Pronounced conformational changes following agonist activation of the M(3) muscarinic acetylcholine receptor.
The conformational changes that convert G protein-coupled receptors (GPCRs) activated by diffusible ligands from their resting into their active states are not well understood at present. To address this issue, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system, employing a recently developed disulfide cross-linking strategy that allows the formation of disulfide bonds using Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. In the present study, we generated and analyzed 30 double Cys mutant M(3) receptors, all of which contained one Cys substitution within the C-terminal portion of transmembrane domain (TM) VII (Val-541 to Ser-546) and another one within the C-terminal segment of TM I (Val-88 to Phe-92). Following their transient expression in COS-7 cells, all mutant receptors were initially characterized in radioligand binding and second messenger assays (carbachol-induced stimulation of phosphatidylinositol hydrolysis). This analysis showed that all 30 double Cys mutant M(3) receptors were able to bind muscarinic ligands with high affinity and retained the ability to stimulate G proteins with high efficacy. In situ disulfide cross-linking experiments revealed that the muscarinic agonist, carbachol, promoted the formation of cross-links between specific Cys pairs. The observed pattern of disulfide cross-links, together with receptor modeling studies, strongly suggested that M(3) receptor activation induces a major rotational movement of the C-terminal portion of TM VII and increases the proximity of the cytoplasmic ends of TM I and VII. These findings should be of relevance for other family A GPCRs.
Han
Sung-Jun
SJ
Molecular Signaling Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
Hamdan
Fadi F
FF
Kim
Soo-Kyung
SK
Jacobson
Kenneth A
KA
Brichta
Lars
L
Bloodworth
Lanh M
LM
Li
Jian H
JH
Wess
Jürgen
J
D000595:Amino Acid Sequence; D000818:Animals; D001285:Atropine / Q000494:pharmacology; D015153:Blotting, Western; D019556:COS Cells; D002217:Carbachol / Q000737:chemistry / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015951:Factor Xa / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D010716:Phosphatidylinositols / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000737:chemistry* / Q000235:genetics*; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D014633:Valine / Q000737:chemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D008024:Ligands; D010716:Phosphatidylinositols; D043587:Receptor, Muscarinic M3; D012694:Serine; D001285:Atropine; D002217:Carbachol; D015951:Factor Xa; D014633:Valine; D003545:Cysteine; D010100:Oxygen