Ligand-stimulated beta 2-adrenergic receptor internalization via the constitutive endocytic pathway into rab5-containing endosomes.
The small GTPase rab5 appears to be rate-limiting for the constitutive internalization of transferrin receptor and for fluid-phase endocytosis. However, it is unknown whether rab5 regulates receptors whose internalization is stimulated by the binding of ligand, and whether such receptors change the underlying rate of the endocytic pathways they utilize. As a model for ligand-stimulated endocytosis, we used transfected HEK293 cells expressing high levels of an epitope-tagged human beta 2-adrenergic receptor. Nearly all receptors were on the cell surface in the absence of agonist, but within ten minutes of agonist addition > 50% of receptors internalized and colocalized extensively with rab5. Hypertonic sucrose blocked beta 2-adrenergic receptor internalization, as well as that of transferrin receptor, suggesting a clathrin-mediated process. In contrast, an inhibitor of potocytosis had little effect upon beta 2-adrenergic receptor internalization, suggesting that this process did not require active caveolae. Consistent with this finding, caveolin was not detectable in the 12 beta 6 line, as assessed by western blotting with a polyclonal anti-caveolin antibody. Stimulated receptor internalization did not affect the rate or capacity of the constitutive endocytic pathway since there was no detectable increase in fluid-phase endocytosis after addition of beta-agonist, nor was there a significant change in the amount of surface transferrin receptor. Altogether, these data suggest that beta 2-adrenergic receptors internalize by a clathrin-mediated and rab5-regulated constitutive endocytic pathway. Further, agonist-stimulated receptor internalization has no detectable effect upon the function of this pathway.
Department of Pediatrics, Baylor College of Medicine, Houston VA Medical Center, TX 77030, USA.
R H
RH
Moore
N
N
Sadovnikoff
S
S
Hoffenberg
S
S
Liu
P
P
Woodford
K
K
Angelides
J A
JA
Trial
N D
ND
Carsrud
B F
BF
Dickey
B J
BJ
Knoll
D058666:Adrenergic beta-2 Receptor Agonists; D000595:Amino Acid Sequence; D002460:Cell Line; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000737:chemistry*; D020558:GTP Phosphohydrolases / Q000032:analysis*; D019204:GTP-Binding Proteins / Q000032:analysis*; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D011990:Receptors, Transferrin / Q000032:analysis; D013194:Staining and Labeling; D014162:Transfection; D020696:rab5 GTP-Binding Proteins
D058666:Adrenergic beta-2 Receptor Agonists; D011990:Receptors, Transferrin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins