Intramolecular interactions in muscarinic acetylcholine receptors studied with chimeric m2/m5 receptors.

Current models of the three-dimensional structures of muscarinic acetylcholine receptors and other G protein-coupled receptors are based primarily on high-resolution electron diffraction data obtained with bacteriorhodopsin, the molecular structure of which is characterized by the presence of seven alpha-helical transmembrane domains (TM I-VII). However, bacteriorhodopsin does not couple to G proteins and its primary sequence lacks a series of amino acids that are conserved among virtually all G protein-coupled receptors. Therefore, it remains to be shown experimentally whether the molecular structures of these functionally different proteins are in fact identical. To address this question, we have analyzed the pharmacological properties of a series of hybrid human m2/m5 muscarinic receptors. Initially, we identified several chimeric constructs that, upon transient expression in COS-7 cells, were unable to bind significant amounts of the muscarinic antagonists N-[3H]methylscopolamine and [3H]quinuclidinyl benzilate. A common structural feature of these constructs was the presence of m2 receptor sequence in TM VII and of m5 receptor sequence in TM I. The ligand-binding activity of these "pharmacologically inactive" hybrid receptors could be restored by replacing TM I (consisting of m5 receptor sequence) with the corresponding m2 receptor domain. These data provide the first direct experimental evidence that the molecular architecture of muscarinic receptors (and, most likely, that of other G protein-coupled receptors) resembles that of bacteriorhodopsin, in that the seven TM helices are arranged in a ring-like fashion such that TM I lies directly adjacent to TM VII.