Mutational analysis of the relative orientation of transmembrane helices I and VII in G protein-coupled receptors.
Currently, detailed structural information about the arrangement of the seven transmembrane helice (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 TM 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 (m5Thr37--> m2Ala30) or TM VII (m2Thr423--> m5His478). All our experimental data are consistent with the notion that the two altered threonine residues face each other at the TM 1/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).
Liu
J
J
Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Schöneberg
T
T
van Rhee
M
M
Wess
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011976:Receptors, Muscarinic; D019204:GTP-Binding Proteins