Received for publication November 12, 2007.
Revised May 28, 2008.
Accepted for publication June 2, 2008.

DIFFERENT INTERNALIZATION PROPERTIES OF THE 1a AND 1b-ADRENERGIC RECEPTOR SUBYTPES: THE POTENTIAL ROLE OF RECEPTOR INTERACTION WITH ARRESTINS AND AP50

Abstract

The internalization properties of the 1a and 1b-adrenergic receptors (AR) subtypes transiently expressed in 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 some constitutive internalization and recycling. We investigated the interaction of the 1-AR subtypes with arrestin 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both co-immunoprecipitation experiments and arrestin translocation assays indicated that the agonist-induced interaction of the 1a-AR with 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 arrestins despite its binding to AP50. In contrast, the 1b-AR mutant R8, lacking AP50 binding, bound arrestins efficiently and displayed delayed endocytosis. RNA interference showed the 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 suggest that the lack of significant endocytosis of the 1a-AR might be linked to its poor interaction with 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/arrestin interaction and that this interaction is the main event triggering receptor internalization.

Key words: Adrenergic, Sequestration/Internalization, GRKs, barrestins, Recycling