β2-Adrenergic Receptor Desensitization, Internalization, and Phosphorylation in Response to Full and Partial Agonists

Previous studies indicated that partial agonists cause less desensitization of the β2-adrenergic receptor (βAR) than full agonists; however, the molecular basis for this in intact cells has not been investigated. In the present work, we have determined the rates of desensitization, internalization, and phosphorylation caused by a series of βAR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the βAR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified βAR behaved identically to the wild type receptor with regard to agonistKd, coupling efficiency, and desensitization. The coupling efficiencies for βAR agonist activation of adenylyl cyclase relative to epinephrine (100%) were 42% for fenoterol, 4.9% for albuterol, 2.5% for dobutamine, and 1.1% for ephedrine. At concentrations of these agonists yielding >90% receptor occupancy, the rate and extent (0–30 min) of agonist-induced desensitization of βAR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine ≥ fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the βAR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, βAR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11–13-fold increases in the level of βAR phosphorylation after just 1 min, whereas the weak agonists dobutamine and ephedrine caused only 3–4-fold increases, similar to levels induced by cAMP-dependent protein kinase activation with forskolin. With longer treatment times, the level of βAR phosphorylation declined with strong agonists, but it progressively increased with the weaker partial agonists, such that after 30 min the -fold elevation with epinephrine (6.2 ± 0.82) was not appreciably different from ephedrine (5.0 ± 0.96) and significantly less than that caused by albuterol (10.4 ± 1.7). In summary, our results demonstrate an excellent proportionality between the agonist strength and agonist-induced desensitization, internalization, and the rapid initial phase of phosphorylation. The data support the hypothesis that increasing agonist-coupling efficiency primarily affects desensitization by increasing the rate of βARK phosphorylation of the βAR.