MUTATIONAL ANALYSIS OF THE RELATIVE ORIENTATION OF TRANSMEMBRANE HELIX-I AND HELIX-VII IN G-PROTEIN-COUPLED RECEPTORS

Currently, detailed structural information about the arrangement of the seven transmembrane helices (TM I-VII) present in all G protein-coupled receptors is still lacking. We demonstrated previously that hybrid m2/m5 muscarinic acetylcholine receptors which contain m5 sequence in TRI I and m2 sequence in TM VII were unable to bind significant amounts of muscarinic radioligands (Pittel, Z., and Wess, J. (1994) Mol. Pharmacol. 45, 61-64). By using immunocytochemical and enzyme-linked immunosorbent assay techniques, we show in the present study that these pharmacologically inactive mutant receptors are present (at high levels) on the surface of transfected COS-7 cells. Strikingly, all misfolded m2/m5 hybrid receptors could be pharmacologically rescued by introduction of a single point mutation into either TM I (m5Thr(37) --> m2Ala(30)) or TM VII (m2Thr(423) --> m5His(478)). All experimental data are consistent with the notion that the two altered threonine residues face each other at the TM I/TM VII interface in the pharmacologically inactive m2/m5 hybrid receptors, thus interfering with proper helix-helix packing. Our data provide the first experimental evidence as to how TM I and TM VII are oriented relative to each other and also strongly suggest that the TM helices in G protein-coupled receptors are arranged in a counterclockwise fashion (as viewed from the extracellular membrane surface).