Agonist-induced sorting of human beta2-adrenergic receptors to lysosomes during downregulation.
During prolonged exposure to agonist, beta2-adrenergic receptors undergo downregulation, defined by the loss of radioligand binding sites. To determine the cellular basis for beta2-adrenergic receptor downregulation, we examined HEK293 cells stably expressing beta2-adrenergic receptors with an N-terminal epitope tag. Downregulation was blocked by leupeptin, a cysteine protease inhibitor, but not by pepstatin, an inhibitor of aspartate proteases. Immunofluorescence microscopy of cells treated with agonist for 3-6 hours in the presence of leupeptin showed beta2-adrenergic receptors, but not transferrin receptors, localizing with the lysosomal protease cathepsin D, and with lysosomes labeled by uptake of a fluorescent fluid-phase marker. No localization of beta2-adrenergic receptors with lysosomal markers was observed in the absence of leupeptin, most likely due to proteolysis of the epitope. The proton pump inhibitor, bafilomycin A1, significantly inhibited this agonist-induced redistribution of beta2-adrenergic receptors into lysosomes, causing receptors to accumulate in the rab11-positive perinuclear recycling compartment and slowing the rate of beta2-adrenergic receptor recycling. Control experiments showed that leupeptin had no nonspecific effects on the cellular trafficking of either beta2-adrenergic receptors or transferrin receptors. Although cAMP alone caused a small decline in receptor levels without redistributing beta2-adrenergic receptors from the plasma membrane, this effect was additive to that seen with agonist alone, suggesting that agonist-induced beta2-adrenergic receptor downregulation resulted largely from cAMP-independent mechanisms. These results indicate that during agonist-induced downregulation, a significant fraction of beta2-adrenergic receptors are specifically sorted to lysosomes via the endosomal pathway, where receptor degradation by cysteine proteases occurs. These results provide a cellular explanation for the loss of radioligand binding sites that occurs during prolonged exposure to agonist.
Moore
R H
RH
Departments of Pediatrics (Pulmonary), Molecular Physiology & Biophysics, Medicine (Pulmonary) and Cell Biology, Baylor College of Medicine, Houston VA Medical Center, Houston, TX 77030, USA.
Tuffaha
A
A
Millman
E E
EE
Dai
W
W
Hall
H S
HS
Dickey
B F
BF
Knoll
B J
BJ
D058666:Adrenergic beta-2 Receptor Agonists; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002402:Cathepsin D / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism*; D018942:Macrolides; D018613:Microscopy, Confocal; D011480:Protease Inhibitors / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000032:analysis / Q000378:metabolism*; D013997:Time Factors; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
D058666:Adrenergic beta-2 Receptor Agonists; D000900:Anti-Bacterial Agents; D002402:Cathepsin D; D019204:GTP-Binding Proteins; D018942:Macrolides; D011480:Protease Inhibitors; D018343:Receptors, Adrenergic, beta-2; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C040929:bafilomycin A1