Coupling of the alpha(2A)-adrenergic receptor to the G -proteins G(i2) and G(i3).

Abstract

The $\alpha\sb2$-adrenergic receptor (AR) couples to the pertussis toxin-sensitive G$\sb{\rm i}$ class of G-proteins. $\alpha\sb2$-AR stimulation has many cellular effects including inhibition of adenylyl cyclase, activation of Na$\sp+$/H$\sp+$ antiport, regulation of K$\sp+$ channels and intracellular Ca$\sp{++}$. The $\alpha\sb2$-AR has at least three subtypes ($\alpha\sb{\rm 2A}, \alpha\sb{\rm 2B}, \alpha\sb{\rm 2C}$). There are three known G$\sb{\rm i}$ subtypes (G$\sb{\rm i1}$, G$\sb{\rm i2}$, G$\sb{\rm i3}$). A fundamental mechanistic question of signal transduction concerns that of coupling of receptors to G-proteins, and of G-proteins to effectors. Specifically, does a single receptor subtype couple to a single G-protein to produce a unique response, or alternatively is coupling promiscuous? To answer this scientific question, I tested the hypothesis: the $\alpha\sb{\rm 2A}$-AR couples to multiple G-proteins. Prior to answering this question, I developed two novel reagents. First, I characterized p-iodoclonidine (PIC), the only known ($\sp{125}$I) $\alpha\sb2$-AR agonist (Gerhardt et al., Mol. Pharmacol, 38:214, 1990). Second, I transfected and isolated a CHO-K1 clone (MAG-2 cells) which stably expresses 2.3 pmol $\alpha\sb{\rm 2A}$-AR/mg membrane protein. Western blots of MAG-2 membrane proteins demonstrated the presence of G$\sb{\rm i2}$ and G$\sb{\rm i3}$. To test the hypothesis, antisera to the receptor-coupling domain of G$\sb{\rm i1}$/G$\sb{\rm i2}$ or G$\sb{\rm i3}$ were used in binding and functional assays to ascertain which subtype(s) couples to the receptor. Both antisera decreased high-affinity, GppNHp- sensitive specific binding of ($\sp3$H) -bromoxidine and ($\sp{125}$I) -PIC by 30-50% over control (non-immune serum). In the adenylyl cyclase assay, each antiserum reduced bromoxidine or PIC mediated inhibition 30-60%; when used in combination, there was complete reversal of $\alpha\sb{\rm 2A}$-AR inhibition. The antisera effect was concentration-dependent and was not competitive. Radioligand affinity was unaltered by the antisera. In conclusion, the $\alpha\sb{\rm 2A}$-AR couples to the G$\sb{\rm i2}$ and G$\sb{\rm i3}$ G-proteins. Furthermore, both G$\sb{\rm i2}$ and G$\sb{\rm i3}$ inhibit adenylyl cyclase (Gerhardt and Neubig, Mol. Pharmacol. 40:707-711, 1991).