Vol. 58, Issue 2, 421-430, August 2000

Kinetic Analysis of Agonist-Induced Down-Regulation of the ₂-Adrenergic Receptor in BEAS-2B Cells Reveals High- and Low-Affinity Components

Bruce R. Williams, Roger Barber, and Richard B. Clark

Department of Integrative Biology and Pharmacology, The University of Texas Medical School, Houston, Texas

We examined the interrelationships of internalization and down-regulation of the ₂-adrenergic receptor in response to treatment of the BEAS-2B human epithelial cell line with both a series of agonists at high occupancy and with various concentrations of fenoterol that gave occupancies from 0.93 to 0.001. We found that the extent of internalization measured after a 30-min treatment increased as a function of coupling efficiency, with ephedrine, dobutamine, albuterol, fenoterol, and epinephrine giving 0, 7, 17, 48, and 55% internalization, respectively. With the exception of dobutamine, the rates of down-regulation (k_deg) also showed a dependence on agonist coupling efficiency, giving (in terms of fraction of receptors lost/h) 0.082 with ephedrine, 0.250 with dobutamine, 0.148 with albuterol, 0.194 with fenoterol, and 0.212 with epinephrine. Comparison of down-regulation to internalization showed that weak agonists caused down-regulation in the absence of significant internalization. The extent of internalization caused by fenoterol over a 1000-fold range of occupancy was proportional to agonist occupancy. However, although no internalization was observed with the low concentrations (0.2 and 2 nM fenoterol), these concentrations did cause significant down-regulation. Thus, as with partial agonists, it was clear that down-regulation occurred in the absence of measurable internalization. The kinetics of agonist-induced down-regulation are consistent with a scheme in which down-regulation proceeds by two pathways; a high-affinity, low-capacity component (EC₅₀ = 0.5 nM) clearly dissociated from internalization and a low-affinity, high-capacity component (EC₅₀ = 160 nM) closely correlated with internalization.