Receptors & signal transduction最新文献

Ontogeny of responsiveness to hormones is mainly regulated by the presence of receptors; their type, number, and location in the hormone target cells. Some of these parameters have been used to study the responsiveness of rat vagina to estradiol. The estrogen binding sites (EBS) in the cytosol of rat vagina are present immediately at birth, however the animal becomes responsive to the hormone only after 25 +/- 5 d of age. The authors demonstrate how the serum and tissue levels of estradiol affect the EBS in this tissue during the postnatal development of the rat. The various responses observed after a single i.p. injection of estradiol in the immature rats are explained based on the status of the binding sites for estrogen in this tissue.

Recent evidence indicates that the N-terminal extracellular domain of receptors in the secretin-glucagon receptor family is responsible for ligand recognition. In this report, the N-terminal ectodomain of the human secretin receptor (HSR) was expressed in Escherichia coli, and the ability of this recombinant protein to interact with secretin was investigated by functional assays. The cDNA region encoding the N-terminal ectodomain of HSR linked to the polyhistidine fusion partner was expressed in E. coli. The resulting fusion protein was purified and used for competitive studies. A permanently transfected cell line with the HSR expressed was used in this study. The cell line was able to respond to secretin leading to the elevation of both intracellular cAMP and protein kinase-A activity. Using this cell line, incubation of secretin with the recombinant protein led to a dose-dependent inhibition of both cAMP production and protein kinase-A activity. These findings strongly suggested that the N-terminal ectodomain of HSR alone can act as a functional domain that provides a means to study ligand-receptor interactions of this receptor. The His-tagged recombinant HSR ectodomain may also be used for screening secretin-specific agonists and antagonists by affinity chromatography in the future.

3-methylcholanthrene (MC), a potent promutagen and procarcinogen, is also an inducer of mammalian CYPIAI (cytochrome P1-450) gene. The CYPIAI enzyme is responsible for the detoxification of MC and its oxidation into reactive epoxide intermediates. Through its epoxide metabolites, MC functions also as an inducer of drug-metabolizing enzyme glutathione S-transferase (GST) gene expression. Induction of murine GST Ya gene by MC and a variety of other chemical agents is mediated by a regulatory element composed of two adjacent AP-1-like sites, and activated by the Fos/Jun heterodimeric complex (AP-1). In cultured cells, MC causes the induction of AP-1 activity, which is the result of an increased expression of c-Fos and c-Jun proteins. The mechanisms involved in MC activation of c-fos and c-jun gene expression were examined in the present study. Evidence is presented that stimulation of c-fos transcription by MC involves a signal transduction pathway, which includes activation of the small G protein Ras, Raf-1 kinase, and the mitogen-activated protein (MAP) kinases, ERK1 and ERK2. Furthermore, we find that phorbol 12-myristate 13-acetate, which uses both protein kinase C and protein-tyrosine kinase activities to induce c-fos promoter, may share a common pathway with MC downstream of Ras. The signal transduction pathway induced by MC to stimulate c-jun promoter involves Ras activation and the JNK group of MAP-kinases.

(R,S)-[125I]IQNB has been used extensively in in vivo studies in rats, and has been of utility in demonstrating the in vivo subtype selectivity of nonradioactive ligands in competition studies. Because of the implications for the study of Alzheimer's disease (AD), those ligands that demonstrate m2 selectivity are of particular interest. Radiolabelled Z- and E-(-,-)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z- and E-(-,-)-[125I]IQNP) are analogs of (R,S)-[125I]IQNB. Rat brain regional dissection studies and in vivo autoradiographic comparison of the time-courses of (R,S)-[125I]IQNB, Z-(-,-)-[125I]IQNP, and E-(-,-)-[125I]IQNP have indicated that Z- and E-(-,-)-[125I]IQNP, in general, are distributed similarly to (R,S)-[125I]IQNB. Z-(-,-)-[125I]IQNP binds to the muscarinic receptors in those brain regions enriched in the m2 subtype with approximately a two- to fivefold higher % dose/g compared with (R,S)-[125I]IQNB. Thus, as we show here autoradiographically, using QNB as the competing nonradioactive ligand in in vivo competition studies against Z-(-,-)-[125I]IQNP provides a sensitive and accurate probe for demonstrating the in vivo m2 selectivity of nonradioactive ligands.

Muscarinic acetylcholine receptors contain two highly conserved tyrosine residues that are located within or at the extracellular border of the second transmembrane domain and are unique to this subfamily of G protein-coupled receptors. These tyrosine residues are located at positions 82 and 85 of the sequence of the m1 subtype of muscarinic receptors. In this article, we studied the involvement of these two residues in ligand binding to and agonist-induced activation of this receptor subtype using site-directed mutagenesis. Our data suggest for the first time an important role of these two tyrosines in muscarinic receptor function. Evidence is also provided that although the aromatic moiety of these tyrosine residues plays a role in antagonist binding, both this moiety and the tyrosine phenolic hydroxyl group are involved in agonist binding and receptor activation. The results are discussed in terms of a possible relationship of these two tyrosine residues and other conserved tyrosine moieties located in different transmembrane segments. All of these residues might contribute in concert, albeit to different degrees, to the process of ligand binding and receptor activation. The present findings are expected to further our current understanding of the muscarinic receptor domains involved in these processes.

The platelet-activating factor (PAF) receptor (PAFR) is a G protein-coupled receptor (GPCR) that mediates a diverse array of biological responses to PAF. Recently, we provided evidence that the third intracellular domain (3i) of the rat PAFR (rPAFR) is a critical determinant in its coupling to phosphoinositide phospholipase C (PI PLC)-activating G proteins. In the present study, we assessed the potential role of a conserved alanine in the carboxyl-terminal region of 3i of the rPAFR in rPAFR signaling activity. Previous studies with the m5 muscarinic acetylcholine and human PAF receptors revealed that substitution of a carboxyl-terminal alanine was found to impair receptor-mediated PI PLC activation. Here we report the effects of the analogous nonconservative substitution of glutamate for alanine 230 of the rPAFR (rPAFR A230E) on receptor-mediated agonist binding and PI PLC activation following transient expression of the receptor cDNA. BHK cells transfected with a cDNA encoding the rPAFR A230E exhibited PAF-stimulated increases in inositol phosphate (IP) accumulation with no increase in basal levels of IPs. PAF-stimulated IP production in rPAFR transfectants was dependent on the amount of DNA transfected, although PAF provoked a larger increase in IPs in rPAFR transfectants than in rPAFRA230E transfectants in cells transfected with equal amounts of receptor cDNA. This latter finding apparently reflects differences in the transfection efficiency or expression of the wild-type and rPAFR A230E cDNAs because PAF produced indistinguishable effects on IP accumulation in rPAFR and rPAFR A230E transfectants expressing equivalent numbers of receptors. These results provide evidence for a nonconserved role of this conserved alanine in coupling of group I GPCRs to PI PLC-activating G proteins and also suggest that this residue has differential roles in regulating expression and signaling by rat and human PAFRs.

An LH/CG receptor-enriched fraction was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testicular homogenates and solubilized with Triton X-100. To confirm the functional nature of the detergent-solubilized LH/CG receptor, the extract was first incorporated by lipid hydration into phospholipid vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. LH/CG receptor incorporation was then determined by measurement of specific binding of [125I]hCG. Specific binding of [125I]hCG by the reconstituted receptor was saturable, time-dependent, and thermally stable at room temperature. Scatchard analysis of competitive binding data indicated the presence of a single class of high-affinity (6.9 x 10(10)M-1), low-capacity (17.5 fmol hCG/mg protein) binding sites. The reconstituted receptor was functionally coupled to adenylyl cyclase and responded to both LH and NaF with increased cyclic AMP (cAMP) production. Stimulation of LH/CG receptor-enriched proteoliposomes with LH resulted in concentration-dependent uptake of external calcium (as 45Ca2+), which was hormone-specific, saturable, and sensitive to blockade by voltage-dependent and voltage-independent calcium channel antagonists. Similar uptake could not be induced by sodium fluoride, (Bu)2 cAMP, GTP-gamma-S, cholera toxin, or pertussis toxin. These results indicate that the reconstituted LH/CG receptor, as is the membrane-associated receptor, is functionally coupled to signal transduction pathways involving both adenylyl cyclase activation and calcium mobilization, and is a reliable working model that will facilitate further examination of the molecular mechanisms of LH action.

17-beta-Estradiol (beta E) causes granulocytic differentiation and neutrophilia in mice. However, the presence of estrogen receptors in myeloblasts and granulocytic progenitor cells has not been reported. beta E can be converted to a bioreactive species, estradiolquinone. We have previously shown that hydroquinone (HQ), via conversion to bioreactive p-benzoquinone (BQ), causes neutrophilia in mice and induces granulocytic differentiation in myeloblasts through interaction with the leukotriene D4 (LTD4) receptor. Therefore, we tested whether beta E could be oxidized by a myeloperoxidase-mediated reaction to a bioreactive intermediate, which might, in turn, induce granulocytic differentiation in mouse myeloblasts by activating the LTD4 receptor, thus obviating the need for LTD4, the downstream intracellular mediator of granulocyte colony-stimulating factor (G-CSF)-induced signal transduction. The interleukin (IL)-3-dependent, G-CSF-inducible normal mouse myeloblastic cell line, 32D cl 3(G), was used to determine the ability of beta E to induce terminal granulocytic differentiation in myeloblasts. Morphological analysis of stage-specific granulocytic differentiation indicated that beta E was capable of the concentration- (10(-8)-10(-4)M) and time-(6d) dependent induction of a complete program of terminal granulocytic differentiation in myeloblasts similar to that seen with G-CSF or LTD4. beta E-induced granulocytic differentiation was prevented by the peroxidase inhibitor, indomethacin, and was completely and competitively inhibited in the presence of a specific LTD4 receptor antagonist, MK-571, suggesting that a bioreactive form of estradiol, such as estradiolquinone, is interacting with the receptor. beta E was shown to cause a similar concentration-dependent induction of granulocytic differentiation in human HL-60 myeloblasts that was also inhibited by the receptor antagonist. Biological effects of beta E in nontarget tissues may result from the interaction of bioreactive estradiolquinone with critical cellular macromolecules involved in normal cellular signaling pathways.

Chronic exposure of humans to benzene (BZ), a Class I carcinogen, causes acute myelogenous leukemia, possibly via its bone marrow metabolite, hydroquinone (HQ). The ability to alter cytokine-dependent growth and differentiation in hematopoietic stem or progenitor cells appears to be a property of agents with leukemogenic potential. We have previously reported that BZ and HQ specifically stimulate granulopoiesis in mice and cause granulocytic differentiation in normal murine interleukin (IL)-3-dependent, granulocyte colony-stimulating factor (G-CSF)-inducible 32D myeloblasts. BZ induces granulocytic differentiation by upregulating the production of leukotriene D4 (LTD4), an essential intracellular mediator of G-CSF signaling. We report here that HQ (0.5-4.0 microM), as well as LTD4 (1 nM-10 microM), causes a concentration-dependent induction of granulocytic differentiation in 32D myeloblasts. Unlike LTD4, which induces terminal granulocytic differentiation, HQ undergoes a myeloperoxidase-dependent oxidation to bioreactive p-benzoquinone (BQ), which induces differentiation predominantly to the myelocyte stage. Studies with the highly specific LTD4 receptor antagonist, MK-571, suggest that BQ induces granulocytic differentiation in myeloblasts by activating the LTD4 receptor, thus obviating the requirement for LTD4. This was confirmed by the demonstration that HQ, in the presence of LTD4, shifts the stage-specific pattern of terminal differentiation induced by LTD4 to the incomplete (myelocyte) profile induced by HQ. The inability of HQ to induce a complete program of terminal granulocytic differentiation in myeloblasts, as well as its ability to compete with induction by LTD4, may have a bearing on the leukemogenic potential of BZ.

Phosphoinositides bind to profilin and regulate actin-based cytoskeletal protein assembly. We report here that profilin is phosphorylated in vitro by protein kinase C (PKC) in the presence of phosphoinositides and micromolar concentrations of calcium. PKC-mediated phosphorylation of profilin was observed only in the presence of phosphoinositides; phosphatidylserine and diacylglycerol (known activators of PKC) and other lipids, including phosphatidic acid and phosphatidylglycerol phosphate, did not activate the phosphorylation. The activation of PKC-mediated phosphorylation of profilin by phosphoinositides was as follows: phosphatidylinositol (PI) 4-phosphate (K(m) = 18 microM) > PI 4,5-bisphosphate (K(m) = 30 microM) > PI (no activation). About 0.5 mol phosphate was incorporated per mol of profilin. Phosphorylation of profilin by PKC was not affected by the presence of various concentrations of actin. Phospho-amino acid analysis showed serine to be the only amino acid phosphorylated. The amino acid sequence of a phosphopeptide from CNBr-digested profilin corresponded to the COOH-terminal peptide of profilin (Ala-Ser-His-Leu-Arg-Ser-Gln-Tyr). Further digestion of this phosphopeptide by trypsin generated two phosphopeptides (Arg-Ser-Gln-Tyr and Ser-Gln-Tyr), thereby confirming that the phosphorylation site was the antepenultimate Ser (Ala-Ser-His-Leu-Arg-Arg-Ser(P)-Gln-Tyr).