The proposed existence of subtypes of muscarinic cholinergic receptors (MR) has been based primarily on pharmacological data obtained with a single atypical MR antagonist, pirenzepine. The goal of this research proposal is to provide more unambiguous information on the existence and properties of MR subtypes. Two cell lines (1321N1 human astrocytoma and NG108-15 neuroblastoma x glioma cells) that express very different biochemical responses to MR stimulation have been studied extensively in this laboratory. Although both cell lines express hormone receptors that inhibit adenylate cyclase and stimulate phosphoinositide hydrolysis, activation of MR of NG108-15 cells only results in inhibition of adenylate cyclase whereas activation of MR of 1321N1 cells only results in increases in inositol phosphate formation. In addition, the MR of NG108-15 cells interact with the inhibitory guanine nucleotide regulatory protein, G1, while the MR of 1321N1 cells interact with an unidentified guanine nucleotide regulatory protein that is not G1. The most parsimonious interpretation of these data is that the two cell lines express different MR that selectively interact with one or the other aforementioned second messenger response systems. As such they will be used as model systems that are uncompromised by the complexities introduced by the cellular and receptor heterogeneity of mammalian tissues. The pharmacological specificities of the MR of the two cell lines will be defined based on: (a) their interaction with antagonists reported to be selective for one of the putative MR subtypes; (b) their inactivation by irreversibly acting ligands; (c) their agonist-induced interaction with guanine nucleotide regulatory proteins. Differences in the biochemical properties of the MR of the two cell lines will be identified based on: (a) the relative mobilities during SDS-PAGE of the MR covalently labeled with 3H-propylbenzylcholine mustard; (b) the pI values of the labeled MR; (c) the peptide fingerprints of the labeled proteins after protease and CNBr-cleavage. Differences in agonist-induced modification of the MR of the two cell lines will be established by: (a) comparing the general properties of the second messenger response system of each MR during exposure of the cells to agonist; (b) determining if an agonist-induced covalent modification of MR occurs; (c) identifying the type, e.g. phosphorylation, of covalent modification and delineating its mechanism of occurrence and importance in agonist-induced changes in MR responsiveness and cellular distribution; and (d) comparing peptide fingerprints of the covalently-modified MR from each cell line. It is important to establish clearer understanding of the existence of MR subtypes and the use of model cell lines offers unique opportunities to this end. Rational therapy of a number of pathophysiological conditions, e.g. Alzheimer's disease, can ultimately benefit from this knowledge.
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