Structure of the m4 cholinergic muscarinic receptor gene and its promoter

Ian C. Wood, Avtar Roopra, Christina (Chris) Harrington, Noel J. Buckley

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

Cholinergic muscarinic receptor genes are members of the G-protein receptor gene superfamily. In this study we describe the structure of the gene and promoter of the rat m4 muscarinic receptor gene. A rat cosmid clone containing the coding region for the m4 gene and 25 kilobases of upstream sequence was isolated. This clone directed expression of the rat m4 gene when introduced in IMR32 cells, a human neuroblastoma that expresses m4, but did not drive expression when introduced into Chinese hamster ovary cells, a line that does not express the m4 gene. S1 nuclease, modified 5'-rapid amplification of cDNA ends and polymerase chain reaction analysis of rat cosmid DNA and cDNA showed that the gene consists of a 2.6-kilobase coding exon, extending 34 base pairs (bp) upstream from the initiating ATG, separated from a 460-493 bp noncoding exon by a 4.8-kilobase intron. DNA sequence analysis shows that the noncoding exon is GC-rich and that the promoter does not contain a TATA or CAAT box and has several consensus sequences for enhancer elements including five Sp-1 binding sites, one AP-2 site, one AP-3 binding site and two E-boxes within the proximal 600 bp. A reporter construct consisting of 1440 bp of flanking DNA and 80 bp of the first exon cloned into a luciferase reporter plasmid, drove cell specific expression in transient transfection assays. Removal of 1088 bp of the 5' end of this construct resulted in expression in non-m4 expressing cell lines suggesting there is a repressor element in this region.

Original languageEnglish (US)
Pages (from-to)30933-30940
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number52
DOIs
StatePublished - Dec 29 1995
Externally publishedYes

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Cholinergic Receptors
Muscarinic Receptors
Cholinergic Agents
Genes
Base Pairing
Rats
Exons
Cosmids
Muscarinic M4 Receptors
Complementary DNA
Clone Cells
Binding Sites
Cells
Cell Line
Polymerase chain reaction
DNA sequences
DNA
Consensus Sequence
Cricetulus
Luciferases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure of the m4 cholinergic muscarinic receptor gene and its promoter. / Wood, Ian C.; Roopra, Avtar; Harrington, Christina (Chris); Buckley, Noel J.

In: Journal of Biological Chemistry, Vol. 270, No. 52, 29.12.1995, p. 30933-30940.

Research output: Contribution to journalArticle

Wood, Ian C. ; Roopra, Avtar ; Harrington, Christina (Chris) ; Buckley, Noel J. / Structure of the m4 cholinergic muscarinic receptor gene and its promoter. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 52. pp. 30933-30940.
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