JCVI: Site-directed Mutagenesis of the Rat M1 Muscarinic Acetylcholine Receptor. Role of Conserved Cysteines In Receptor Function
 
 
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Savarese, T. M., Wang, C. D., Fraser, C. M.

Site-directed Mutagenesis of the Rat M1 Muscarinic Acetylcholine Receptor. Role of Conserved Cysteines In Receptor Function

J Biol Chem. 1992 Jun 05; 267(16): 11439-48.

PubMed Citation

Abstract

There are 9 cysteine residues in the rat m1 muscarinic acetylcholine receptor (mAChR) that are conserved among all five mammalian mAChR subtypes sequenced to date. To study the role of these cysteines in rat m1 mAChR function, site-directed mutagenesis was used to convert each Cys residue to Ser, and the mutant receptor genes were transfected into mAChR-deficient Chinese hamster ovary (CHO) cells. Substitution of Cys391 (extracellular loop III) or Cys421 and Cys435 (carboxyl terminus) produces receptors with wild type phenotype. Cells transfected with Ser98 or Ser178 (extracellular loops I and II, respectively) receptor genes display no carbachol-mediated hydrolysis of phosphoinositides (PI), and membranes prepared from these cells do not bind the muscarinic antagonist [3H] quinuclidinyl benzilate, even though the cells express transcripts of the m1 mAChR as determined by RNA hybridization analysis. Since biochemical evidence suggests that these cysteines form a disulfide bridge (Curtis, C. A. M., Wheatley, M., Bansal, S., Birdsall, N. J. M., Eveleigh, P., Pedder, E. K., Poyner, D., and Hulme, E. C. (1989) 264, 489-495), our findings imply that this disulfide linkage may be critical for formation of the ligand binding domain or for proper protein folding. The Ser394 mAChR (extracellular loop III) exhibits a 44% decrease in efficacy for carbachol-mediated stimulation of PI hydrolysis relative to the wild type receptor, but displays normal ligand binding affinities. The Ser407 m1 mAChR (transmembrane helix VII) displays a decreased efficacy for eliciting carbachol-mediated PI hydrolysis (39% that of CHO cells transfected with the wild type receptor) and a 4-fold shift to the right in the carbachol dose-response curve, which is consistent with the 4-fold decrease in carbachol affinity at the Ser407 m1 mAChR. In contrast, the Ser417 m1 mAChR (transmembrane helix VII) displays an increase in carbachol affinity and a shift to the left in the carbachol dose-response curve for PI hydrolysis. These findings suggest that cysteine residues in the seventh transmembrane helix of the m1 mAChR may influence agonist binding and the efficiency of receptor activation.