Control elements of muscarinic receptor gene expression (original) (raw)
Related papers
Journal of Biological Chemistry, 1996
We describe here the characterization of the rat m4 muscarinic acetylcholine receptor gene and the identification of its regulatory region. Two 5'-noncoding exons are located approximately 5 kilobases upstream from the coding exon, and at least two alternatively spliced variants of m4 mRNA are expressed in the neuronal cell line PC12D. There are two transcription initiation sites. The promoter region is GC-rich, contains no TATA-box, but has two potential CAAT boxes and several putative binding sites for transcription factors Sp1 and AP-2. We assessed the m4 promoter activity functionally in transient expression assays using luciferase as a reporter. The proximal 435-base pair (bp) sequence of the 5'-flanking region produced luciferase activity in both m4-expressing neuronal cell lines (PC12D and NG108-15) and non-neuronal cell lines (L6 and 3Y1B). A longer fragment containing an additional 638-bp sequence produced luciferase activity only in m4-expressing neuronal cell lines. These data suggest that the proximal 435-bp sequence contains a constitutive promoter and that a 638-bp sequence farther upstream contains a cell type-specific silencer element. A consensus sequence for the neural-restrictive silencer element is found within this 638-bp segment.
FEBS Letters, 1988
Chimaeric muscarinic acetylcholine receptors (mAChR) in which corresponding portions of mAChR I and mAChR II are replaced with each other have been produced in Jfenopus oocytes by expression of cDNA constructs encoding them. Functional analysis of the chimaeric mAChRs indicates that a region mostly comprising the putative cytoplasmic portion between the proposed transmembrane segments V and VI is involved in selective coupling of mAChR I and mAChR II with different effector systems. In contrast, the exchange of this region between mAChR I and mAChR II does not significantly affect the antagonist binding properties of the two mAChR subtypes. Muscarinic acetylcholine receptor subtype; Chimaeric receptor; cDNA expression; Effector coupling; Selective antagonist; (Xenopus oocyte) 1. INTRODUCTION The muscarinic acetylcholine receptor mediates a variety of cellular responses through the action of guanine nucleotide-binding regulatory proteins (G-proteins) [11. Accumulating evidence indicates that molecularly distinct mAChR subtypes are selectively coupled with different effector systems, albeit not exclusively. Expression in Xenopus oocytes of cloned DNAs encoding four individual mAChR subtypes has revealed that mAChR I and mAChR III mediate activation of a Ca2+-dependent C1-current, whereas mAChR II and mAChR IV principally induce activation of Na ÷ and K ÷ currents in a Ca2+-independent manner [2-4]. Expression studies in mammalian cells have further shown that mAChR I and mAChR III are coupled efficiently with phosphoinositide hydrolysis [5-7], intracellular Ca 2÷ release [8], ac
Molecular analysis of the regulation of muscarinic receptor expression and function
Life Sciences, 1999
We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined
Molecular events associated with the regulation of signaling by M2 muscarinic receptors
Life Sciences, 1999
Multiple events are associated with the regulation of signaling by the M, muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M, mAChRs appears to involve agonist-dependent phosphorylation of M, mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-3 11 from TVSTS to AVAAA in this domain of the human M, mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M, mAChRs on the 3ti intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M, mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on GPy. RGS proteins appear to increase the concentration of free G/3y in addition to acting as GAPS. Thus multiple mechanisms acting at either the level of the M, mAChRs or the G-proteins can contribute to the regulation of signaling via the M, mAChRs.
Structure of the human M2 muscarinic acetylcholine receptor gene and its promoter
Gene, 2001
The M 2 muscarinic receptor inhibits the release of acetylcholine from cholinergic ®bers in the lungs and elsewhere. In airway parasympathetic neurons, M 2 receptor expression is decreased by viral infections and by interferon-g, increasing actylcholine release. Dexamethasone increases M 2 receptor expression, decreasing acetylcholine release. We carried out 5 H rapid ampli®cation of cDNA ends beginning with mRNA from human heart and IMR32 human neuroblastoma cells. This demonstrated a 5 H UTR of 100 BP, corresponding to two sequences on chromosome 7, separated by a 22.6 kB intron. The splice acceptor site is at 245 relative to the initiating atg. The 3000 BP upstream of 5 H RACE product were subcloned into a pGL3 luciferase reporter vector. Deletional constructs were expressed in IMR32 cells. These demonstrated that 412 BP provided full expression of the reporter gene, and suggested a repressor element between 21848 and 21510.
Regulation of muscarinic acetylcholine receptor signaling
Pharmacology & Therapeutics, 2003
Multiple mechanisms regulate the signaling of the five members of the family of the guanine nucleotide binding protein (G protein)coupled muscarinic acetylcholine (ACh) receptors (mAChRs). Following activation by classical or allosteric agonists, mAChRs can be phosphorylated by a variety of receptor kinases and second messenger-regulated kinases. The phosphorylated mAChR subtypes can interact with b-arrestin and presumably other adaptor proteins as well. As a result, the various mAChR signaling pathways may be differentially altered, leading to short-term or long-term desensitization of a particular signaling pathway, receptor-mediated activation of the mitogenactivated protein kinase pathway downstream of mAChR phosphorylation, as well as long-term potentiation of mAChR-mediated phospholipase C stimulation. Agonist activation of mAChRs may also induce receptor internalization and down-regulation, which proceed in a highly regulated manner, depending on receptor subtype and cell type. In this review, our current understanding of the complex regulatory processes that underlie signaling of mAChR is summarized. D