Impaired M₃ Muscarinic Acetylcholine Receptor Signal Transduction Through Blockade of Binding of Multiple Proteins to its Third Intracellular Loop

Several motifs found in the third intracellular loop of the M(3) muscarinic receptor are critical for G protein activation and scaffold protein interaction. However, how multiprotein complexes form is not fully understood. A minigene encoding the third intracellular loop of the M(3) muscarinic receptor was constructed to explore whether peptides from this intracellular region could act as inhibitors of the muscarinic multiprotein complex formation and signaling. We found that this construct, when co-expressed with the M(3) receptor, has the ability to act as a competitive antagonist of G protein receptors and receptor-scaffold/accessory proteins. Transient transfection of human embryonic kidney-293 cells with DNA encoding the human M(3) and M(5) receptor subtypes results in a carbachol-dependent increase of inositol phosphate. Co-expression of the M(3) third cytoplasmic loop minigene dramatically reduces both carbachol-mediated G protein activation and inositol phosphate accumulation. Minigene expression also abrogates activation of M(3) and M(5) receptor mitogen-activated protein kinases pathway. Furthermore, minigene expression led to reduced AKT activation. These data, together with results of co-immunoprecipitation of different scaffold and kinase proteins, provide experimental evidence for the role for the third cytoplasmic loop of the human M(3) muscarinic receptor in G-protein activation and multiprotein complex formation.