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Different Internalization Properties of the 1a- and 1b-Adrenergic Receptor Subtypes: The Potential Role of Receptor Interaction with β-Arrestins and AP50

Département de Pharmacologie et Toxicologie, Faculté de Biologie et de Médecine, Lausanne, Switzerland

The internalization properties of the 1a- and 1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the 1b-AR displayed robust agonist-induced endocytosis, the 1a-AR did not. Constitutive internalization of the 1a-AR was negligible, whereas the 1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the 1-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 1a-AR with β-arrestins was much weaker than that of the 1b-AR. In addition, the 1a-AR did not bind AP50. The 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 β-arrestins despite its binding to AP50. In contrast, the 1b-AR mutant R8, lacking AP50 binding, bound β-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that β-arrestin 2 plays a prominent role in 1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the 1a- and 1b-AR and provide evidence that the lack of significant endocytosis of the 1a-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 1b-AR is important for receptor/β-arrestin interaction and that this interaction is the main event triggering receptor internalization.

Address correspondence to: Prof. Susanna Cotecchia, Département de Pharmacologie et de Toxicologie, Rue du Bugnon 27, CH-1005 Lausanne. E-mail: susanna.cotecchia{at}unil.ch