Interaction of the muscarinic acetylcholine receptor M2 subtype with G protein G{alpha}i/o isotypes and G{beta}{gamma} subunits as studied with the maltose-binding protein-M2-G{alpha}i/o fusion proteins expressed in Escherichia coli

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 (Gαi1–i3/o) at its N- and C-terminals, respectively (MBP-M2-Gαi/o), in Escherichia coli, and examined the effect of G protein β subunits (Gβ) on the receptor–Gα interaction as assessed by agonist- and GDP-dependent [35S]GTPS binding of the fusion proteins. We found that (i) Gβ promoted both the agonist-dependent and -independent [35S]GTPS binding with little effect on the guanine nucleotide-sensitive high-affinity agonist binding, (ii) the specific [35S]GTPS binding activity was much greater for MBP-M2-GαoA than for MBP-M2-Gαi1–i3 in the absence of Gβ, whereas Gβ preferentially promoted the agonist-dependent decrease in the affinity for GDP of MBP-M2-Gαi1–i3 rather than of MBP-M2-GαoA, and (iii) the proportion of agonist-dependent [35S]GTPS binding was roughly 50% irrespective of species of Gα and the presence or absence of Gβ. These results demonstrate that receptor-Gα fusion proteins expressed in E. coli could be useful for studies of receptor–G interaction.
Source: Journal of Biochemistry - Category: Biochemistry Authors: Tags: Regular Papers Source Type: research