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 α1a- and α1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the α1b-AR displayed robust agonist-induced endocytosis, the α1a-AR did not. Constitutive internalization of the α1a-AR was negligible, whereas the α1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the α1-AR subtypes with β-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and β-arrestin translocation assays indicated that the agonistinduced interaction of the α1a-AR with β-arrestins was much weaker than that of the α1b-AR. In addition, the α1a-AR did not bind AP50. The α1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding β-arrestins despite its binding to AP50. In contrast, the α1b-AR mutant ΔR8, lacking AP50 binding, bound β-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that β-arrestin 2 plays a prominent role in α1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the α1a- and α1b-AR and provide evidence that the lack of significant endocytosis of the α1a-AR is linked to its poor interaction with β-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the α1b-AR is important for receptor/β-arrestin interaction and that this interaction is the main event triggering receptor internalization.