Regulation of expression and function of muscarinic receptors.

The regulation of expression and function of the muscarinic acetylcholine receptor has been studied using several different systems. The role of glycosylation of the m2 receptor was examined by removal of glycosylation sites using site-directed mutagenesis followed by expression in stably transfected cells. The results demonstrated that glycosylation was not required for the synthesis and appearance of the receptors on the cell surface or for the coupling of the receptors to inhibition of adenylyl cyclase activity. Site-directed mutagenesis also was used to demonstrate that the single cysteine in the carboxy terminal domain of the m2 receptor was not required for receptor function, thus rendering unlikely a model suggesting a requirement for palmitoylation of this cysteine in receptor function. The muscarinic receptors expressed in embryonic chick heart were identified by molecular cloning. Two genes were initially identified which are expressed in chick heart and correspond to the chick m2 and m4 receptors. Experiments using the polymerase chain reaction to identify low abundance mRNAs indicate that at least one addition receptor gene is expressed in chick heart. In cell culture, activation of the muscarinic receptors decreases the levels of mRNA encoding the cm2 and cm4 receptors. This probably results from decreased gene transcription due to both mAChR-mediated inhibition of adenylyl cyclase and mAChR-mediated stimulation of phospholipase C. The elucidation of the factors which regulate the expression and function of muscarinic acetylcholine receptors (mAChR) is of obvious importance in understanding the mechanisms underlying cholinergic transmission. In this chapter, we will describe studies on the expression and function of wild type and mutant muscarinic receptors, the molecular characterization of mAChR expressed in chick heart, and the regulation of mAChR gene expression in response to muscarinic receptor activation.

Hormonal activation of gene transcription in ras-transformed NIH3T3 cells overexpressing RII alpha and RII beta subunits of the cAMP-dependent protein kinase.

ras-Transformed NIH3T3 (R3T3) cells were transfected with expression vectors for the RII alpha and RII beta regulatory subunits of the type II isozyme of cAMP-dependent protein kinase, and the effects on gene activation by corticotropin-releasing factor (CRF) and prostaglandin E1 (PGE1) were analyzed. In RII alpha and RII beta-overexpressing cells, type II isozyme levels were increased, and type I isozyme levels were eliminated, demonstrating that both RII regulatory subunits compete efficiently with RI for catalytic subunit. The type II isozyme separated into three peaks on high performance liquid chromatography, referred to as A, B, and C. Western blot analysis strongly suggests that peak A and peak C correspond to holoenzymes containing RII beta and RII alpha, respectively. Overexpression of RII alpha resulted in the loss of peak A and a dramatic reduction in RII beta protein with no change in RII beta mRNA, indicating that the level of RII beta protein is controlled posttranscriptionally and that RII beta protein may become unstable when displaced from C. The role of type I and II kinases in transcriptional activation was investigated by comparing the response of control and RII expressing clones to site-selective cAMP analogs and the hormones, CRF and PGE1. The site-selective analogs demonstrated that either type I or type II kinase could activate the cAMP-responsive alpha-subunit promoter. The response to various concentrations of CRF or PGE1 was identical in control cells and transfected clones containing very little type I kinase. These experiments suggest that in the CRF and PGE1 response pathways leading to gene induction, the magnitude and sensitivity of the response are not influenced by the presence or absence of type I cAMP-dependent protein kinase.