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

Abstract

Previous studies indicated that partial agonists cause less desensitization of the ␤ 2 -adrenergic receptor (␤AR) 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 ␤AR agonists displaying a 95-fold range of coupling efficiencies. These studies were performed with HEK-293 cells overexpressing the ␤AR with hemagglutinin and 6-histidine epitopes introduced into the N and C termini, respectively. This modified ␤AR behaved identically to the wild type receptor with regard to agonist K d , coupling efficiency, and desensitization. The coupling efficiencies for ␤AR 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 ␤AR activation of adenylyl cyclase followed the same order as coupling efficiency, i.e. epinephrine > fenoterol > albuterol > dobutamine > ephedrine. The rate of internalization of the ␤AR with respect to these agonists also followed the same order as the desensitization and exhibited a slight lag. Like internalization and desensitization, ␤AR phosphorylation exhibited a dependence on agonist strength. The two strongest agonists, epinephrine and fenoterol, provoked 11-13-fold increases in the level of ␤AR 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 ␤AR 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 ␤ARK phosphorylation of the ␤AR.The adenylyl cyclase-coupled ␤AR 1 system has served as a model system for the study of the phenomenon of desensitization of G-protein-coupled receptors (1-5). Desensitization is functionally defined as an attenuation of hormonal responsiveness upon agonist stimulation. There are four currently known mechanisms of agonist-induced desensitization that appear to have physiological significance: receptor sequestration/internalization (1), ␤AR kinase (␤ARK) phosphorylation of serines and threonines on the ␤AR C terminus (3, 5), cAMP-dependent protein kinase (PKA) phosphorylation of ␤AR ...

Intracellular Domains of CXCR3 That Mediate CXCL9, CXCL10, and CXCL11 Function

“…Cells were incubated for 3 h followed by stimulation by 100 nM CXCL10 or CXCL11. Extracts were made, and immunoprecipitations were performed as described previously (36). Briefly, cells were lysed in 0.15 M NaCl, 20 mM HEPES, pH 7.4, 5 mM EDTA, 3 mM EGTA, 4 mg/ml dodecyl-␤-maltoside, 0.2 mg/ml cholesteryl hemisuccinate, plus protease inhibitors and phosphatase inhibitors.…”

Desensitization of β2-Adrenergic Receptors with Mutations of the Proposed G Protein-coupled Receptor Kinase Phosphorylation Sites

“…Construction of the Mutant ␤ARs-The construction of the plasmid containing the HA and six histidine-tagged ␤AR has been described previously (8). This plasmid is designated here as WT␤AR.…”

“…The GRK/ PKA mutants (designated as GRK2 Ϫ or GRK5 Ϫ ), as well as a mutant ␤AR containing only the PKA substitutions (PKA Ϫ ), were constructed in the WT␤AR that had been modified by placement of the hemagglutinin (HA) antigen at the amino terminus and six histidine residues at the carboxyl terminus. We recently established that the desensitization, internalization, and phosphorylation of this double epitope-modified ␤AR, stably transfected into HEK 293 cells, was indistinguishable from the wild type receptor (8). Furthermore, the HEK 293 cell line offers a system in which the effects of overexpressed GRK2 on ␤AR phosphorylation and internalization have been studied (9) and in which endogenous GRK2 expression has been shown (10).…”

Influence of β2-adrenoceptor gene polymorphisms on β2-adrenoceptor expression in human lung

“…14 kinase) that are thought to be involved in receptor desensitization [34][35][36]. Consequently, this could result in a relative increase in β 2 -adrenoceptors in preparations expressing the less frequent allele at position 164.…”

“…Exposure to β 2 -agonists could lead to β 2 -adrenoceptor down-regulation [18,[34][35][36][37] and corticosteroids have been shown to increase β 2 -adrenoceptor expression in a variety of test systems [38][39][40][41]. In the present study, tissue was donated anonymously and drug histories were, therefore, unavailable.…”