Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remain ing Cys residues Cys-140, Cys-220, and Cys-532) and in which the central po rtion of the third intracellular loop had been replaced with a factor Xa cl eavage site. Radioligand binding and second messenger assays showed that th e m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus gener ating 10 double Cys mutant receptors, All 10 mutant receptors contained a C ys residue at position 169 at the beginning of the second intracellular loo p and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493, Radioligand binding studies and phosphatidylin ositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the dif ferent mutant receptor constructs were incubated with factor Xa protease an d the oxidizing agent Cu(II)-(1,10-phenanthroline)(3), and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitor ed by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 d ouble Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [S-35]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cro ss-links prevented or strongly inhibited receptor-mediated G protein activa tion, suggesting that the highly dynamic character of the cytoplasmic recep tor surface is a prerequisite for efficient receptor-G protein interactions . This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled recep tor.