Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor.
In this study, we employed an in situ disulfide cross-linking strategy to gain insight into the structure of the inactive and active state of the M(3) muscarinic acetylcholine receptor. Specifically, this study was designed to identify residues in TM I that are located in close to Cys532 (position 7.42), an endogenous cysteine residue present in the central portion of TM VII. Cysteine residues were substituted, one at a time, into 10 consecutive positions of TM I (Ala71-Val80) of a modified version of the M(3) muscarinic receptor that lacked most endogenous cysteine residues and contained a factor Xa cleavage site within the third intracellular loop. Following their expression in COS-7 cells, the 10 resulting cysteine mutant receptors were oxidized in their native membrane environment, either in the absence or in the presence of muscarinic ligands. Disulfide cross-link formation was monitored by examining changes in the electrophoretic mobility of oxidized and factor Xa-digested receptors on SDS gels. When molecular iodine was used as the oxidizing agent, the L77C receptor (position 1.42) was the only mutant receptor that displayed significant disulfide cross-linking, either in the absence or in the presence of muscarinic agonists or antagonists. On the other hand, when the Cu(II)-(1,10-phenanthroline)(3) complex served as the redox catalyst, muscarinic ligands inhibited disulfide cross-linking of the L77C receptor, probably because of impaired access of this relatively bulky oxidizing agent to the ligand binding crevice. The iodine cross-linking data suggest that M(3) muscarinic receptor activation is not associated with significant changes in the relative orientations of the outer and/or central segments of TM I and VII. In bovine rhodopsin, the residues present at the positions corresponding to Cys532 and Leu77 in the rat M(3) muscarinic receptor are not located directly adjacent to each other, raising the possibility that the relative orientations of TM I and VII are not identical among different class I GPCRs. Alternatively, dynamic protein backbone fluctuation may occur, enabling Cys532 to move within cross-linking distance of Leu77 (Cys77).
Hamdan
Fadi F
FF
Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 8 Center Drive, Bethesda, Maryland 20892, USA.
Ward
Stuart D C
SD
Siddiqui
Nasir A
NA
Bloodworth
Lanh M
LM
Wess
Jürgen
J
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D019556:COS Cells; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives* / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D008024:Ligands; D008969:Molecular Sequence Data; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D019832:N-Methylscopolamine / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010449:Peptide Mapping / Q000379:methods*; D010716:Phosphatidylinositols / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000187:drug effects / Q000235:genetics; D051381:Rats; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014162:Transfection; D014316:Tritium
D003432:Cross-Linking Reagents; D004220:Disulfides; D008024:Ligands; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D013438:Sulfhydryl Compounds; C093171:methanethiosulfonate ethylammonium; D014316:Tritium; D005020:Ethyl Methanesulfonate; D003545:Cysteine; D000409:Alanine; D019832:N-Methylscopolamine