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Journal of Cell Science, Vol 112, Issue 3 329-338, Copyright © 1999 by Company of Biologists
JOURNAL ARTICLES |
RH Moore, A Tuffaha, EE Millman, W Dai, HS Hall, BF Dickey and BJ Knoll
Departments of Pediatrics (Pulmonary), Molecular Physiology & Biophysics, Medicine (Pulmonary) and Cell Biology, Baylor College of Medicine, Houston VA Medical Center, Houston, TX 77030, USA.
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.
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