Publication Date:
2014-10-29
Description:
We expressed the fusion proteins of the muscarinic acetylcholine receptor M 2 subtype (M 2 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-M 2 -Gα i/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 [ 35 S]GTPS binding of the fusion proteins. We found that (i) Gβ promoted both the agonist-dependent and -independent [ 35 S]GTPS binding with little effect on the guanine nucleotide-sensitive high-affinity agonist binding, (ii) the specific [ 35 S]GTPS binding activity was much greater for MBP-M 2 -Gα oA than for MBP-M 2 -Gα i1–i3 in the absence of Gβ, whereas Gβ preferentially promoted the agonist-dependent decrease in the affinity for GDP of MBP-M 2 -Gα i1–i3 rather than of MBP-M 2 -Gα oA , and (iii) the proportion of agonist-dependent [ 35 S]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.
Print ISSN:
0021-924X
Electronic ISSN:
1756-2651
Topics:
Biology
,
Chemistry and Pharmacology
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