Rh. Moore et al., Specific changes in beta(2)-adrenoceptor trafficking kinetics and intracellular sorting during downregulation, EUR J PHARM, 369(1), 1999, pp. 113-123
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.