THE 2ND INTRACELLULAR LOOP OF THE ALPHA(2)-ADRENERGIC RECEPTORS DETERMINES SUBTYPE-SPECIFIC COUPLING TO CAMP PRODUCTION

The alpha(2)-adrenergic receptors (alpha(2)-ARs), which primarily coup le to inhibition of cAMP production, have been reported to have a stim ulating effect on adenylyl cyclase activity in certain cases. When exp ressed in Spodoptera frugiperda Sf9 cells the alpha(2A) subtype showed only inhibition of forskolin-stimulated cAMP production when activate d by norepinephrine (NE), whereas the alpha(2B) subtype displayed a bi phasic dose-response curve with inhibition at low concentrations of NE and a potentiation at higher concentrations. To further investigate t he subtype-specific coupling, we expressed a set of chimeric alpha(2A) -/alpha(2B)-ARs at similar expression levels in Sf9 cells to determine the structural domain responsible for the difference between the two subtypes. When the third intracellular loops were interchanged between alpha(2A) and alpha(2B) subtypes, the coupling specificity remained u n changed, indicating that this loop does not confer selectivity towar d a stimulating response. A biphasic dose-response curve, typical for the alpha(2B) subtype, could be seen when the second intracellular loo p of the alpha(2B) subtype was inserted into the alpha(2A) subtype, su ggesting that this loop is important for determining the subtype-speci fic coupling of alpha(2)-ARs to cAMP production. Site-directed mutagen esis of non-conserved amino acids in the second intracellular loop of the alpha(2A) subtype indicated that several residues are involved in the coupling specificity.