Structure of the human M2 muscarinic acetylcholine receptor gene and its promoter (original) (raw)
Related papers
Structure and Transcription of the Human m3 Muscarinic Receptor Gene
American Journal of Respiratory Cell and Molecular Biology, 2002
We have isolated and characterized the human m3 muscarinic receptor gene and its promoter. Using 5 rapid amplification of cDNA ends (RACE), internal polymerase chain reaction (PCR), and homology searching to identify EST clones, we determined that the cDNA encoding the m3 receptor comprises 4,559 bp in 8 exons, which are alternatively spliced to exclude exons 2, 4, 6, and/or 7; the receptor coding sequence occurs within exon 8. Analysis of P1 artificial chromosome (PAC) and bacterial artificial chromosome (BAC) clones and of PCRamplified genomic DNA, and homology searching of human chromosome 1 sequence provided from the Sanger Centre (Hinxton, Cambridge, UK) revealed that the m3 muscarinic receptor gene spans at least 285 kb. A promoter fragment containing bp-1240 to ؉ 101 (relative to the most 5 transcription start site) exhibited considerable transcriptional activity during transient transfection in cultured subconfluent, serum-fed canine tracheal myocytes, and 5 deletion analysis of promoter function revealed the presence of positive transcriptional regulatory elements between bp-526 and-269. Sequence analysis disclosed three potential AP-2 binding sites in this region; five more AP-2 consensus binding motifs occur between bp-269 and ؉ 101. Cotransfection with a plasmid expressing human AP-2 ␣ substantially increased transcription from m3 receptor promoter constructs containing 526 or 269 bp of 5 flanking DNA. Furthermore, m3 receptor promoter activity was enhanced by long-term serum deprivation of canine tracheal myocytes, a treatment that is known to increase AP-2 transcription-promoting activity in these cells. Together, these data suggest that expression of the human m3 muscarinic receptor gene is regulated in part by AP-2 in airway smooth muscle. Muscarinic acetylcholine receptors are a family of G-protein linked seven transmembrane receptors. They are present throughout the body in smooth muscle, cardiac muscle, exocrine glands, and neurons of the central and peripheral nervous systems. Five receptor subtypes, termed m1-m5, have been identified, and these are encoded by distinct genes (1-3). The m1, m3, and m5 receptors interact with pertussis toxin-insensitive Gq proteins, thereby activating phospholipase C, whereas the m2 and m4 receptors preferentially interact with pertussis toxin-sensitive Gi proteins, and so inhibit adenylyl cyclase. Both m2 and m3 muscarinic receptors are present on airway smooth muscle cells, with m2 receptors comprising the majority (4-6). However, the m3 receptor is the predom-(
Life Sciences, 1992
The coding sequence of the rat m3, m4 and m5 subtypes of muscarmic acetylchohne receptor (mAChR) genes was amplified by the polymerase chain reaction (PCR), cloned, and expressed m the murme fibroblast (B82) cell line. Sequencing of the cloned genes revealed some nucleotide differences when compared with the DNA sequence published in the hterature When the different sequence appeared m only one clone obtained by PCR, it was considered an error of the polymerase The overall error frequency m the 25 cycles of PCR with either Taq polymerase or Replinase was 1 nucleotide m 1,692 base pmrs In order to evaluate the different nucleotlde sequence from a PCR product as an error or as an allelic variant, at least three different clones were sequenced The cloned genes were each stably expressed in a B82 cell line and pharmacologically evaluated The affimty of the different antagonists to the muscannic receptor subtypes was determined by [3H](-)MQNB/hgand inhibitmn experiments In the m3, m4 and m5 transfected cells, carbachol appeared to stimulate [3H]inomtol monophosphate (IP1) accumulation Carbachol, at 3 pM, appeared to suppress the forskohn-stlmulated cAMP formation in the m4 transfected cells These findings suggest these mAChRs amphfied by PCR, cloned, and expressed in the B82 cell lines exhibit the pharmacological characteristics of the muscarimc receptor subtypes
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.
Overproduction of human M₃ muscarinic acetylcholine receptor: an approach toward structural studies
Biotechnology progress
Human M3 muscarinic acetylcholine receptor (M3R), present in both the central and the peripheral nervous system, is involved in several neurodegenerative and autoimmune diseases. Recently, M3R overexpression has been suggested to play a role in certain forms of cancer, showing promise as a new potential pharmacological target. However, the lack of structural information hampered to develop a new potent selective and potent antagonist. We describe here different strategies for overexpressing functional M3R on the perspective of future biophysical studies. To achieve this goal, four tagged M3R genes were engineered and codon optimized. Different heterologous expression systems, including mammalian cells and viral transfection, were employed to overexpress M3R. Although codon optimization resulted in only twofold to threefold increase of M3R expression, we found that epitope tagging of the synthetic M3R, especially with hemagglutinin and Flag epitope tags, could improve M3R expression levels. On the other hand, viral transfection led to a yield of 27 pmol/mg protein that is the highest level reported so far for this receptor subtype in mammalian cells. Taking together several of the strategies used can help increasing M3R expression, not only to start purification efforts but also for secondary structural analysis trial and functional analyses. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Journal of Pharmacology and Experimental Therapeutics, 2010
We investigated the functional role of a conserved motif, F(x) 6 LL, in the membrane proximal C-tail of the human muscarinic M 1 (hM 1 ) receptor. By use of site-directed mutagenesis, several different point mutations were introduced into the C-tail sequence 423 FRDTFRLLL 431 . Wild-type and mutant hM 1 receptors were transiently expressed in Chinese hamster ovary cells, and the amount of plasma membrane-expressed receptor was determined by use of intact, whole-cell [ 3 H]N-methylscopolamine binding assays. The plasma membrane expression of hM 1 receptors possessing either L430A or L431A or both point mutations was significantly reduced compared with the wild type. The hM 1 receptor possessing a L430A/L431A doublepoint mutation was retained in the endoplasmic reticulum (ER), and atropine treatment caused the redistribution of the mutant
Non-Neuronal Functions of the M2 Muscarinic Acetylcholine Receptor
Genes, 2013
Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M 2 muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M 2 receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M 2 receptor in human pathological conditions such as autoimmune diseases and cancer.