Agonist-induced sorting of human beta(2)-adrenergic receptors to lysosomesduring downregulation

During prolonged exposure to agonist, beta(2)-adrenergic receptors undergo downregulation, defined by the loss of radioligand binding sites. To determ ine the cellular basis for beta(2)-adrenergic receptor downregulation, we e xamined HEK293 cells stably expressing beta(2)-adrenergic receptors with an N-terminal epitope tag. Downregulation was blocked by leupeptin, a cystein e protease inhibitor, but not by pepstatin, an inhibitor of aspartate prote ases, Immunofluorescence microscopy of cells treated with agonist for 3-6 h ours in the presence of leupeptin showed beta(2)-adrenergic receptors, but not transferrin receptors, localizing with the lysosomal protease cathepsin D, and with lysosomes labeled by uptake of a fluorescent fluid-phase marke r, No localization of beta(2)-adrenergic receptors with lysosomal markers w as observed in the absence of leupeptin, most likely due to proteolysis of the epitope, The proton pump inhibitor, bafilomycin A(1), significantly inh ibited this agonist-induced redistribution of beta(2)-adrenergic receptors into lysosomes, causing receptors to accumulate in the rab11-positive perin uclear recycling compartment and slowing the rate of beta(2)-adrenergic rec eptor recycling. Control experiments showed that leupeptin had no nonspecif ic effects on the cellular trafficking of either beta(2)-adrenergic recepto rs or transferrin receptors, Although cAMP alone caused a small decline in receptor levels without redistributing beta(2)-adrenergic receptors from th e plasma membrane, this effect was additive to that seen with agonist alone , suggesting that agonist-induced beta(2)-adrenergic receptor downregulatio n resulted largely from cAMP-independent mechanisms, These results indicate that during agonist-induced downregulation, a significant fraction of beta (2)-adrenergic receptors are specifically sorted to lysosomes via the endos omal pathway, where receptor degradation by cysteine proteases occurs. Thes e results provide a cellular explanation for the loss of radioligand bindin g sites that occurs during prolonged exposure to agonist.