Deletion analysis of the m4 muscarinic acetylcholine receptor. Molecular determinants for activation of but not coupling to the Gi guanine-nucleotide-binding regulatory protein regulate receptor internalization

Eur J Biochem. 1994 Jun 1;222(2):525-31. doi: 10.1111/j.1432-1033.1994.tb18894.x.


In order to investigate whether coupling to and/or activation of guanine-nucleotide-binding proteins (G proteins) is involved in agonist-induced internalization of m4 muscarinic acetylcholine receptors (mAChRs), a deletion mutant [des-(264-394)mAChR] was constructed that lacks a substantial portion of the putative third intracellular loop. The wild-type receptor and des-(264-394)mAChR stably expressed in Chinese hamster ovary cells in essentially comparable amounts, exhibited identical antagonist-binding affinities. Consistent with the reported importance of the third cytoplasmic loop for Gi protein activation, the des-(264-394)mAChR showed a drastically reduced potential to mediate agonist-induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to Gi proteins was not impaired, as demonstrated by a similar guanine-nucleotide-sensitive and pertussis-toxin-sensitive high-affinity agonist-receptor binding for both mAChRs. In contrast, des-(264-394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of Gi proteins rather than ineffective coupling to Gi proteins. Internalization of wild-type receptor and des-(264-394)mAChR was observed with similar agonist concentrations and showed similar maximal values. However, des-(264-394)mAChR displayed a significantly reduced rate of receptor internalization. A similar attenuation of wild-type mAChR internalization was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR involved in Gi-protein coupling and activation are not identical and that activation of, but not coupling to, Gi proteins regulates m4 mAChR internalization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenylyl Cyclases / metabolism
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • CHO Cells
  • Carbachol / pharmacology
  • Colforsin / pharmacology
  • Cricetinae
  • Dose-Response Relationship, Drug
  • GTP-Binding Proteins / metabolism*
  • Guanosine Triphosphate / metabolism
  • Kinetics
  • Mice
  • Molecular Sequence Data
  • Mutagenesis
  • Oligodeoxyribonucleotides
  • Protein Structure, Secondary
  • Receptors, Muscarinic / chemistry
  • Receptors, Muscarinic / genetics
  • Receptors, Muscarinic / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Sequence Deletion*
  • Transfection


  • Oligodeoxyribonucleotides
  • Receptors, Muscarinic
  • Recombinant Proteins
  • Colforsin
  • Guanosine Triphosphate
  • Carbachol
  • GTP-Binding Proteins
  • Adenylyl Cyclases