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1.
Psychopharmacology (Berl) ; 141(1): 83-92, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9952069

ABSTRACT

The D4 dopamine receptor, a member of the D2-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D4 receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D4.2, D4.4, or D4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D4 receptor variants. D4 dopamine receptor-mediated sensitization was blocked by the D4 antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for Gi/Go proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D4 receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of Gialpha without altering the abundance of Gsalpha, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D4 receptor may involve mechanisms that do not contribute to short-term sensitization.


Subject(s)
Adenylyl Cyclases/metabolism , Receptors, Dopamine D2/metabolism , Cells, Cultured , Colforsin/pharmacology , Cyclic AMP/metabolism , Dopamine Agonists/pharmacology , GTP-Binding Proteins/biosynthesis , Humans , Receptors, Dopamine D2/agonists , Receptors, Dopamine D4
2.
J Neurosci ; 18(21): 8692-9, 1998 Nov 01.
Article in English | MEDLINE | ID: mdl-9786976

ABSTRACT

D2L dopamine receptor activation results in rapid inhibition and delayed heterologous sensitization of adenylate cyclase in several host cell types. The D2L dopamine receptor was stably transfected into NS20Y neuroblastoma cells to examine inhibition and sensitization in a neuronal cell environment and to identify the particular G-proteins involved. Acute activation of D2L receptors with the selective D2 agonist quinpirole inhibited forskolin-stimulated cAMP accumulation, whereas prolonged incubation (2 hr) with quinpirole resulted in heterologous sensitization (more than twofold) of forskolin-stimulated cAMP accumulation in NS20Y-D2L cells. To unambiguously identify the pertussis toxin (PTX)-sensitive G-proteins responsible for inhibition and sensitization, we used viral-mediated gene delivery to assess the ability of genetically engineered PTX-resistant G-proteins (Galphai1*, Galphai2*, Galphai3*, and Galphao*) to rescue both responses after PTX treatment. The expression and function of individual recombinant G-proteins was confirmed with Western blotting and inhibition of GTPgammaS-stimulated adenylate cyclase, respectively. To assess the specificity of D2L-Galpha coupling, cells were infected with herpes simplex virus (HSV) recombinants expressing individual PTX-resistant G-protein alpha subunits and treated with PTX, and quinpirole-induced responses were measured. Infection of NS20Y-D2L cells with HSV-Galphao* rescued both inhibition and sensitization in PTX-treated cells, whereas infection with HSV-Galphai1*, HSV-Galphai2*, or HSV-Galphai3* failed to rescue either response. In summary, the current study provides strong evidence that the D2L dopamine receptor couples to Galphao in neuronal cells, and that this coupling is responsible for both the acute and subacute effects of D2 receptor activation on adenylate cyclase activity.


Subject(s)
GTP-Binding Proteins/physiology , Neurons/physiology , Receptors, Dopamine D2/physiology , Adenylate Cyclase Toxin , Animals , Colforsin/pharmacology , Cyclic AMP/metabolism , Enzyme Activation , GTP-Binding Proteins/metabolism , Genetic Vectors , Mice , Neuroblastoma , Pertussis Toxin , Quinpirole/pharmacology , Receptors, Dopamine D2/chemistry , Receptors, Dopamine D2/genetics , Simplexvirus/genetics , Transfection , Tumor Cells, Cultured , Virulence Factors, Bordetella/pharmacology
3.
Immunology ; 93(2): 213-20, 1998 Feb.
Article in English | MEDLINE | ID: mdl-9616371

ABSTRACT

The transcriptional activation of germline T-cell receptor (TCR) and immunoglobulin (Ig) genes has been proposed to promote the rearrangement of these genes. Here we report the identification of distal TCR promoters (PDs), located upstream of the previously characterized promoters in the mouse V beta 5.1 and V beta 8.1 gene segments, that are active in germline TCR genes in fetal thymus and liver in vivo. We also identified an immature T-cell clone, SL12.4, that expresses both endogenous and transfected PDs in a regulated manner in vitro. We propose that the transcription of these distal promoters in germline TCR genes may be important for inducing TCR gene rearrangements during T-cell development. Northern blot, RNase protection and reverse transcription-polymerase chain reaction (RT-PCR) analyses demonstrated that PDs are also transcribed from fully rearranged TCR genes in adult thymus, lymph node, and spleen. Although the functional significance of this expression is not known, our sequence analysis of the 5' leader in PD-derived V beta 5.1 and V beta 8.1 transcripts revealed the presence of several open reading frames (ORFs) that may encode novel polypeptides or regulate the efficiency of TCR beta translation.


Subject(s)
Lymphoid Tissue/immunology , Promoter Regions, Genetic , Receptors, Antigen, T-Cell, alpha-beta/genetics , Transcription, Genetic/immunology , Animals , Base Sequence , Blotting, Northern , Cell Culture Techniques , Fetus/immunology , Lymphoid Tissue/embryology , Mice , Mice, Inbred BALB C , Mice, Inbred Strains , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , RNA, Messenger/genetics
4.
J Immunol ; 151(12): 6801-14, 1993 Dec 15.
Article in English | MEDLINE | ID: mdl-7903099

ABSTRACT

The expression of TCR-beta mRNAs competent to encode functional V(D)JC beta proteins requires the activation of programmed DNA rearrangement events. It is not known whether other regulatory mechanisms control the steady-state levels of mature TCR-beta transcripts during thymic ontogeny. In this report, we demonstrate that TCR-beta pre-mRNAs accumulate in T cells, thus implicating RNA splicing as another potential level of regulation. Three methods were used to characterize the intron content of these pre-mRNA: Northern blot analysis, ribonuclease H mapping, and reverse transcription polymerase chain reaction analysis. Using these methods, we demonstrate that intron-containing TCR-beta transcripts derived from both the JC beta 1 and JC beta 2 loci accumulate in murine fetal and adult thymus. (VD)JC beta 1 pre-mRNAs that accumulate in the thymus possess unusually long poly(A) tails (> or = 300 nucleotides) and contain different combinations of four introns: the large intron between the J beta 1 and C beta 1 elements and the three introns within the C beta 1 element. The presence of an unusual transcript possessing IVS2C beta 1 at the 5' terminus suggests that cleavage of its splice acceptor is inefficient or negatively regulated. The profile of incompletely spliced TCR-beta transcripts present in the thymus in vivo is identical in intron content to those that we previously showed accumulate in the nucleus of the immature SL12.4 T lymphoma cell clone. An unstable negative regulatory protein may control TCR-beta expression in this cell clone because fully spliced TCR-beta transcripts are dramatically induced in the cytoplasm after treatment with any of five different protein synthesis inhibitors (cycloheximide, anisomyosin, emetine, puromycin, and pactamycin), all of which act by distinct mechanisms to inhibit protein synthesis.


Subject(s)
RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/metabolism , Animals , Base Sequence , Cell Differentiation , Clone Cells/immunology , Clone Cells/metabolism , DNA Probes , Embryonic and Fetal Development , Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Introns , Mice , Molecular Sequence Data , Poly A/genetics , Poly A/metabolism , Polymerase Chain Reaction , Protein Synthesis Inhibitors/pharmacology , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing/drug effects , Ribonuclease H , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolism , Transcription, Genetic
5.
Mol Cell Biol ; 13(3): 1686-96, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8441406

ABSTRACT

The expression of functional T cell receptor-beta (TCR-beta) transcripts requires the activation of programmed DNA rearrangement events. It is not clear whether other mechanisms dictate TCR-beta mRNA levels during thymic ontogeny. We examined the potential role of RNA splicing as a regulatory mechanism. As a model system, we used an immature T cell clone, SL12.4, that transcribes a fully rearranged TCR-beta gene but essentially lacks mature 1.3-kb TCR-beta transcripts in the cytoplasm. Abundant TCR-beta splicing intermediates accumulate in the nucleus of this cell clone. These splicing intermediates result from inefficient or inhibited excision of four of the five TCR-beta introns; the only intron that is efficiently spliced is the most 5' intron, IVSL. The focal point for the regulation appears to be IVS1C beta 1 and IVS2C beta 1, since unusual splicing intermediates that have cleaved the 5' splice site but not the 3' splice site of these two introns accumulate in vivo. The block in 3' splice site cleavage is of interest since sequence analysis reveals that these two introns possess canonical splice sites. A repressional mechanism involving a labile repressor protein may be responsible for the inhibition of RNA splicing since treatment of SL12.4 cells with the protein synthesis inhibitor cycloheximide reversibly induces a rapid and dramatic accumulation of fully spliced TCR-beta transcripts in the cytoplasm, concomitant with a decline in TCR-beta pre-mRNAs in the nucleus. This inducible system may be useful for future studies analyzing the underlying molecular mechanisms that regulate RNA splicing.


Subject(s)
RNA Splicing , RNA, Messenger/biosynthesis , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/immunology , Animals , Base Sequence , Cell Compartmentation , Cell Nucleus/metabolism , Clone Cells , Consensus Sequence , Cycloheximide/pharmacology , Cytoplasm/metabolism , DNA Probes , Introns/genetics , Mice , Molecular Sequence Data , Nucleic Acid Hybridization , Oligonucleotides , Polymerase Chain Reaction , RNA Precursors/metabolism , RNA Splicing/drug effects , Repressor Proteins/metabolism
6.
Nucleic Acids Res ; 20(20): 5345-50, 1992 Oct 25.
Article in English | MEDLINE | ID: mdl-1437551

ABSTRACT

The vast majority of mammalian genes are interrupted by non-coding segments of DNA termed introns. Introns are spliced out of RNA transcripts as lariat structures, and then are typically debranched and rapidly degraded. Here, we described an unusual spliced intron from the constant region of the T cell receptor-beta (TCR-beta) locus that is relatively stable in mammalian cells. This intron, IVS1C beta 1, accumulates as a set of lariat RNA structures with different length tails in the nucleus of T cells. The accumulation of this spliced intron is developmentally regulated during murine thymocyte ontogeny. The property of stability appears to be evolutionarily conserved since the human version of this intron also accumulates in T cells. The stability is selective since other spliced TCR-beta introns do not detectably accumulate in T cells. The unusual stability of this intron does not depend on T cell specific factors since non-T cells transfected with TCR-beta gene constructs also accumulate spliced IVS1C beta 1. The discovery of a mammalian intron that accumulates as a lariat in vivo provides an opportunity to elucidate mechanisms that regulate intron debranching, stability, and nuclear localization.


Subject(s)
Introns/physiology , RNA Splicing/physiology , RNA, Messenger/metabolism , Receptors, Antigen, T-Cell, alpha-beta/genetics , T-Lymphocytes/metabolism , Animals , Base Sequence , Blotting, Northern , Cell Nucleus/metabolism , Cloning, Molecular , HeLa Cells , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Rats
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