title: Location of agonist-dependent-phosphorylation sites in the third intracellular loop of muscarinic acetylcholine receptors (m2 subtype).
abstract: 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.
authors.affiliation: Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Nakata
authors.forename: K
authors.initials: K
authors.lastname: Kameyama
authors.forename: K
authors.initials: K
authors.lastname: Haga
authors.forename: T
authors.initials: T
authors.lastname: Haga
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D054769:G-Protein-Coupled Receptor Kinase 2; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D010766:Phosphorylation; D011487:Protein Conformation; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine
chemical_list: D011976:Receptors, Muscarinic; D011993:Recombinant Fusion Proteins; D020794:Receptor Protein-Tyrosine Kinases; C080577:muscarinic receptor kinase; D054769:G-Protein-Coupled Receptor Kinase 2