Location of agonist-dependent-phosphorylation sites in the third intracellular loop of muscarinic acetylcholine receptors (m2 subtype)

Eur J Biochem. 1994 Feb 15;220(1):29-36. doi: 10.1111/j.1432-1033.1994.tb18595.x.


Muscarinic acetylcholine receptors (mAChR, human m2 subtype) expressed in Sf9 (Spodoptera frugiperda) cells using the baculovirus system were purified and subjected to phosphorylation by a mAChR kinase, which was partially purified from porcine cerebrum. Two bands with apparent molecular masses of 59 kDa and 39 kDa as determined by SDS/PAGE were found to be phosphorylated in an agonist-dependent manner. Both bands were labeled by the irreversible muscarinic ligand [3H]propylbenzilylcholine mustard. Molecular masses of the [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled bands decreased following treatment with N-glycanase. The 59-kDa and 39-kDa bands were converted to 52-kDa and 32-kDa bands, respectively, indicating that both the 59-kDa and 39-kDa bands contain the amino-terminal region where glycosylation sites are present. The ratio of incorporated [32P]phosphate and bound [3H]propylbenzilylcholine mustard was essentially the same for the 59-kDa and 39-kDa bands, indicating that all the phosphorylation sites reside in the sequence of 39 kDa from the amino-terminal region. The amounts of incorporated [32P]phosphate were estimated to be 10-11/receptor, with 7-8 serine and 3-4 threonine, but no phosphorylated tyrosine residues. Further treatment of [32P]phosphorylated or [3H]propylbenzilylcholine-mustard-labeled receptors with V8 protease indicated that the phosphorylation sites were not present in 30-kDa amino-terminal segment. These results indicate that the phosphorylation sites are localized in the range 30-39 kDa from the amino terminus, which consists of primarily the central part of the third intracellular loop. Consistent with this conclusion, a fusion protein containing glutathione S-transferase linked to a peptide corresponding to residues 227-324 of the central part of the third intracellular loop was found to be phosphorylated by the mAChR kinase in a heparin-sensitive manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Baculoviridae / genetics
  • Binding Sites / genetics
  • Cloning, Molecular
  • G-Protein-Coupled Receptor Kinase 2
  • Humans
  • Hydrolysis
  • In Vitro Techniques
  • Molecular Sequence Data
  • Molecular Weight
  • Moths
  • Phosphorylation
  • Protein Conformation
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Receptors, Muscarinic / chemistry
  • Receptors, Muscarinic / genetics
  • Receptors, Muscarinic / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Swine


  • Receptors, Muscarinic
  • Recombinant Fusion Proteins
  • Receptor Protein-Tyrosine Kinases
  • muscarinic receptor kinase
  • G-Protein-Coupled Receptor Kinase 2