Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation.
G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.
Jäger
Dorothea
D
Pharmacology and Toxicology Section, Institute of Pharmacy, Rheinische Friedrich-Wilhelms-University, D-53121 Bonn, Germany.
Schmalenbach
Caroline
C
Prilla
Stefanie
S
Schrobang
Jasmin
J
Kebig
Anna
A
Sennwitz
Matthias
M
Heller
Eberhard
E
Tränkle
Christian
C
Holzgrabe
Ulrike
U
Höltje
Hans-Dieter
HD
Mohr
Klaus
K
D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D043585:Receptor, Muscarinic M2 / Q000737:chemistry*; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism*; D012243:Rhodopsin / Q000737:chemistry
D043585:Receptor, Muscarinic M2; D043562:Receptors, G-Protein-Coupled; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012243:Rhodopsin