α2A- and α2C-Adrenergic Receptors Form Homo- and Heterodimers: The Heterodimeric State Impairs Agonist-Promoted GRK Phosphorylation and β-Arrestin Recruitment

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Abstract
Dimerization of seven transmembrane-spanning receptors diversifies their pharmacologic and physiologic properties. The α2-adrenergic receptor (α2AR) subtypes A and C are both expressed on presynaptic nerves and act to inhibit norepinephrine release via negative feedback. However, in vivo and in vitro studies examining the roles of the two individual α2A- and α2CAR subtypes are not readily reconciled. We tested the hypothesis that the receptors form homo- and heterodimers and that the α2A−α2C heterodimer has unique properties. SDS−PAGE of epitope-tagged receptors revealed potential oligomers including dimers. BRET of live HEK-293 cells transfected with the subtypes fused to Rluc or YFP revealed that both subtypes form dimers and the heterodimer. A lower BRET50 for the α2A−α2C heterodimer (0.79 ± 0.20) compared to that of the α2A or α2C homodimer (2.331 ± 0.44 or 3.67 ± 0.69, respectively) suggests that when both subtypes are expressed, there is a greater likelihood that the two receptors will form the heterodimer than homodimers. Co-immunoprecipitation studies confirmed homo- and heterodimer formation. The presence of the α2CAR within the heterodimer resulted in a marked reduction in the level of GRK2-mediated α2AAR phosphorylation, which was accompanied by a qualitative attenuation of β-arrestin recruitment. Signaling of the α2A−α2C heterodimer to the β-arrestin-dependent activation of Akt was decreased compared to that of the α2AAR homodimer, while p44/p42 MAP kinase activation was unaffected. Thus, the α2CAR alters α2AAR signaling by forming oligomers, and these complexes, which appear to be preferred over the homodimers, should be considered a functional signaling unit in cells in which both subtypes are expressed.

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