Am. J. Respir. Cell Mol. Biol., Volume 26, Number 3, March, 2002 298-305

Structure and Transcription of the Human m3 Muscarinic Receptor Gene

Sean M. Forsythe,^* Paul C. Kogut,^* John F. McConville, Yiping Fu, Joel A. McCauley, Andrew J. Halayko, Hong Wei Liu, Allen Kao, Darren J. Fernandes, Shashi Bellam, Elaine Fuchs, Satrajit Sinha, Graeme I. Bell, Blanca Camoretti-Mercado, and Julian Solway

Section of Pulmonary and Critical Care Medicine, Department of Medicine; Department of Biochemistry and Molecular Biology; Department of Molecular Genetics and Cell Biology; and Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois

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 PCR- amplified 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.

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