Agonist-induced sorting of human β₂-adrenergic receptors to lysosomes during downregulation

During prolonged exposure to agonist, β₂-adrenergic receptors undergo downregulation, defined by the loss of radioligand binding sites. To determine the cellular basis for β₂-adrenergic receptor downregulation, we examined HEK293 cells stably expressing β₂-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 β₂-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 β₂-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 β2-adrenergic receptors into lysosomes, causing receptors to accumulate in the rab11-positive perinuclear recycling compartment and slowing the rate of β₂-adrenergic receptor recycling. Control experiments showed that leupeptin had no nonspecific effects on the cellular trafficking of either β₂-adrenergic receptors or transferrin receptors. Although cAMP alone caused a small decline in receptor levels without redistributing β₂-adrenergic receptors from the plasma membrane, this effect was additive to that seen with agonist alone, suggesting that agonist-induced β₂-adrenergic receptor downregulation resulted largely from cAMP-independent mechanisms. These results indicate that during agonist-induced downregulation, a significant fraction of β₂-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.