid hormones but also actively regulates testosterone signaling through putative plasma membrane receptors and by local expression of androgen-binding proteins apparently to reach local elevated testosterone concentrations in specific androgen target tissues. Circulating SHBG levels are influenced by metabolic and hormonal factors, and they are reduced in obesity and insulin resistance, suggesting that SHBG may have a broader clinical utility in assessing the risk for cardiovascular diseases. Importantly, plasma SHBG levels are strongly correlated with testosterone concentrations, and in men, low testosterone levels are associated with an adverse cardiometabolic profile. Although obesity and insulin resistance are associated with an increased incidence of cardiovascular disease, whether they lead to abnormal expression of circulating SHBG or its interaction with androgen signaling remains to be elucidated. SHBG is produced mainly in the liver, but it can also be expressed in several tissues including the brain, fat tissue, and myocardium. Expression of SHBG is controlled by peroxisome proliferator-activated receptor γ (PPARγ) and AMP-activated protein kinase (AMPK). AMPK/PPAR interaction is critical to regulate hepatocyte nuclear factor-4 (HNF4), a prerequisite for SHBG upregulation. In cardiomyocytes, testosterone activates AMPK and PPARs. Therefore, the description of local expression of cardiac SHBG and its circulating levels may shed new light to explain physiological and adverse cardiometabolic roles of androgens in different tissues. According to emerging clinical evidence, here, we will discuss the potential mechanisms with cardioprotective effects and SHBG levels to be used as an early metabolic and cardiovascular biomarker in men.

Putative Membrane Receptors Contribute to Activation and Efficient Signaling of Mitogen-Activated Protein Kinase Cascades during Adaptation of Aspergillus fumigatus to Different Stressors and Carbon Sources.

The high-osmolarity glycerol (HOG) response pathway is a multifunctional signal transduction pathway that specifically transmits ambient osmotic signals. Saccharomyces cerevisiae Hog1p has two upstream signaling branches, the sensor histidine kinase Sln1p and the receptor Sho1p. The Sho1p branch includes two other proteins, the Msb2p mucin and Opy2p. Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Here, we investigated the roles played by A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p putative homologues during the activation of the mitogen-activated protein kinase (MAPK) HOG pathway. The shoA, msbA, and opyA singly and doubly null mutants are important for the cell wall integrity (CWI) pathway, oxidative stress, and virulence as assessed by a Galleria mellonella model. Genetic interactions of ShoA, MsbA, and OpyA are also important for proper activation of the SakAHog1p and MpkASlt2 cascade and the response to osmotic and cell wall stresses. Comparative label-free quantitative proteomics analysis of the singly null mutants with the wild-type strain upon caspofungin exposure indicates that the absence of ShoA, MsbA, and OpyA affects the osmotic stress response, carbohydrate metabolism, and protein degradation. The putative receptor mutants showed altered trehalose and glycogen accumulation, suggesting a role for ShoA, MsbA, and OpyA in sugar storage. Protein kinase A activity was also decreased in these mutants. We also observed genetic interactions between SlnA, ShoA, MsbA, and OpyA, suggesting that both branches are important for activation of the HOG/CWI pathways. Our results help in the understanding of the activation and modulation of the HOG and CWI pathways in this important fungal pathogen.IMPORTANCEAspergillus fumigatus is an important human-pathogenic fungal species that is responsible for a high incidence of infections in immunocompromised individuals. A. fumigatus high-osmolarity glycerol (HOG) and cell wall integrity pathways are important for the adaptation to different forms of environmental adversity such as osmotic and oxidative stresses, nutrient limitations, high temperatures, and other chemical and mechanical stresses that may be produced by the host immune system and antifungal drugs. Little is known about how these pathways are activated in this fungal pathogen. Here, we characterize four A. fumigatus putative homologues that are important for the activation of the yeast HOG pathway. A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p are genetically interacting and are essential for the activation of the HOG and cell wall integrity pathways. Our results contribute to the understanding of A. fumigatus adaptation to the host environment.

Hybrid Antigens Expressing Surface Loops of ZnuD From Acinetobacter baumannii Is Capable of Inducing Protection Against Infection.

Acinetobacter baumannii is an important human pathogen causing substantial mortality in hospitalized patients for which treatment with antibiotics has become problematic due to growing antibiotic resistance. In an attempt to develop alternative strategies for dealing with these serious infections surface antigens are being considered as targets for vaccines or immunotherapy. The surface receptor proteins required for zinc acquisition in Gram-negative bacterial pathogens have been proposed as vaccine targets due to their crucial role for growth in the human host. In this study we selected the putative ZnuD outer membrane receptor from A. baumannii as a target for vaccine development. Due to challenges in production of an integral outer membrane protein for vaccine production, we adopted a recently described hybrid antigen approach in which surface epitopes from the Neisseria meningitidis TbpA receptor protein were displayed on a derivative of the C-lobe of the surface lipoprotein TbpB, named the loopless C-lobe (LCL). A structural model for ZnuD was generated and four surface loops were selected for hybrid antigen production by computational approaches. Hybrid antigens were designed displaying the four selected loops (2, 5, 7, and 11) individually or together in a single hybrid antigen. The hybrid antigens along with ZnuD and the LCL scaffold were produced in the E. coli cytoplasm either as soluble antigens or as inclusion bodies, that were used to generate soluble antigens upon refolding. Mice were immunized with the hybrid antigens, ZnuD or LCL and then used in an A. baumannii sepsis model to evaluate their ability to protect against infection. As expected, the LCL scaffold did not induce a protective immune response, enabling us to attribute observed protection to the displayed loops. Immunization with the refolded ZnuD protein protected 63% of the mice while immunization with hybrid antigens displaying individual loops achieved between 25 and 50% protection. Notably, the mice immunized with the hybrid antigen displaying the four loops were completely protected from infection.

Cell signaling dependence of rapid glucocorticoid-induced endocannabinoid synthesis in hypothalamic neuroendocrine cells

Glucocorticoids induce a rapid synthesis of endocannabinoid in hypothalamic neuroendocrine cells by activation of a putative membrane receptor. Somato-dendritically released endocannabinoid acts as a retrograde messenger to suppress excitatory synaptic inputs to corticotropin-releasing hormone-, oxytocin-, and vasopressin-secreting cells. The non-genomic signaling mechanism responsible for rapid endocannabinoid synthesis by glucocorticoids has yet to be fully characterized. Here we manipulated cell signaling molecules pharmacologically using an intracellular approach to elucidate the signaling pathway activated by the membrane glucocorticoid receptor in hypothalamic neuroendocrine cells. We found that rapid glucocorticoid-induced endocannabinoid synthesis in magnocellular neuroendocrine cells requires the sequential activation of multiple kinases, phospholipase C, and intracellular calcium mobilization. While there remain gaps in our understanding, our findings reveal many of the critical players in the rapid glucocorticoid signaling that culminates in the retrograde endocannabinoid modulation of excitatory synaptic transmission.

Purity and stability of the membrane-limited glucocorticoid receptor agonist dexamethasone-BSA

Cellular effects of glucocorticoids can be separated into classical transcriptional regulation via activation of the canonical nuclear glucocorticoid receptor and rapid actions mediated by activation of one or more putative membrane-associated glucocorticoid receptors that regulate both transcriptional and non-transcriptional signaling. Dexamethasone-bovine serum albumin (Dex-BSA) is one of several membrane-limited steroid receptor agonists. Dex-BSA and other steroid conjugates such as corticosterone-, estradiol- and testosterone-BSA have been used to study rapid steroid effects initiated by putative membrane receptors. The purity and stability of the steroid-BSA conjugate is crucial, therefore, since any steroid that is not bound to or that dissociates from the BSA conjugate could penetrate into the intracellular compartment and confound the experiment. We used fluorine NMR to determine if free Dex could be detected in a commercially available Dex-BSA dissolved in H2O. Non-covalently bound Dex was detected in the Dex-BSA solution, but the level of free Dex remained constant over time and with increasing temperature, indicating that the free Dex was not a result of instability of the Dex-BSA conjugate. The free Dex was lost when the Dex-BSA was denatured and subjected to dialysis, which suggested that it was trapped in the Dex-BSA three-dimensional structure and not covalently bound to the BSA. The purified, renatured Dex-BSA retained its rapid activity, which confirmed that the observed effects of Dex-BSA are not caused by non-covalently-bound Dex. Therefore, the Dex contaminant found in the Dex-BSA solution is likely to be tightly, but non-covalently, bound to BSA, and the Dex-BSA activity remains membrane-limited. Our findings indicate that Dex-BSA remains a suitable membrane-restricted glucocorticoid receptor agonist, but suggest that denaturing purification is a useful control for the study of membrane-initiated steroid-BSA actions.

Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands.

The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands.

Molecular mechanism involved in the response to hydrogen peroxide stress in Acinetobacter oleivorans DR1.

Two-dimensional gel electrophoresis was conducted to investigate the effect of H2O2 on whole protein expression in Acinetobacter oleivorans DR1. Functional classification of 13 upregulated proteins using MALDI-TOF mass spectrometry showed relationships with oxidative stress, energy production and conversion, nucleotide and amino acid metabolism, membrane-related, ion transport, and chaperone-related functions. Alignment of OxyR-binding regions from Pseudomonas aeruginosa and Escherichia coli with promoters of identified proteins revealed that only ahpC, ahpF, and trxB (thioredoxin-disulfide reductase) genes, along with a newly found oprC (putative outer membrane receptor protein) gene, have OxyR-binding sites. The oxyR and ahpC mutants were more sensitive to H2O2 and showed growth defects in both nutritional and n-hexadecane-amended media. Four catalases present in the genome of A. oleivorans DR1 were not detected, which led us to confirm the expression and activity of those catalases in the presence of H2O2. The expression patterns of the four catalase genes differed at different concentrations of H2O2. Interestingly, the promoters of both known OxyR-controlled katG gene (AOLE_17390) and putative small catalase gene (AOLE_09800) have OxyR-binding sites. Gel-shift assay confirmed OxyR binding to the promoter regions of newly identified OxyR-controlled genes encoding OprC and a putative catalase. Hierarchical expression and OxyR-binding of several OxyR-controlled genes suggested that concentration is an important factor in inducing the set of genes under H2O2 stress.

Expression of corticosteroid-binding globulin CBG in the human heart.

Glucocorticoids are known to be involved in myocardial regeneration and destruction. Cardiomyocytes are mostly devoid of nuclear glucocorticoid receptors (GRs) and it is generally assumed that effects of adrenal steroids in heart are mediated through the mineralocorticoid receptor (MR). Here we used immunocytochemistry to study localization of corticosteroid binding globulin (CBG) in semithin sections of human cardiac tissue samples. With staining of consecutive sections we examined colocalization with GR and MR immunoreactivities. While GR staining was almost undetectable, a portion of myocytes with MR immunostained nuclei was found. Almost all cardiomyocytes exhibited CBG immunostaining in cytoplasm and on the cell membrane. Most pronounced CBG immunoreactivities were found in Purkinje fibers and in smooth muscle cells of arterial walls. With RT-PCR, we found in homogenates of cardiac tissue detectable levels of CBG encoding mRNA. Our findings indicate that CBG is expressed in human heart. Known cardiac effects of adrenal steroids may in part be mediated through the binding globulin and its putative membrane receptor in addition to nuclear steroid receptors and direct genomic action. Highlights of our study: Human cardiomyocytes express mineralocorticoid receptors, but are mostly free of nuclear glucocorticoid receptors. CBG is expressed in myocardium and in Purkinje fibers. CBG in heart is colocalized with mineralocorticoid receptor. Endothelia and smooth muscle cells of arterial walls show colocalization of CBG and MR.

Genome-Wide Detection of Fitness Genes in Uropathogenic Escherichia coli during Systemic Infection

Uropathogenic Escherichia coli (UPEC) is a leading etiological agent of bacteremia in humans. Virulence mechanisms of UPEC in the context of urinary tract infections have been subjected to extensive research. However, understanding of the fitness mechanisms used by UPEC during bacteremia and systemic infection is limited. A forward genetic screen was utilized to detect transposon insertion mutants with fitness defects during colonization of mouse spleens. An inoculum comprised of 360,000 transposon mutants in the UPEC strain CFT073, cultured from the blood of a patient with pyelonephritis, was used to inoculate mice intravenously. Transposon insertion sites in the inoculum (input) and bacteria colonizing the spleen (output) were identified using high-throughput sequencing of transposon-chromosome junctions. Using frequencies of representation of each insertion mutant in the input and output samples, 242 candidate fitness genes were identified. Co-infection experiments with each of 11 defined mutants and the wild-type strain demonstrated that 82% (9 of 11) of the tested candidate fitness genes were required for optimal fitness in a mouse model of systemic infection. Genes involved in biosynthesis of poly-N-acetyl glucosamine (pgaABCD), major and minor pilin of a type IV pilus (c2394 and c2395), oligopeptide uptake periplasmic-binding protein (oppA), sensitive to antimicrobial peptides (sapABCDF), putative outer membrane receptor (yddB), zinc metallopeptidase (pqqL), a shikimate pathway gene (c1220) and autotransporter serine proteases (pic and vat) were further characterized. Here, we report the first genome-wide identification of genes that contribute to fitness in UPEC during systemic infection in a mammalian host. These fitness factors may represent targets for developing novel therapeutics against UPEC.

Jasmonic acid and glucose synergistically modulate the accumulation of glucosinolates in Arabidopsis thaliana.

The interplay of plant hormones and glucose (Glu) in regulating glucosinolate accumulation in Arabidopsis thaliana was investigated in this study. Glucose-induced glucosinolate biosynthesis was enhanced significantly by the addition of jasmonic acid (JA), whereas the synergistic effect of salicylic acid (SA) and Glu was less obvious. The enhanced glucosinolate accumulation is associated with elevated expression of genes in glucosinolate biosynthetic pathway, as well as the transcription factors involved in their regulation, such as MYB28, MYB29, MYB34, and MYB122. The induction of indolic and aliphatic glucosinolates after treatment with JA and Glu in JA-insensitive mutants, coi1, jar1, and jin1, was compromised. Moreover, the effect of JA and Glu on glucosinolate contents was dramatically reduced in Glu-insensitive mutants, rgs1-2 and abi5-7. These results indicate a crosstalk between JA and Glu signalling in the regulation of glucosinolate biosynthesis. JA signalling, RGS1 (the putative membrane receptor of Glu signalling), and ABI5, are involved in the synergistic effect of JA and Glu on glucosinolate accumulation.

Characterization of membrane receptor binding activity for cortisol in the liver and kidney of the euryhaline teleost, Mozambique tilapia (Oreochromis mossambicus)

Glucocorticoids (GCs) regulate an array of physiological responses in vertebrates. Genomic GC actions mediated by nuclear steroid receptors require a lag time on the order of hours to days to generate an appreciable physiological response. Experimental evidence has accumulated that GCs, can also act rapidly through a nongenomic mechanism to modulate cellular physiology in vertebrates. Causal evidence in the Mozambique tilapia (Oreochromis mossambicus) suggests that the GC cortisol exerts rapid, nongenomic actions in the gills, liver, and pituitary of this euryhaline teleost, but the membrane receptor mediating these actions has not been characterized. Radioreceptor binding assays were conducted to identify a putative GC membrane receptor site in O. mossambicus. The tissue distribution, binding kinetics, and pharmacological signature of the GC membrane-binding activity were characterized. High affinity (Kd=9.527±0.001nM), low-capacity (Bmax=1.008±0.116fmol/mg protein) [3H] cortisol binding was identified on plasma membranes prepared from the livers and a lower affinity (Kd=30.08±2.373nM), low capacity (Bmax=4.690±2.373fmol/mg protein) binding was found in kidney membrane preparations. Competitors with high binding affinity for nuclear GC receptors, mifepristone (RU486), dexamethasone, and 11-deoxycorticosterone, displayed no affinity for the membrane GC receptor. The association and dissociation kinetics of [3H] cortisol binding to membranes were orders of magnitude faster (t1/2=1.7–2.6min) than those for the intracellular (nuclear) GC receptor (t1/2=10.2h). Specific [3H] cortisol membrane binding was also detected in the gill and pituitary but not in brain tissue. This study represents the first characterization of a membrane GC receptor in fishes and one of only a few characterized in vertebrates.

Understanding the Direct Synaptic Effects of Estradiol

More than 35 years ago, Kelly and coworkers (1) demonstrated that iontophoretic application of an estradiol ester altered the firing rates of a subset of hypothalamic neurons in a matter of milliseconds, an effect that appeared to be stereospecific in that responses were observed to esters of 17 -estradiol, but not its 17 isomer. These observations accompanied the almost simultaneous demonstration by Pietras and Szego (2) that endometrial cells had estradiol recognition sites on their plasma membranes. Kelly et al (1) proposed that neurons might also express stereospecific membrane receptor sites, capable of mediating rapid electrophysiological responses to changes in ovarian estradiol secretion. Progress in characterizing these putative membrane receptors was initially slow, because most work continued to focus on estrogen receptor (ER) actions in the cell nucleus and the regulation of transcriptional response mechanisms. This situation changed dramatically in the early 1990s with the demonstration of rapid effects of estradiol on a number of intracellular signaling pathways (3, 4) as well as direct evidence of ER-like immunoreactivity in dendrites, axon terminals, and postsynaptic densities in the brain (5–7) (reviewed in Blaustein, Ref. 8). Over the ensuing 20 years, multiple potential receptor systems for estradiol have been identified in the central nervous system, signaling via a number of different intracellular pathways (reviewed in Micevych and Christensen, Ref. 9). Studies in cells transfected with vectors expressing ER or ER have shown that whereas newly synthesized receptors concentrate in the cell nucleus, they also incorporate into the plasma membrane (10) where they can initiate estradiol-dependent changes in ERK phosphorylation (11), suggesting that plasma membrane and nuclear ER or ER may be derived from a common source. The demonstration that ER immunoreactivity is present in multiple neuronal cellular compartments raises the question of how the distribution of extranuclear ER is controlled. Are ERs associated with specific cellular organelles or do synaptic and dendritic ERs represent receptors en passant to the plasma membrane? Studies in nonneuronal systems have demonstrated that that ER is trafficked to the plasma membrane via a caveolin-dependent process (12) where it can be anchored in place by palmitoylation (13). Is the same true for ER in neurons? Is the density of ER in the plasma membrane regulated independently of nuclear receptor concentrations? Is ER distributed evenly throughout neuronal cell membranes or is it concentrated specifically in target sites where it can both respond to locally synthesized steroids and exert rapid effects on neurotransmission? The paper by Tabatadze et al (14) in this issue of Endocrinology begins to answer some of these questions. Using tissue fractionationtechniques, theauthorsdemonstrate that ER is concentrated in synaptosomes, particularly in association with synaptic vesicles and neuropeptide-containing large dense-core vesicles. Immunoisolation experiments showthatER is associatedwithbothgammaaminobutryic acid (GABA)and glutamate-containing vesicles. On Western blots, synaptosomal ER immunoreactivity migrates as two bands at approximately 66 and 70 kDa, the ratio of the higher to lower molecular mass forms being greatly decreased by phosphatase treatment, suggesting that the 70kDa band represents a phosphorylated form of the receptor. Finally, treatment of hippocampal slices in vitro was found to decrease the quantity of ER associated with synaptosomal membranes, via a depalmitoylation-dependent mechanism. Overall, the results suggest that ER associates with synaptosomal membranes via a palmitoyla-

Zebrafish Dkk3a Protein Regulates the Activity of myf5 Promoter through Interaction with Membrane Receptor Integrin α6b

Myogenic regulatory factor Myf5 plays important roles in muscle development. In zebrafish myf5, a microRNA (miR), termed miR-3906 or miR-In300, was reported to silence dickkopf-3-related gene (dkk3r or dkk3a), resulting in repression of myf5 promoter activity. However, the membrane receptor that interacts with ligand Dkk3a to control myf5 expression through signal transduction remains unknown. To address this question, we applied immunoprecipitation and LC-MS/MS to screen putative membrane receptors of Dkk3a, and Integrin α6b (Itgα6b) was finally identified. To further confirm this, we used cell surface binding assays, which showed that Dkk3a and Itgα6b were co-expressed at the cell membrane of HEK-293T cells. Cross-linking immunoprecipitation data also showed high affinity of Itgα6b for Dkk3a. We further proved that the β-propeller repeat domains of Itgα6b are key segments bound by Dkk3a. Moreover, when dkk3a and itgα6b mRNAs were co-injected into embryos, luciferase activity was up-regulated 4-fold greater than that of control embryos. In contrast, the luciferase activities of dkk3a knockdown embryos co-injected with itgα6b mRNA and itgα6b knockdown embryos co-injected with dkk3a mRNA were decreased in a manner similar to that in control embryos, respectively. Knockdown of itgα6b resulted in abnormal somite shape, fewer somitic cells, weaker or absent myf5 expression, and reduced the protein level of phosphorylated p38a in somites. These defective phenotypes of trunk muscular development were similar to those of dkk3a knockdown embryos. We demonstrated that the secreted ligand Dkk3a binds to the membrane receptor Itgα6b, which increases the protein level of phosphorylated p38a and activates myf5 promoter activity of zebrafish embryos during myogenesis.

1α,25-Dihydroxyvitamin D3 Signaling Pathways on Calcium Uptake in 30-Day-Old Rat Sertoli Cells

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.

Estradiol Meets Notch Signaling in Developing Neurons

The transmembrane receptor Notch, a master developmental regulator, controls gliogenesis, neurogenesis, and neurite development in the nervous system. Estradiol, acting as a hormonal signal or as a neurosteroid, also regulates these developmental processes. Here we review recent evidence indicating that estradiol and Notch signaling interact in developing hippocampal neurons by a mechanism involving the putative membrane receptor G protein-coupled receptor 30. This interaction is relevant for the control of neuronal differentiation, since the downregulation of Notch signaling by estradiol results in the upregulation of neurogenin 3, which in turn promotes dendritogenesis.

Oenocytoid cell lysis to release prophenoloxidase is induced by eicosanoid via protein kinase C

Eicosanoids mediate insect cellular immune responses, which depend largely on phenoloxidase (PO) activity. In plasma, PO is activated by the proteolytic cleavage of proPO, which is stored in oenocytoids, a specific hemocyte type, of the beet armyworm, Spodoptera exigua. Eicosanoids induce an acute cell lysis of oenocytoids, which releases proPO into the plasma. We investigated an intracellular signal pathway following a functional interaction of eicosanoid(s) to a putative membrane receptor. U-73122 (a specific inhibitor of phospholipase C) inhibited oenocytoid lysis of S. exigua significantly after bacterial infection. We concluded that oenocytoid lysis required a certain level of calcium ion because EGTA (a calcium chelator) treatment inhibited cell lysis. Two protein kinase C (PKC) inhibitors (staurosporine and calphostin C) significantly inhibited the oenocytoid lysis. Oenocytoid lysis was likely induced by Na + entry and subsequent osmotic shock because juvenile hormone analog, pyriproxyfen, which activates Na +-K + ATPase and induces subsequent cell shrinkage, antagonized the effect of eicosaniod on cell lysis. Furthermore, ouabain (a specific Na + pump inhibitor) significantly inhibited oenocytoid lysis. These results suggest that eicosanoid mediates oenocytoid lysis by activating the intracellular PKC pathway.

Nongenomic steroid actions: completing the puzzle. Aldosterone as an example.

In the common unidimensional theory of steroid action, steroids bind to intracellular receptors and modulate nuclear transcription and thus protein synthesis. These genomic steroid effects, being characterized by their delayed onset of action and their dependence on transcription and protein synthesis, have been known for several decades. In contrast, very rapid actions of steroids, which are considered to be of nongenomic origin, have been recognized more widely and characterized in detail only during the past ten years. Specific rapid effects of steroids and related hormones like vitamin D3 and thyroid hormones on cellular function involve a conventional second messenger cascade which in most cases includes phospholipase C, phosphoinositide turnover, intracellular pH and intracellular calcium ([Ca2+]i), and protein kinase C. Furthermore, binding sites in membranes have been characterized exposing binding features compatible with an involvement in rapid-steroid signaling. Characteristics of putative membrane receptors are completely different from those of classic intracellular steroid receptors; this also includes the inability of classic steroid receptor antagonists to inhibit those rapid nongenomic steroid actions. The physiological and pathophysiological relevance of these effects is still largely unclear, but their existence has been proven recently even under in vivo conditions. New drugs modulating nongenomic steroid actions may find applications in various areas such as the cardiovascular and central nervous systems, infertility and electrolyte homeostasis. This short review focuses mainly on the nongenomic actions of aldosterone and their cardiovascular implications.

Pharmacological characterization of intracellular, membrane, and plasma binding sites for corticosterone in house sparrows

The diversity and specificity of glucocorticoid effects are dependent on cell-specific receptor mechanisms. Three known corticosteroid receptors mediate tissue effects of glucocorticoids in vertebrates: two intracellular receptors that act primarily as ligand-activated transcription factors, and a membrane-associated receptor. The intracellular receptor sub-types have been well characterized in mammals, however relatively little is known about them across non-mammalian vertebrates. The membrane-associated receptors are poorly characterized in most vertebrate taxa. To explore the basis for glucocorticoid action in birds, we pharmacologically characterized the three putative corticosteroid receptors in the brain, as well as a plasma corticosterone binding globulin, in the house sparrow ( Passer domesticus). We found that house sparrow brain cytosol contained two distinguishable binding sites for corticosterone. A high affinity, mineralocorticoid-like receptor had subnanomolar affinity for corticosterone ( K d ∼ 0.2 nM). However, this ‘MR-like’ high-affinity receptor did not bind RU28318 or canrenoic acid, two compounds that bind mammalian MR with high affinity. A lower-affinity, glucocorticoid-like receptor in brain cytosol bound corticosterone with an average K d = 5.61 nM. This GR-like receptor showed subnanomolar affinity for RU 486. MR- and GR-like receptors were found in equal numbers in whole brain assays (average B max = 69 and 62 fmol/mg protein, respectively). House sparrow brain membranes contain a single binding site specific for glucocorticoids, with characteristics consistent with a steroid/receptor interaction. Corticosterone affinity for this putative membrane receptor was approximately 24 nM, with apparent B max = 177 fmol/mg protein. House sparrow plasma contained a single binding site for [ 3H]corticosterone. Specific binding to plasma sites was inhibited by glucocorticoids, progesterone, and testosterone. Testosterone binding to this corticosteroid binding globulin is noteworthy as sex steroid-specific binding globulins have not been identified in birds. Taken together, these data extend our ability to evaluate the comparative actions of glucocorticoids, increase our understanding of mechanisms behind the tissue specificity of glucocorticoid action, and offer insight into the evolution of glucocorticoid action in vertebrates.