Different internalization properties of the α1a- and α1b-adrenergic receptor subtypes:: The potential role of receptor interaction with β-arrestins and AP50

The internalization properties of the alpha 1a- and alpha 1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha 1b-AR displayed robust agonist-induced endocytosis, the alpha 1a-AR did not. Constitutive internalization of the alpha 1a-AR was negligible, whereas the alpha 1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha 1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonist-induced interaction of the alpha 1a-AR with beta-arrestins was much weaker than that of the alpha 1b-AR. In addition, the alpha 1a-AR did not bind AP50. The alpha 1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha 1b-AR mutant Delta R8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha 1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha 1a- and alpha 1b-AR and provide evidence that the lack of significant endocytosis of the alpha 1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha 1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.