title: Interaction of the muscarinic acetylcholine receptor M₂ subtype with G protein Gα(i/o) isotypes and Gβγ subunits as studied with the maltose-binding protein-M₂-Gα(i/o) fusion proteins expressed in Escherichia coli.
abstract: We expressed the fusion proteins of the muscarinic acetylcholine receptor M2 subtype (M2 receptor) with a maltose-binding protein (MBP) and various G protein α subunits (Gα(i1-i3/o)) at its N- and C-terminals, respectively (MBP-M2-Gα(i/o)), in Escherichia coli, and examined the effect of G protein βγ subunits (Gβγ) on the receptor-Gα interaction as assessed by agonist- and GDP-dependent [(35)S]GTPγS binding of the fusion proteins. We found that (i) Gβγ promoted both the agonist-dependent and -independent [(35)S]GTPγS binding with little effect on the guanine nucleotide-sensitive high-affinity agonist binding, (ii) the specific [(35)S]GTPγS binding activity was much greater for MBP-M2-Gα(oA) than for MBP-M2-Gα(i1-i3) in the absence of Gβγ, whereas Gβγ preferentially promoted the agonist-dependent decrease in the affinity for GDP of MBP-M2-Gα(i1-i3) rather than of MBP-M2-Gα(oA), and (iii) the proportion of agonist-dependent [(35)S]GTPγS binding was roughly 50% irrespective of species of Gα and the presence or absence of Gβγ. These results demonstrate that receptor-Gα fusion proteins expressed in E. coli could be useful for studies of receptor-G interaction.
authors.affiliation: Faculty of Science, Institute for Biomolecular Science, Gakushuin University, 1-5-1 Mejiro, Toshima-Ku, Tokyo 171-8588, Japan susumuG4ichiyama@gmail.com.
authors.forename: Susumu
authors.initials: S
authors.lastname: Ichiyama
authors.affiliation: Faculty of Science, Institute for Biomolecular Science, Gakushuin University, 1-5-1 Mejiro, Toshima-Ku, Tokyo 171-8588, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Nemoto
authors.affiliation: Faculty of Science, Institute for Biomolecular Science, Gakushuin University, 1-5-1 Mejiro, Toshima-Ku, Tokyo 171-8588, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tanabe
authors.affiliation: Faculty of Science, Institute for Biomolecular Science, Gakushuin University, 1-5-1 Mejiro, Toshima-Ku, Tokyo 171-8588, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Haga
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D044385:GTP-Binding Protein alpha Subunits / Q000737:chemistry / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D043585:Receptor, Muscarinic M2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C487078:CHRM2 protein, human; D044385:GTP-Binding Protein alpha Subunits; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D058619:Maltose-Binding Proteins; D043585:Receptor, Muscarinic M2; D011993:Recombinant Fusion Proteins
keywords: G protein; G protein-coupled receptor (GPCR); muscarinic acetylcholine receptor M2 subtype