Acetylcholine is believed to be a neurotransmitter, a communicator between excitable cells. It is thought to stimulate receptors in order to communicate a specific message to cells. One receptor that is of interest to this laboratory is the muscarinic receptor that inhibits adenylate cyclase, an enzyme that synthesizes the intracellular messenger, cyclic AMP. This laboratory uses primary cultures of cerebellar granule cells as a neuronal model to study these receptors. We have found this receptor to desensitize, or lose its ability to inhibit adenylate cyclase, within thirty minutes after being exposed to drugs that mimic acetylcholine. This phenomena of desensitization, though known for some other receptor types, is not seen with two other receptors (adenosine A, and GABAB) that also inhibit adenylate cyclase. The mechanism of desensitization for this muscarinic receptor is unknown. Preliminary results have shown that the number of receptors, as determined by labeled ligand binding methods, does not change after desensitization. Because receptor internalization is a common mechanism of down-regulating receptors, it was important to rule this out as a possible mechanism of desensitization. Phosphorylation of receptive proteins (such as the beta-adrenergic receptor) is a -known mechanism of receptor desensitization. This research proposal will test the hypothesis that the desensitization of these receptors results from the phosphorylation of the receptive protein. To study phosphorylation of muscarinic receptors, it is necessary to partially purify the receptor proteins so that the protein labeled with radioactive phosphate can be detected. In. preliminary studies, we have found a protein to be phosphorylated following a agonist treatment. This protein was identified by purification with affinity column chromatography specific for muscarinic receptors. This protein has the same molecular weight as a muscarinic protein described by others. However, this protein needs to be further indentified as being a muscarinic receptor. This proposal outlines studies that will: 1. Further characterize these muscarinic receptors and their desensitization; 2. Isolate the muscarinic receptors by affinity chromatography and determine the extent of their phosphorylation; and 3. Verify the muscarinic subtype that is phosphorylated as a result of agonist exposure.
|Fohrman, E B; de Erausquin, G; Costa, E et al. (1993) Muscarinic m3 receptors and dynamics of intracellular Ca2+ cerebellar granule neurons. Eur J Pharmacol 245:263-71
|Longone, P; Mocchetti, I; Riva, M A et al. (1993) Characterization of a decrease in muscarinic m2 mRNA in cerebellar granule cells by carbachol. J Pharmacol Exp Ther 265:441-6
|Holopainen, I; Wojcik, W J (1993) A specific antisense oligodeoxynucleotide to mRNAs encoding receptors with seven transmembrane spanning regions decreases muscarinic m2 and gamma-aminobutyric acidB receptors in rat cerebellar granule cells. J Pharmacol Exp Ther 264:423-30
|Contrera, J G; Mcleskey, S W; Holopainen, I et al. (1993) Muscarinic m2 receptors in cerebellar granule cell cultures from rat: mechanism of short-term desensitization. J Pharmacol Exp Ther 265:433-40
|McLeskey, S W; Wojcik, W J (1992) Propylbenzilylcholine mustard has greater specificity for muscarinic m2 receptors than for m3 receptors present in cerebellar granule cell culture from rat. J Pharmacol Exp Ther 263:703-7