Robert H. Moore

Salmeterol-induced desensitization, internalization and phosphorylation of the human β2-adrenoceptor

Partial agonists of the β2‐adrenoceptor which activate adenylyl cyclase are widely used as bronchodilators for the relief of bronchoconstriction accompanying many disease conditions, including bronchial asthma. The bronchodilator salmeterol has both a prolonged duration of action in bronchial tissue and the ability to reassert this activity following the temporary blockade of human β2‐adrenoceptors with antagonist. We have compared the activation and desensitization of human β2‐adrenoceptor stimulation of adenylyl cyclase induced by salmeterol, adrenaline and salbutamol in a human lung epithelial line, BEAS‐2B, expressing β2‐adrenoceptor levels of 40–70 fmol mg−1, and in human embryonic kidney (HEK) 293 cell lines expressing 2–10 pmol mg−1. The efficacy observed for the stimulation of adenylyl cyclase by salmeterol was only ≈percnt;10% of that observed for adrenaline in BEAS‐2B cells expressing low levels of β2‐adrenoceptor, but similar to adrenaline in HEK 293 cells expressing very high levels of receptors. Salmeterol pretreatment of these cells induced a rapid and stable activation of adenylyl cyclase activity which resisted extensive washing and β2‐adrenoceptor antagonist blockade, consistent with binding to a receptor exosite and/or to partitioning into membrane lipid. The desensitization and internalization of β2‐adrenoceptors induced by the partial agonists salmeterol and salbutamol were considerably reduced relative to the action of adrenaline. Consistent with these observations, the initial rate of phosphorylation of the receptor induced by salmeterol and salbutamol was much reduced in comparison to adrenaline. Our data suggest that the reduction in the rapid phase of desensitization of β2‐adrenoceptors after treatment with salmeterol or salbutamol is caused by a decrease in the rate of β2‐adrenoceptor kinase (βARK) phosphorylation and internalization. In contrast, the rate of cyclic AMP‐dependent protein kinase (PKA)‐mediated phosphorylation by these partial agonists appears to be similar to adrenaline.

Rab11 regulates the recycling and lysosome targeting of β2-adrenergic receptors

The pericentriolar recycling endosome (RE) may be an alternative compartment through which some β2-adrenergic receptors (β2ARs) recycle from early endosomes to the cell surface during prolonged exposure to agonist. For the transferrin receptor, CXCR2, and the M4-muscarinic acetylcholine receptor, trafficking through the RE and receptor recycling is regulated by the small GTPase rab11. The precise role of the RE and rab11 in regulating the cellular trafficking of the β2AR is not understood. We therefore monitored trafficking of β2ARs in HEK293 cells following the modulation of rab11 activity. Expression of a rab11 mutant deficient in GTP binding (as a fusion between enhanced green fluorescent protein (EGFP) and the rab11S25N mutant) significantly slowed receptor recycling to the cell surface from dispersed transferrin-positive peripheral vesicles following a brief exposure to agonist. The agonist was applied at a time when receptors have undergone only one or two rounds of endocytosis and recycling. In cells overexpressing wild-type rab11, β2ARs localized to a rab11-positive compartment and the rate of β2AR recycling to the cell surface was reduced, but only after prolonged exposure to agonist and multiple rounds of receptor endocytosis and recycling. This effect was associated with impaired β2AR trafficking to lysosomes and receptor proteolysis, whereas the sorting of low-density lipoprotein from transferrin-positive vesicles to late endosomes and lysosomes was not affected. These data highlight a pivotal role for rab11 in regulating the traffic of a G protein-coupled receptor at the level of the RE, where modulation of rab11 activity dictates the balance between receptor recycling and downregulation during prolonged exposure to agonist.

Role of the G Protein-coupled Receptor Kinase Site Serine Cluster in β2-Adrenergic Receptor Internalization, Desensitization, and β-Arrestin Translocation

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of β2-adrenergic receptors (β2ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in β-arrestin-dependent β2AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced ∼2-fold increases in the EC50 for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 μm isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-β-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-β-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent β-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced β-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, β-arrestin binding, and internalization of β2ARs.

Characterization of β2-adrenergic receptor dephosphorylation: comparison with the rate of resensitization

Dephosphorylation of the cyclic AMP-dependent protein kinase (PKA) site phosphoserine 262 and the G protein-coupled receptor kinase (GRK) site phosphoserines 355 and 356 of the β2-adrenergic receptor (β2AR) were characterized in both intact human embryonic kidney 293 cells and subcellular fractions and were correlated with the rate of resensitization of isoproterenol stimulation of adenylyl cyclase after treatment with isoproterenol and blockade by antagonist. Dephosphorylation of the PKA site after stimulation with 300 pM isoproterenol occurred with a t½ of 9 min (k = 0.08 ± 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 μM isoproterenol for 5 min exhibited a lag phase of ≈ 5 min, after which dephosphorylation proceeded slowly with a t½ of 18 min (k = 0.039 ± 0.006/min). Consistent with the slow rate of GRK site dephosphorylation, the phosphatase inhibitors calyculin A and okadaic acid failed to augment phosphorylation in intact cells during continuous agonist stimulation indicating that GRK site dephosphorylation was minimal. However, both inhibited dephosphorylation of the GRK sites after the addition of antagonist. Slow GRK site dephosphorylation after antagonist treatment was also demonstrated by the relative stability of internalized phosphorylated β2AR in cells as observed both by immunofluorescence microscopy using a phospho-site-specific antibody and by studies of the subcellular localization of the GRK-phosphorylated β2AR on sucrose gradients that revealed nearly equivalent levels of GRK site phosphorylation in the plasma membrane and vesicular fractions. In addition, dephosphorylation of the GRK sites by intrinsic phosphatase activity occurred only in the heavy vesicle fractions. In contrast to the slow rates of dephosphorylation, the rate of resensitization of isoproterenol stimulation of adenylyl cyclase was 5- and 10-fold faster (k = 0.43 ± 0.009/min; t½ = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.

Regulation of G-Protein-Coupled Receptor Activity by Rab GTPases

The regulation of G-protein-coupled receptor (GPCR) activity involves a diversity of vesicular transport processes. Receptor desensitization and resensitization are intimately connected with membrane trafficking events such as endocytosis, intracellular sorting, transport to lysosomes, and recycling to the plasma membrane. Ras-related GTPases of the rab family are known to regulate these processes, including a subset of rab proteins that are specific for endosomes and lysosomes. While much study has been given to endocytic rabs using standard models such as transferrin receptors, less is known about how rabs regulate signal-transducing receptors. This article reviews recent work concerning rab GTPases and their regulation of GPCR activity via membrane transport mechanisms.

Rapid Recycling of β2‐Adrenergic Receptors is Dependent on the Actin Cytoskeleton and Myosin Vb

For the β2‐adrenergic receptor (β2AR), published evidence suggests that an intact actin cytoskeleton is required for the endocytosis of receptors and their proper sorting to the rapid recycling pathway. We have characterized the role of the actin cytoskeleton in the regulation of β2AR trafficking in human embryonic kidney 293 (HEK293) cells using two distinct actin filament disrupting compounds, cytochalasin D and latrunculin B (LB). In cells pretreated with either drug, β2AR internalization into transferrin‐positive vesicles was not altered but both agents significantly decreased the rate at which β2ARs recycled to the cell surface. In LB‐treated cells, nonrecycled β2ARs were localized to early embryonic antigen 1‐positive endosomes and also accumulated in the recycling endosome (RE), but only a small fraction of receptors localized to LAMP‐positive late endosomes and lysosomes. Treatment with LB also markedly enhanced the inhibitory effect of rab11 overexpression on receptor recycling. Dissociating receptors from actin by expression of the myosin Vb tail fragment resulted in missorting of β2ARs to the RE, while the expression of various CART fragments or the depletion of actinin‐4 had no detectable effect on β2AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of β2ARs, a process that likely is dependent on myosin Vb.

Anti-Inflammatory Activities of α2-Agonists

Beta2-adrenergic agonists (beta2-agonists) play a pivotal role in the acute and chronic management of asthma. Their major action on the airways is the relaxation of smooth muscle cells. In addition to their bronchodilator properties, beta2-agonists may have other effects through their activation of beta2-receptors expressed on resident airway cells such as epithelial cells and mast cells and circulating inflammatory cells such as eosinophils and neutrophils. These non-bronchodilator activities of beta2-agonists may enhance their efficacy in the management of asthma. In pre-clinical studies, the anti-inflammatory effects of beta2-agonists are demonstrated through their stabilizing effect on mast cells and their inhibition of mediator release from eosinophils, macrophages T-lymphocytes, and neutrophils. In addition, beta2-agonists may inhibit plasma exudation in the airway, the release of neuropeptides from sensory nerves, and mediator release from epithelial cells. These in vitro observations are not as clearly demonstrated in clinical trials, which may be explained by the rapid desensitization of beta2-adrenergic receptors on airway inflammatory cells. The regular use of short-acting beta2-agonists alone has been shown to have deleterious effects on asthma control. Therefore, short-acting agents should only be used when needed for rescue of acute symptoms. Monotherapy with long-acting beta2-agonists has also been associated with poor asthma control. However, when given concomitantly with inhaled corticosteroids, beta2-agonists may potentiate the anti-inflammatory effect of corticosteroids, improve asthma control and prevent exacerbations.

Specific changes in β2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation

Agonist-activated beta2-adrenoceptors rapidly internalize and then recycle to the cell surface, however chronic agonist eventually causes receptor downregulation. To characterize beta2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation, human embryonic kidney cells expressing epitope-tagged receptors were examined by radioligand binding with (+/-)-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole- 2-on hydrochloride ([3H]CGP12177) and immunofluorescence microscopy. The first-order receptor recycling rate constant declined after 18 h of agonist compared with 15 min (0.05 min(-1) vs. 0.12 min(-1)), thus increasing the intracellular 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 receptors. Although the intracellular receptor fraction did not increase between 15 min and 18 h of agonist, some receptors moved irreversibly into a protease-containing compartment while retaining radioligand binding activity. Our results indicate that beta2-adrenoceptor downregulation is associated principally with an increased intracellular transit time during recycling. This could promote the diversion of receptors into protease-containing compartments, where there is an irreversible commitment to downregulation prior to loss of radioligand binding activity.

Intravenous injection of pharmaceutical tablets presenting as multiple pulmonary nodules and declining pulmonary function in an adolescent with cystic fibrosis.

Here we present the unusual case of an adolescent with cystic fibrosis presenting with declining pulmonary function and diffuse micronodular pulmonary disease. This case illustrates the radiographic and pathologic findings associated with the intravenous injection and pulmonary arterial embolization of insoluble pharmaceutical-tablet constituents. The number of first-time users reporting nonmedical use of prescription pain relievers is increasing dramatically, especially in adolescents. Recognition of both the diagnostic imaging features and histologic features on lung biopsy are critical steps for early diagnosis, intervention, and potential prevention of sudden death in these at-risk patients.

Endosome sorting of β2‐adrenoceptors is GRK5 independent

We have investigated the role of G protein‐coupled receptor kinase 5 (GRK5) in the regulation of endosome sorting of human β2‐adrenoceptors. Expressing GRK5 at a high level significantly increased the extent of internalization of wild‐type β2‐adrenoceptors and of an internalization‐defective mutant receptor, and increased receptor phosphorylation at serines 355 and 356 in the cytoplasmic tail. Overexpressing GRK5 did not alter β2‐adrenoceptor recycling as assessed by immunofluorescence microscopy and radioligand binding assays nor was there any change in receptor downregulation. These data indicate that GRK5 does not regulate the sorting of β2‐adrenoceptors in the endocytic pathway.