Specific changes in beta(2)-adrenoceptor trafficking kinetics and intracellular sorting during downregulation

Agonist-activated beta(2)-adrenoceptors rapidly internalize and then recycl e to the cell surface, however chronic agonist eventually causes receptor d ownregulation. To characterize beta(2)-adrenoceptor trafficking kinetics an d intracellular sorting during downregulation, human embryonic kidney cells expressing epitope-tagged receptors were examined by radioligand binding w ith (+/-)-[H-3]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride ([H-3]CGP12177) and immunofluorescence microscopy. The first -order receptor recycling rate constant declined after 18 h of agonist comp ared with 15 min (0.05 min(-1) vs. 0.12 min(-1)), thus increasing the intra cellular transit time (20.0 min vs. 8.3 min). There was also a reduction in the rate of receptor endocytosis and a decline in the total number of rece ptors. Although the intracellular receptor fraction did not increase betwee n 15 min and 18 h of agonist, some receptors moved irreversibly into a prot ease-containing compartment while retaining radioligand binding activity. O ur results indicate that beta(2)-adrenoceptor downregulation is associated principally with an increased intracellular transit time during recycling. This could promote the diversion of receptors into protease-containing comp artments, where there is an irreversible commitment to downregulation prior to loss of radioligand binding activity. (C) 1999 Elsevier Science B.V. Al l rights reserved.