Functional Interactions of Recombinant α₂ Adrenergic Receptor Subtypes and G Proteins in Reconstituted Phospholipid Vesicles

The functional interaction of the recombinant α₂ adrenergic receptor subtypes, α₂-C10 (the human platelet α₂ receptor, equivalent to the α₂A subtype) and α₂-C4 (an α₂ receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of G_s (adenylyl cyclase stimulatory) and members of the inhibitory family, including G_i1 G_i2, and G_i3, and G₀. The cloned α subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the α subunits with βγ subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both α₂ receptor subtypes could couple to the inhibitory G proteins but not to G_s, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the α₂-C10 adrenergic receptor subtype showed the following preference: G_i3 > G_i1 > G_i2 > G₀. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (G_i3) to 1.5-fold (G₀). The preference of G-protein interaction for the α₂-C4 receptor subtype was the same as that observed for the α₂-C10, but the extent of activation was slightly lower. The results show that in vitro each of the α₂ adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that α₂-C10 is coupled more efficiently to G-protein activation than is α₂-C4.