Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50

• Published in: Molecular pharmacology Vol. 74; no. 3; p. 562
• Main Authors: Stanasila, Laura, Abuin, Liliane, Dey, Julien, Cotecchia, Susanna
• Format: Journal Article
• Language: English
• Published: United States 01-09-2008
• Subjects: Adaptor Protein Complex 2 - metabolism; Adaptor Protein Complex mu Subunits - metabolism; Animals; Arrestins - metabolism; beta-Arrestin 2; beta-Arrestins; Biotinylation; Cell Line; Cricetinae; Endocytosis; Gene Silencing; Humans; Immunoprecipitation; Mutant Proteins - metabolism; Protein Binding; Protein Transport; Rats; Receptors, Adrenergic, alpha-1 - chemistry; Receptors, Adrenergic, alpha-1 - metabolism; Recombinant Fusion Proteins - metabolism; Structure-Activity Relationship
• ISSN: 1521-0111
• DOI: 10.1124/mol.107.043422

The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-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 agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-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 alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-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 alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.