Molecular analysis of the regulation of muscarinic receptor expression and function.

We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined the developmental regulation of mAChR expression in the chick retina. Early in development, the M4 receptor is the predominant mAChR while the levels of the M2 and M3 receptors increase later in development. The level of M2 receptor is also initially very low in retinal cultures and undergoes a dramatic increase over several days in vitro. The level of M2 receptor can be increased by a potentially novel, developmentally regulated, secreted factor produced by retinal cells. The promoter for the chick M2 receptor gene has been isolated and shown to contain a site for GATA-family transcription factors which is required for high level cardiac expression. The M2 promoter also contains sites which mediate induction of transcription in neural cells by neurally active cytokines. We have generated knockout mice lacking the M1 receptor and shown that these mice do not exhibit pilocarpine-induced seizures and muscarinic agonist-induced suppression of the M-current potassium channel in sympathetic neurons.

Tyrosines 905 and 915 of gp130 are required for maximum induction of m2 muscarinic acetylcholine receptor and VIP gene transcription by cytokines in neuronal cells.

1. Leukemia inhibitory factor action is mediated by a heterodimeric receptor consisting of two subunits, gp130 and the low-affinity leukemia inhibitory factor receptor (LIFR). 2. We used chimeric receptors containing the intracellular domain of either the LIFR or gp130 to identify regions of the receptors required for induction of the m2 muscarinic acetylcholine receptor gene in IMR-32 and SN56 neuronal cells. 3. While chimeric receptors containing the intracellular domain of gp130 were able to induce transcription from both the m2 and the vasoactive intestinal peptide (VIP) gene promoters, chimeric receptors containing the intracellular domain of the LIFR were incapable of mediating induction of the m2 gene despite being able to induce VIP transcription. 4. Deletion and mutagenesis studies identified two tyrosines, Y905 and Y915, which were required for maximal induction of the m2 and VIP genes. 5. Because Y905 and Y915 are reported to be the only tyrosine residues in gp130 that bind Stat1, these results suggest that this transcription factor plays a key role in the induction of transcription of both the m2 and the VIP genes.

Disruption of a neuropeptide gene, flp-1, causes multiple behavioral defects in Caenorhabditis elegans.

Neuropeptides serve as important signaling molecules in the nervous system. The FMRFamide (Phe-Met-Arg-Phe-amide)-related neuropeptide gene family in the nematode Caenorhabditis elegans is composed of at least 18 genes that may encode 53 distinct FMRFamide-related peptides. Disruption of one of these genes, flp-1, causes numerous behavioral defects, including uncoordination, hyperactivity, and insensitivity to high osmolarity. Conversely, overexpression of flp-1 results in the reciprocal phenotypes. On the basis of epistasis analysis, flp-1 gene products appear to signal upstream of a G protein-coupled second messenger system. These results demonstrate that varying the levels of FLP-1 neuropeptides can profoundly affect behavior and that members of this large neuropeptide gene family are not functionally redundant in C. elegans.

GATA factor-dependent regulation of cardiac m2 muscarinic acetylcholine gene transcription.

The m2 subtype is the predominant muscarinic acetylcholine receptor subtype expressed in heart and regulates the rate and force of cardiac contraction. We have previously reported the isolation of the promoter region for the chick m2 receptor gene and defined a region of the chick m2 promoter sufficient for high level expression in cardiac primary cultures. In this manuscript we demonstrate transactivation of cm2 promoter by the GATA family of transcription factors. In addition, we define the GATA-responsive element in the chick m2 promoter and demonstrate that this element is required for expression in cardiac primary cultures. Finally, we demonstrate specific binding of both a chick heart nuclear protein and of cloned chick GATA-4, -5, and -6 to the GATA-responsive element.

Isolation and characterization of the chicken m2 acetylcholine receptor promoter region: induction of gene transcription by leukemia inhibitory factor and ciliary neurotrophic factor.

We have isolated the promoter region and determined the start sites of transcription for the gene encoding the chicken m2 (cm2) muscarinic acetylcholine receptor. Transfection experiments, using cm2-luciferase reporter gene constructs, demonstrated that a 789-bp genomic fragment was sufficient to drive high level expression in chicken heart primary cultures, while an additional 1.2-kb region was required for maximal expression in mouse septal/ neuroblastoma (SN56) cells. Treatment of SN56 cells with the cytokines ciliary neurotrophic factor and leukemia inhibitory factor increases expression of endogenous muscarinic acetylcholine receptors and results in a 4- to 6-fold induction of cm2 promoter driven luciferase expression. We have mapped a region of the cm2 promoter that is necessary for induction by cytokines.

The flp-1 propeptide is processed into multiple, highly similar FMRFamide-like peptides in Caenorhabditis elegans.

Previously, we described a gene, flp-1, that encodes seven FMRFamide-like peptides from two alternatively spliced transcripts in the nematode Caenorhabditis elegans. To determine whether all or a subset of the predicted peptides coded for by flp-1 are produced in vivo, we undertook the isolation of FMRFamide-like peptides from C. elegans. Six FLRFamide-containing peptides, all contained within the putative translation products of the flp-1 gene, were isolated from extracts of mixed stage animals. By quantitative PCR analysis of RNA from mixed stage animals, we found that the shorter transcript of flp-1 has a higher level of expression than the longer transcript.

Alternatively spliced transcripts of the flp-1 gene encode distinct FMRFamide-like peptides in Caenorhabditis elegans.

We have isolated and characterized several cDNAs and the corresponding genomic region of a gene encoding multiple FMRFamide-like neuropeptides from the nematode Caenorhabditis elegans. The gene, named flp-1, consists of six exons of which four encode FMRFamide-like peptides. The cDNA and genomic sequences revealed that two distinct transcripts are generated by the use of an alternative 3' splice acceptor site between exons 3 and 4. This alternative splice results in the substitution of AGSDPNFLRFG for one of the copies of SADPNFLRFG found in the other translation product. Based on PCR analysis of RNA from mixed-stage animals, both transcripts are expressed. This gene is the first example of a distinct FMRFamide-like peptide being derived from alternative splicing, suggesting a unique role for the substituted peptide in the animal.