FUNCTIONAL INTERACTIONS OF RECOMBINANT-ALPHA-2 ADRENERGIC-RECEPTOR SUBTYPES AND G-PROTEINS IN RECONSTITUTED PHOSPHOLIPID-VESICLES

The functional interaction of the recombinant alpha-2 adrenergic receptor subtypes, alpha-2-C10 (the human platelet alpha-2 receptor, equivalent to the alpha-2A subtype) and alpha-2-C4 (an alpha-2 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 alpha-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 alpha-subunits with beta-gamma-subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha-2 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 alpha-2-C10 adrenergic receptor subtype showed the following preference: G(i3) > G(i1) greater-than-or-equal-to 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 alpha-2-C4 receptor subtype was the same as that observed for the alpha-2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha-2 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 alpha-2-C10 is coupled more efficiently to G-protein activation than is alpha-2-C4.