Transcriptional regulation of the human muscarinic M2 receptor gene

Abstract

Muscarinic M2 receptors are important regulators of airway smooth muscle tone and alteration in M2 receptor function has been described in asthmatic patients. Information regarding transcriptional regulatory control of muscarinic M2 receptor expression in human airway smooth muscle cells is not available in the scientific literature. This project aimed to study the transcriptional regulation of human muscarinic M2 receptors and identify potential polymorphic variation, which may contribute to alteration in receptor expression. Total mRNA was extracted from a human airway smooth muscle (HASM) primary cell culture and used as a template for analysis. A 5’ RACE (Rapid Amplification of cDNA Ends) approach was used to identify and characterize the promoter region of the M2 receptor. The promoter activity of pGL3E deletion constructs was subsequently investigated using a luciferase-based reporter gene assay approach in transiently transfected HASM and BEAS-2B cells. Three different regions of transcriptional initiation were identified, with multiple transcription start sites (TSSs) clustered within each region. The distance separating the most 5’ TSS from the coding region exceeds 146kb, and includes multiple exons, some of which are alternatively spliced. Sequencing of genomic DNA revealed the presence of a novel 0.5kb hypervariable region located 648bp upstream of the most 5’ TSS, a CÆA SNP located 136bp upstream of the most 5’ TSS and a multiallelic CA tandem repeat 96bp downstream of the most 5’ TSS. The CA repeat has been shown to influence reporter gene transcriptional activity in transient cell transfectants. This study has elucidated the arrangement of the muscarinic M2 5’ untranslated region, and has defined the key regions likely to be important in transcriptional regulation of the gene in HASM cells. Studies to define potential linkage between the functional tandem CA repeat and asthma are currently underway. This work was funded by the University of Malta and the National Asthma Campaign (UK).