The second intracellular loop of the alpha2-adrenergic receptors determines subtype-specific coupling to cAMP...

The alpha2-adrenergic receptors (alpha2-ARs), which primarily couple to inhibition of cAMP production, have been reported to have a stimulating effect on adenylyl cyclase activity in certain cases. When expressed in Spodoptera frugiperda Sf9 cells the alpha2A subtype showed only inhibition of forskolin-stimulated cAMP production when activated by norepinephrine (NE), whereas the alpha2B subtype displayed a biphasic dose-response curve with inhibition at low concentrations of NE and a potentiation at higher concentrations. To further investigate the subtype-specific coupling, we expressed a set of chimeric alpha2A-/alpha2B-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 alpha2A and alpha2B subtypes, the coupling specificity remained unchanged, indicating that this loop does not confer selectivity toward a stimulating response. A biphasic dose-response curve, typical for the alpha2B subtype, could be seen when the second intracellular loop of the alpha2B subtype was inserted into the alpha2A subtype, suggesting that this loop is important for determining the subtype-specific coupling of alpha2-ARs to cAMP production. Site-directed mutagenesis of non-conserved amino acids in the second intracellular loop of the alpha2A subtype indicated that several residues are involved in the coupling specificity.