Regulation of m4 muscarinic receptor gene expression

In order to identify the mechanisms that regulate the expression of the muscarinic receptors, the regulation of one of these receptors, the m4, was investigated. Four cell lines were identified that express the m4 gene and one of these, the NG108-15 cell line, was chosen as a model system for m4 expression. Pharmacological characterization of the m4 receptor expressed by the NG108-15 and the N18TG2 cell lines, was consistent with that previously reported for the receptor. DNA sequence analysis on the m4 gene expressed in the NO 108-15 indicated that it is of mouse origin. The levels of receptor expressed by the NO 108-15 cell line varied greatly during routine culturing but removal of serum and inhibition of mitosis resulted in a reduction of receptor expression to a constant level. PGE1 differentiation of the cells did not result in any further change in receptor levels. Activation and continued exposure of the muscarinic receptor with the muscarinic agonist, carbachol, resulted in a down-regulation of the receptor in a dose dependent manner. In order to identify cis genomic regions responsible for the transcriptional regulation of the m4 gene, a genomic clone, R3-6, containing the coding region for the m4 gene and approximately 30 kb of 5' sequence was isolated. Transfection of this genomic clone into the IMR32 and CHO cell lines demonstrated that this clone contains the m4 promoter and at least some tissue specific elements. Because a full length cDNA for the m4 gene is not available to aid in locating the transcription initiation site for the m4 gene, several independent techniques were used to locate this sequence within the genomic clone. These included nuclear run on analysis. Northern blot analysis, PCR techniques, primer extension and nuclease protection assays. The data obtained suggests that the upstream sequence for the m4 gene is contained within a single exon of approximately 460 nucleotides and the transcription start site is located between 5.2 and 4.4 kb 5' from the initiating ATG