Receptors & signal transduction最新文献

{"title":"Role of two highly conserved tyrosine residues in the m1 muscarinic receptor second transmembrane domain in ligand binding and receptor function.","authors":"S Y Lee,&nbsp;S Z Zhu,&nbsp;E E el-Fakahany","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 1","pages":"43-52"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19897020","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Functional reconstitution of detergent-solubilized bovine calf testis luteinizing hormone/chorionic gonadotropin receptor into phospholipid vesicles.","authors":"P Grasso","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 2","pages":"53-62"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19974953","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Induction of granulocytic differentiation in myeloblasts by 17-beta-estradiol involves the leukotriene D4 receptor.","authors":"V Dietsch,&nbsp;G F Kalf,&nbsp;B A Hazel","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 2","pages":"63-75"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19974954","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of a glucocorticoid repressor domain in the rat beta 1-adrenergic receptor gene.","authors":"S W Bahouth,&nbsp;E A Park,&nbsp;M Beauchamp,&nbsp;X Cui,&nbsp;C C Malbon","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The expression of the gene encoding the rat beta 1-adrenergic receptor is suppressed by glucocorticoids (Kiely et al., 1994). Within the 3.2-kb 5'-flanking region of the promoter, two potential glucocorticoid response elements (GREs) at -950 and -2791 relative to the translational ATG were identified. Characterization of the glucocorticoid-responsive sequences in the 5'-flanking region of the beta 1-adrenergic receptor gene was explored in rat C6 glioma cells and human HepG2 hepatoma cells using transient expression of beta 1-adrenergic receptor-luciferase fusion genes. The ability of glucocorticoids to suppress luciferase expression was not altered when the most 5'-localized GRE was deleted. Deleting the potential GRE at -950, in contrast, abolished glucocorticoid-induced suppression of the beta 1-adrenergic receptor-luciferase gene transcription. A 25-bp element containing the GRE sequence between nucleotides -950 and -926 confers glucocorticoid-dependent inhibition of transcription to a neutral promoter. Gel mobility shift assays with the alpha-subunit of the human glucocorticoid receptor (hGR alpha) expressed in reticulocyte lysates demonstrated specific binding to the 25-bp sequence harboring the putative GRE. We report an inhibitory GRE in the promoter of the rat beta 1-adrenergic receptor gene that is conserved among the rat, human, and mouse genes.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 3-4","pages":"141-9"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20201005","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Alpha 1 adrenergic receptor activation of proto-oncogene expression in arterial smooth muscle: regulation by nitric oxide and vascular injury.","authors":"M Okazaki,&nbsp;Z W Hu,&nbsp;M Fujinaga,&nbsp;B B Hoffman","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The role of the endothelium in modulating smooth muscle cell growth is unclear. alpha 1 adrenergic receptors activate proto-oncogene expression in smooth muscle. We have now found in rat aorta that carbachol, a muscarinic cholinergic agonist that promotes release of nitric oxide (NO), inhibits expression of c-fos and c-jun mRNA induced by alpha 1 receptors. NO synthase inhibitors blocked the effects of carbachol on c-fos mRNA and a cGMP analog mimicked carbachol. After balloon injury in rat aorta using in situ hybridization, the catecholamine-induced increase in c-fos mRNA expression in the medial layer was inhibited by the alpha 1 receptor antagonists, prazosin and chloroethylclonidine. In the neointima, this response was fully blocked by prazosin; however, chloroethylclonidine only partially inhibited it. These results suggest that NO, acting through a cGMP-dependent mechanism, inhibits expression of the c-fos and c-jun genes in arteries, which may contribute to the growth-inhibiting effects of the endothelium. After endothelial damage, the activation of c-fos in neointima by adrenergic stimulation may involve a subtype of alpha 1 receptor different from that utilized in medial smooth muscle.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 3-4","pages":"165-78"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20201007","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protein kinase C inhibits the Ca(2+)-dependent stimulation of phospholipase C-beta 1 in vitro.","authors":"I Litosch","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Protein kinase C (PKC) inhibited the Ca(2+)-dependent stimulation of a 600-fold purified phospholipase C beta 1 (PLC-beta 1). Inhibition by PKC was time-dependent, and required ATP and diacylglycerol. Inhibition was more pronounced when the PLC assay was conducted with a PIP2 substrate mixture containing phosphatidylserine, then with a substrate mixture containing phosphatidyle-thanolamine. Cyclic AMP-dependent protein kinase A did not inhibit PLC-beta 1 activity. PKC did not affect the rate of PLC-beta 1 activation by Ca2+ or the rate of PLC-beta 1 deactivation by EGTA. PLC-beta 1 purified 1700-fold was less sensitive to inhibition by PKC despite stoichiometric phosphorylation. These results demonstrate that PKC inhibits the Ca(2+)-dependent stimulation of a 600-fold purified PLC-beta 1 in vitro. Furthermore, purification of PLC-beta 1 to homogeneity results in a diminished sensitivity to inhibition by PKC, indicating that other components may participate in mediating the effect of PKC on the Ca(2+)-dependent stimulation of PLC-beta 1 in vitro.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 2","pages":"87-98"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19976765","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vivo autoradiographic and dissection evaluation of isomers of 125I-labeled 1-azabicyclo[2.2.2] oct-3-yl-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (IQNP).","authors":"M R Rayeq,&nbsp;S F Boulay,&nbsp;V K Sood,&nbsp;D W McPherson,&nbsp;F F Knapp,&nbsp;B R Zeeberg","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>(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. Radiolabeled 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. Preliminary rat brain regional dissection studies have indicated that Z- and E-(-,-)-[125I]IQNP, in general, are distributed similarly to (R,S)-[125I]IQNB. An important observation is that Z-(-,-)-[125I]IQNP binds to the muscarinic receptors in those brain regions enriched in the m2 subtype with approximately a two- to fivefold higher percent dose/g compared to (R,S)-[125I]IQNB. These observations are confirmed here by in vivo autoradiographic comparison of the time-courses of (R,S)-[125I]IQNB, Z-(-,-)-[125I]IQNP, and E-(-,-)-[125I]IQNP. Thus, in vivo competition studies against Z-(-,-)-[125I]IQNP would provide a potentially more sensitive and accurate probe for demonstrating the in vivo m2 selectivity of the nonradioactive ligands. In addition, Z-(-,-)-[123I]IQNP would potentially be useful for SPECT imaging of muscarinic receptor loss in neurodegenerative diseases.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 1","pages":"13-34"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"19897018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Effects of hypertonic sucrose and potassium depletion on the binding properties of beta and alpha 1 adrenergic receptors measured on intact cells.","authors":"S J Zhu,&nbsp;L I Hatcher,&nbsp;J C Brown,&nbsp;S M Whittle,&nbsp;M L Toews","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The effects of hypertonic sucrose and of intracellular potassium depletion on the intact-cell-binding properties of beta (beta AR) and alpha 1 (alpha 1AR) adrenergic receptors of DDT1 MF-2 hamster smooth muscle cells were investigated. These treatments are known to block clathrin assembly into coated pits, an early step in the pathway of receptor endocytosis. Conducting intact-cell-binding assays in the presence of 0.4 M sucrose markedly decreased the fraction of both beta ARs and alpha 1ARs converted during the assay to a form exhibiting low apparent affinity for agonists. Intracellular potassium depletion also decreased the fraction of both beta ARs and alpha 1ARs converted to this low-affinity form. In contrast the intact-cell-binding properties of antagonists were unaltered by these treatments. These results suggest a role for receptor internalization in conversion of both beta ARs and alpha 1ARs to their low-affinity forms. A model is proposed in which either an initial agonist-induced receptor sequestration within the plasma membrane or the subsequent endocytosis of receptors into intracellular vesicles allows conversion of these receptors to the form exhibiting low affinity for agonists in binding assays with intact cells.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 3-4","pages":"131-40"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20201004","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Occupancy and composition of proteins bound to the AP-1 sites in the glucocorticoid receptor and c-jun promoters after glucocorticoid treatment and in different cell types.","authors":"T J Barrett,&nbsp;W V Vedeckis","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>The glucocorticoid receptor (GR) and c-jun promoters both contain activator protein-1 (AP-1) sites (GR AP-1 site and c-jun AP-1 site, respectively) that vary from the consensus AP-1 site. Electrophoretic mobility shift assays (EMSAs) were used to monitor GR AP-1 and c-jun AP-1 oligonucleotide binding by nuclear extracts from AtT-20 and L929 cells that were hormone- and vehicle-treated for 1, 6, or 24 h. Both AtT-20 and L929 cell nuclear extracts bound the c-jun AP-1 site somewhat better than the GRAP-1 site and, in the majority of cases, extracts from hormone-treated cells shifted both GRAP-1 and c-jun AP-1 oligonucleotides more than nontreated nuclear extracts. Supershift assays, using Jun and Fos family member-specific antibodies, showed that protein complexes formed by AtT-20 cell nuclear extracts bound to the c-jun AP-1 site were comprised of Jun family members, JunD, JunB, and cJun. No Fos family members were present. However, protein complexes from AtT-20 nuclear extracts that bound the GR AP-1 site were supershifted by JunD, JunB, cJun, and Fra-2 specific antibodies. In L929 cell nuclear extracts, the c-jun AP-1 site is bound by JunD and cJun. No clear association of Fos family members with the c-jun AP-1 site could be demonstrated. The GR AP-1 site bound protein complexes composed of JunD, JunB, Fra-2, and Fra-1 from L929 nuclear extracts. This demonstrates that the composition of the protein complexes that associate with the c-jun AP-1 site differs from those that bind the GR AP-1 site. These data also indicate that the protein complexes that bind the GR and c-jun AP-1 sites are cell-type-specific. Computer analysis also revealed five putative cyclic AMP response elements (CREs) in the GR promoter. Relative mobility shift and binding studies suggest that CRE binding protein (CREB), CREB modulator (CREM), or CREB/CREM may be associated with the c-jun AP-1 and/or GR AP-1 sites, but the association at these sites occurs at a lower binding affinity than for a consensus CRE. Nuclear extracts from AtT-20 and L929 cells were able to shift the CRE, and supershift analysis revealed that Jun family members are part of the protein complexes that bind the CRE. Pan Jun and pan Fos antibodies were able to supershift protein-CRE complexes formed using NIH 3T3 nuclear extracts. These data raise the possibility that the promiscuous binding of CREB and/or CREM to the AP-1 site, and AP-1 transcription factors to one or more CREs, in the GR promoter may contribute to the regulation of GR gene expression.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 3-4","pages":"179-93"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20201008","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pharmacological sequestration of a chimeric beta 3/beta 2 adrenergic receptor occurs without a corresponding amount of receptor internalization.","authors":"S Mostafapour,&nbsp;B K Kobilka,&nbsp;M von Zastrow","doi":"","DOIUrl":"","url":null,"abstract":"<p><p>Many G-protein-coupled receptors undergo sequestration (rapid loss of cell surface receptor binding sites) following exposure to agonists. This process has been assayed traditionally by the use of membrane-impermeant radioligands to measure cell-surface binding sites before and after exposure of cells to agonists. Pharmacological sequestration of the beta 2 adrenergic receptor is associated with internalization of the receptor protein, although it is not known whether receptor internalization is the only mechanism by which ligand-binding sites can be sequestered from the cell surface. Here we show that a chimeric mutant adrenergic receptor, constructed by attaching the carboxyl-terminal cytoplasmic domain from the beta 2 receptor to the beta 3 receptor sequence, exhibits agonist-induced sequestration (measured by 3H-CGP-12177 binding to intact human embryonal kidney [HEK] 293 cells) that is in significant excess to the small amount of receptor internalization measured in the same cells by quantitative flow cytometry. Furthermore, sequestration of the chimeric mutant receptor is reversible at 13 degrees C, a condition that blocks internalization and recycling of adrenergic receptors and has no effect on the sequestration of beta 2 receptors. These data suggest the operation of two distinguishable mechanisms of receptor sequestration in the same cells: agonist-induced internalization and an alternative, biochemically distinguishable mechanism that occurs without a corresponding amount of internalization of the receptor protein.</p>","PeriodicalId":79456,"journal":{"name":"Receptors & signal transduction","volume":"6 3-4","pages":"151-63"},"PeriodicalIF":0.0,"publicationDate":"1996-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"20201006","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}