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Abstract: The internalization of β2-adrenoceptors after agonist activation results in a desensitized and phosphorylated receptor that either resensitizes by recycling to the cell surface or becomes degraded by postendocytic sorting to lysosomes. The duration and physiological effects of agonists therefore depend on β2-adrenoceptor sorting, highlighting the importance of sorting signals. Dileucine motifs within other membrane proteins act as signals for endocytosis and/or postendocytic sorting, and the β2-adrenoceptor has a dileucine motif within helix 8 that might play a role in efficient receptor recycling and/or downregulation. β2-adrenoceptor internalization and sorting were studied in HEK293 cells stably expressing wild type or mutant dialanine L339A,L340A β2-adrenoceptors. The mutant β2-adrenoceptors showed a significantly lower initial rate of phosphorylation at the prominent G-protein coupled receptor kinase (GRK) sites Ser355 and 356 compared to wild type β2-adrenoceptors. Furthermore, the agonist-induced endocytic rate constant for L339A,L340A β2-adrenoceptors was reduced to ~25% that of wild type β2-adrenoceptors, which resulted in a similar reduction in agonist-induced downregulation. Internalized L339A,L340A β2-adrenoceptors recycled to the surface with a rate and extent similar to that of wild type β2-adrenoceptors. Therefore, although the role of L339,L340 in β2-adrenoceptor recycling or postendocytic sorting seems minimal, we conclude that L339,L340 is required for the initial high rate of phosphorylation by G-protein coupled receptor kinases at Ser355,356, which in turn is required for efficient β2-adrenoceptors endocytosis. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The pericentriolar recycling endosome (RE) may be an alternative compartment through which some β2adrenergic receptors (β2ARs) recycle from early endosomes to the cell surface during prolonged exposure to agonist. For the transferrin receptor, CXCR2, and the M4muscarinic 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. [ABSTRACT FROM AUTHOR]

1. We have investigated the role of G protein-coupled receptor kinase 5 (GRK5) in the regulation of endosome sorting of human beta(2)-adrenoceptors. 2. Expressing GRK5 at a high level significantly increased the extent of internalization of wild-type beta(2)-adrenoceptors and of an internalization-defective mutant receptor, and increased receptor phosphorylation at serines 355 and 356 in the cytoplasmic tail. 3. Overexpressing GRK5 did not alter beta(2)-adrenoceptor recycling as assessed by immunofluorescence microscopy and radioligand binding assays nor was there any change in receptor downregulation. 4. These data indicate that GRK5 does not regulate the sorting of beta(2)-adrenoceptors in the endocytic pathway. [ABSTRACT FROM AUTHOR]

1: We have investigated the role of G protein-coupled receptor kinase 5 (GRK5) in the regulation of endosome sorting of human ß2-adrenoceptors. 2: Expressing GRK5 at a high level significantly increased the extent of internalization of wild-type ß2-adrenoceptors and of an internalization-defective mutant receptor, and increased receptor phosphorylation at serines 355 and 356 in the cytoplasmic tail. 3: Overexpressing GRK5 did not alter ß2-adrenoceptor recycling as assessed by immunofluorescence microscopy and radioligand binding assays nor was there any change in receptor downregulation. 4: These data indicate that GRK5 does not regulate the sorting of ß2-adrenoceptors in the endocytic pathway.British Journal of Pharmacology (2004) 141, 277-284. doi:10.1038/sj.bjp.0705504 [ABSTRACT FROM AUTHOR]

Objective: The aim was to determine whether, in a canine model, changes in surface expression of the neutrophil adhesion molecules CD11b/CD18 and L-selectin during and after open heart surgery with cardiopulmonary bypass can be used to identify subjects at risk for postoperative pulmonary dysfunction. Methods: Adult mixed breed dogs underwent cardiopulmonary bypass and were compared to “sham bypass” controls. Flow cytometry was performed on blood from the two groups of dogs and changes in CD11b/CD18 adhesion molecules and L-selectin were investigated. Results: Flow cytometry on blood from bypass dogs showed increased CD18 expression during and after cardiopulmonary bypass and a reciprocal decrease in L-selectin expression. Sham animals showed no significant change. In the bypass animals, changes in adhesion molecule expression were not evenly distributed across the population of circulating neutrophils; however, they were indicative of a percentage of activated cells. There was a significant negative linear relationship between the percentage of activated cells and arterial oxygenation 3 h after bypass (r = −0.80, P < 0.001). From this analysis, 11 animals were identified as “high” responders and seven as “low” responders, with different patterns of cellular activation and oxygenation during and after bypass. High responders had an average of 40(SEM 5)% activated cells during bypass with a persistently raised percentage of activated cells [38(3)%] 3 h later, whereas low responders had only 22(6)% activated cells during bypass and 11(2)% activated cells 3 h after bypass. High responder animals had a marked and continued deterioration in Po2 after bypass [to 25(6)% of baseline 3 h after bypass] whereas low responder animals showed recovery of oxygenation after the first hour postbypass and improved to 80(8)% of baseline at 3 h. Conclusions: Changes in adhesion molecule expression serve as a marker of neutrophil activation during cardiopulmonary bypass. The percentage of activated neutrophils in the circulation within 3 h after cardiopulmonary bypass may be predictive of an ongoing inflammatory process that is linked to pulmonary dysfunction. [ABSTRACT FROM PUBLISHER]

Abstract: Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated β2-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate β2-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the β-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of β2-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of β2-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of β2-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes. [Copyright &y& Elsevier]

For the beta(2)-adrenergic receptor (beta(2)AR), 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 beta(2)AR 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, beta(2)AR internalization into transferrin-positive vesicles was not altered but both agents significantly decreased the rate at which beta(2)ARs recycled to the cell surface. In LB-treated cells, nonrecycled beta(2)ARs 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 beta(2)ARs to the RE, while the expression of various CART fragments or the depletion of actinin-4 had no detectable effect on beta(2)AR sorting. These results indicate that the actin cytoskeleton is required for the efficient recycling of beta(2)ARs, a process that likely is dependent on myosin Vb.

Phosphatidylinositol 3-kinase inhibitors have been shown to affect endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated beta(2)-adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate beta(2)-adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the beta-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of beta(2)-adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of beta(2)-adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of beta(2)-adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.

Salmeterol is a long-acting beta(2)-adrenergic receptor (beta(2)AR) agonist commonly used in the treatment of asthma and chronic obstructive pulmonary disease. It differs from other beta-agonists in that it has a very low intrinisic efficacy, especially when compared with the other available long-acting beta-agonist, formoterol. Receptor desensitization and down-regulation has been described with the chronic use of beta-agonists. This effect may not be the same with all beta-agonists and may be related to their stabilization of altered receptor states. The extreme hydrophobicity and high-affinity quasi-irreversible binding of salmeterol have rendered studies examining the mechanisms by which it mediates receptor desensitization, down-regulation, and internalization difficult. We determined the capacity of salmeterol to induce beta(2)AR endocytosis, G protein-coupled receptor kinase (GRK)-site phosphorylation, degradation, and beta-arrestin2 translocation in HEK293 cells as compared with other agonists of varying intrinsic efficacies. Despite stimulating GRK-mediated phosphorylation of Ser355,356 after 30 min and 18 h to an extent similar to that observed with agonists of high intrinsic efficacy, such as epinephrine and formoterol, salmeterol did not induce significant beta(2)AR internalization or degradation and was incapable of stimulating the translocation of enhanced green fluorescent protein-beta-arrestin2 chimera (EGFP-beta-arrestin2) to the cell surface. Salmeterol-induced receptor endocytosis was rescued, at least in part, by the overexpression of EGFP-beta-arrestin2. Our data indicate that salmeterol binding induces an active receptor state that is unable to recruit beta-arrestin or undergo significant endocytosis or degradation despite stimulating considerable GRK-site phosphorylation. Defects in these components of salmeterol-induced receptor desensitization may be important determinants of its sustained bronchodilation with chronic use.

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 beta(2)-adrenergic receptors (beta(2)ARs). 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 beta-arrestin-dependent beta(2)AR 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 approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum 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)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-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 beta-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 beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.

The pericentriolar recycling endosome (RE) may be an alternative compartment through which some beta2-adrenergic receptors (beta2ARs) 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 beta2AR is not understood. We therefore monitored trafficking of beta2ARs 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, beta2ARs localized to a rab11-positive compartment and the rate of beta2AR 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 beta2AR 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.