Immunological localization of m1–m5 muscarinic acetylcholine receptors in peripheral tissues and brain

Abstract

Knowledge of the distributions and functions of native m1–m5 muscarinic acetylcholine receptors in tissues is limited. To characterize the family of m1–m5 proteins directly, a panel of subtype-selective antibodies was generated against divergent i3 loop-fusion proteins. Each antibody was shown to bind a single cloned receptor specifically. In peripheral tissues and brain, four receptor proteins (m1–m4) were found to account for the vast majority of the muscarinic binding sites using immunoprecipitation studies with the subtype-specific antibodies. The subtypes were differentially distributed, although most tissues were comprised of a complex mixture of receptors. Moreover, within tissues there were major differences in the precise localization of the subtypes, as determined by immunocytochemistry. The immunological methods described offer a novel approach with exquisite sensitivity and specificity for delineating the distribution of m1–m5 receptors in animal and human tissues.