Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.
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.
Département de Pharmacologie et de Toxicologie, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland.
Laura
L
Stanasila
Liliane
L
Abuin
Julien
J
Dey
Susanna
S
Cotecchia
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033983:Adaptor Protein Complex mu Subunits / Q000378:metabolism*; D000818:Animals; D019390:Arrestins / Q000378:metabolism*; D019921:Biotinylation; D002460:Cell Line; D006224:Cricetinae; D004705:Endocytosis; D020868:Gene Silencing; D006801:Humans; D047468:Immunoprecipitation; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
C000605254:ARRB2 protein, human; D033962:Adaptor Protein Complex 2; D033983:Adaptor Protein Complex mu Subunits; C000605257:Arrb2 protein, rat; D019390:Arrestins; D050505:Mutant Proteins; D018340:Receptors, Adrenergic, alpha-1; D011993:Recombinant Fusion Proteins; C438885:adaptor protein complex 2, mu 1 subunit; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins