Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor

EMBO J. 1993 Jan;12(1):331-8.

Abstract

Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]N-methylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242-->Ala, TMD V; Pro505-->Ala, TMD VI; Pro540-->Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201-->Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540-->Ala (TMD VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (Emax approximately 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affinities than the wild type receptor. The Trp-->Phe substitutions (Trp192-->Phe, TMD IV; Trp503-->Phe, TMD VI; Trp530-->Phe, TMD VII) resulted in less pronounced changes (compared with the Pro-->Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G protein-coupled receptors play key roles in receptor expression, ligand binding and receptor activation.

MeSH terms

  • Acetylcholine / metabolism
  • Acetylcholine / pharmacology
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Binding Sites
  • Blotting, Northern
  • Carbachol / metabolism
  • Carbachol / pharmacology
  • Cell Line
  • Codon / genetics
  • GTP-Binding Proteins / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • N-Methylscopolamine
  • Parasympatholytics / metabolism
  • Parasympatholytics / pharmacology
  • Piperidines / metabolism
  • Piperidines / pharmacology
  • Proline*
  • Protein Conformation
  • RNA / genetics
  • RNA / isolation & purification
  • Rats
  • Receptors, Muscarinic / chemistry
  • Receptors, Muscarinic / genetics*
  • Receptors, Muscarinic / metabolism*
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Scopolamine Derivatives / metabolism
  • Transfection
  • Tryptophan*

Substances

  • Codon
  • Parasympatholytics
  • Piperidines
  • Receptors, Muscarinic
  • Recombinant Proteins
  • Scopolamine Derivatives
  • RNA
  • 4-diphenylacetoxy-1,1-dimethylpiperidinium
  • Tryptophan
  • Carbachol
  • Proline
  • GTP-Binding Proteins
  • Acetylcholine
  • N-Methylscopolamine