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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 ...

The neuropeptide somatostatin inhibits secretion from electrically excitable cells in the pituitary, pancreas, gut and brain. In mammalian pituitary tumour cells somatostatin inhibits secretion through two distinct pertussis toxin-sensitive mechanisms. One involves inhibition of adenylyl cyclase, the other an unidentified cyclic AMP-independent mechanism that reduces Ca2+ influx by increasing membrane conductance to potassium. Here we demonstrate that the predominant electrophysiological effect of somatostatin on metabolically intact pituitary tumour cells is a large, sustained increase in the activity of the large-conductance Ca(2+)- and voltage-activated K+ channels (BK). This action of somatostatin does not involve direct effects of Ca2+, cAMP or G proteins on the channels. Our results indicate instead that somatostatin stimulates BK channel activity through protein dephosphorylation.

Foxp3(+) regulatory T cells (Treg) have a central role for keeping the balance between pro- and anti-inflammatory immune responses against chronically encountered antigens at mucosal sites. However, their antigen specificity especially in humans is largely unknown. Here we used a sensitive enrichment technology for antigen-reactive T cells to directly compare the conventional vs. regulatory CD4(+) T-cell response directed against two ubiquitous mucosal fungi, Aspergillus fumigatus and Candida albicans. In healthy humans, fungus-specific CD4(+)CD25(+)CD127(-)Foxp3(+) Treg are strongly expanded in peripheral blood and possess phenotypic, epigenetic and functional features of thymus-derived Treg. Intriguingly, for A. fumigatus, the strong Treg response contrasts with minimal conventional T-cell memory, indicating selective Treg expansion as an effective mechanism to prevent inappropriate immune activation in healthy individuals. By contrast, in subjects with A. fumigatus allergies, specific Th2 cells were strongly expanded despite the presence of specific Treg. Taken together, we demonstrate a largely expanded Treg population specific for mucosal fungi as part of the physiological human T-cell repertoire and identify a unique capacity of A. fumigatus to selectively generate Treg responses as a potentially important mechanism for the prevention of allergic reactions.