Phorbol‐ester‐induced phosphorylation of the β<sub>2</sub>‐adrenergic receptor decreases its coupling to G<sub>s</sub>

Bouvier, Michel; Guilbault, Nathalie; Bonin, Hélène

doi:10.1016/0014-5793(91)80159-z

Cited by 52 publications

(29 citation statements)

References 34 publications

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“…Previous studies have shown that PKA desensitization of the ␤ 2 AR was heterologous; that is, that any stimulation of cAMP and PKA could lead to a desensitization of epinephrine stimulation of adenylyl cyclase. We and others have also shown that activation of PKC with PMA led to a similar desensitization in a number of cell types (Clark et al, 1988Kunkel et al, 1989;Johnson et al, 1990;Bouvier et al, 1991;Hausdorff et al, 1991;Yuan et al, 1994). Both the PKA-and PKC-mediated desensitizations were shown to be ablated with an S261,262A substitution mutant of the ␤ 2 AR (Johnson et al, 1990;Yuan et al, 1994).…”

Section: Resultssupporting

confidence: 53%

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β₂-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

et al. 2004

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Agonist-stimulated desensitization of the ␤ 2 -adrenergic receptor (␤ 2 AR) is caused by both a potent cAMP-dependent protein kinase (PKA)-mediated phosphorylation and a less potent, occupancy-dependent, G protein-coupled receptor kinase (GRK)-mediated phosphorylation that leads to ␤-arrestin binding and internalization. In this study the kinetics of phosphorylation of the third intracellular loop PKA site Ser262 and the putative C-tail GRK sites Ser355, Ser356 of the human ␤ 2 AR overexpressed in human embryonic kidney (HEK) 293 cells were characterized using phosphoserine-specific antibodies. Specificity of the antibodies was shown by their lack of reactivity with mutant ␤ 2 ARs lacking the respective sites. In addition, overexpression of GRK2 and GRK5 increased basal levels of phosphorylation of the GRK sites Ser355, Ser356 in both COS-7 and HEK 293 cells. Epinephrine, prostaglandin E 1 , and forskolin at maximum concentrations stimulated phosphorylation of the ␤ 2 AR PKA site (Ser262) by 4-fold, whereas PMA stimulated it by 2-fold. Epinephrine stimulated PKA site phosphorylation with an EC 50 of 20 to 40 pM. In contrast, epinephrine stimulated GRK site phosphorylation (Ser355,Ser356) with an EC 50 of 200 nM (1-min treatments), which is more than 4000-fold higher relative to PKA site phosphorylation, consistent with an occupancy-driven process. After 10 to 30 min, the EC 50 for epinephrine stimulation of GRK site phosphorylation was reduced to 10 to 20 nM but was still Ϸ200-fold greater than for the PKA site. The EC 50 for internalization correlated with GRK site phosphorylation and showed a similar shift with time of epinephrine stimulation. The kinetics of epinephrine-stimulated GRK site phosphorylation were not altered in a mutant of the ␤ 2 AR lacking the PKA consensus sites. The initial levels (2 min) of a range of agoniststimulated GRK site phosphorylations were correlated with their efficacy for activation of adenylyl cyclase, namely epinephrine Ն formoterol ϭ fenoterol Ͼ terbutaline ϭ zinterol ϭ albuterol Ͼ salmeterol Ͼ Ͼ dobutamine Ն ephedrine. However, after 20 to 30 min of treatment, agonists with intermediate strengths, such as albuterol and salmeterol, stimulate GRK site phosphorylations that are approximately equal to that produced by epinephrine, and the correlation breaks down. The GRK and PKA site antibodies were also effective in detecting phosphorylation of the endogenous ␤ 2 AR expressed in A431 human epidermoid carcinoma cells. To summarize, our results show a remarkable amplification of PKA site phosphorylation relative to the putative GRK site phosphorylation, heterologous stimulation of the PKA site phosphorylation, no dependence of GRK site phosphorylation on PKA sites, and a reasonable correlation of initial levels of GRK site phosphorylation with the strength of a range of agonists.The ␤ 2 -adrenergic receptor (␤ 2 AR) plays significant roles in relaying signals from the autonomic sympathetic nervous system to the cardiovascular and pulmonary systems in particular. There are...

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Section: Resultssupporting

confidence: 53%

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β₂-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

et al. 2004

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“…A potential explanation for this apparent paradox could be that the increased basal cAMP production, resulting from the cholesterol depletion, leads to PKA-mediated receptor desensitization and attenuates ␤ 2 AR/G s coupling. Consistent with this notion, PKA-dependent phosphorylation of the ␤ 2 AR has been shown previously to promote receptor uncoupling (30,45,46).…”

Section: Membrane Cholesterol Regulatessupporting

confidence: 63%

“…HA-␤ 2 AR WT was described previously (29). cDNA of ␤ 2 AR PKAϪ , as described (30), was subcloned into pcDNA3.1 following HindIII/EcoRI digestion. ␤ 2 AR-Luc was described previously (31).…”

Section: Methodsmentioning

confidence: 99%

Cholesterol-dependent Separation of the β2-Adrenergic Receptor from Its Partners Determines Signaling Efficacy

Pontier¹,

Percherancier²,

Galandrin

et al. 2008

Journal of Biological Chemistry

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120

114

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Determining the role of lipid raft nanodomains in G proteincoupled receptor signaling remains fraught by the lack of assays directly monitoring rafts in native membranes. We thus combined extensive biochemical and pharmacological approaches to a nanoscale strategy based on bioluminescence resonance energy transfer (BRET) to assess the spatial and functional influence of cholesterol-rich liquid-ordered lipid nanodomains on ␤ 2 adrenergic receptor (␤ 2 AR) signaling. The data revealed that whereas ␤ 2 AR did not partition within liquid-ordered lipid phase, a pool of G protein and adenylyl cyclase (AC) were sequestered in these domains. Destabilization of the liquid-ordered phase by cholesterol depletion led to a lateral redistribution of G␣ s and AC that favored interactions between the receptor and its signaling partners as assessed by BRET. This resulted in an increased basal and agonist-promoted ␤ 2 AR-stimulated cAMP production that was partially dampened as a result of constitutive protein kinase A-dependent phosphorylation and desensitization of the receptor. This restraining influence of nanodomains on ␤ 2 AR signaling was further substantiated by showing that liquid-ordered lipid phase stabilization using caveolin overexpression or increasing membrane cholesterol amount led to an inhibition of ␤ 2 AR-associated signaling. Given the emerging concept that clustering of receptors and effectors into signaling platforms contributes to the efficacy and selectivity of signal transduction, our results support a model whereby cholesterol-promoted liquid-ordered lipid phase-embedding G s and AC allows their lateral separation from the receptor, thus restraining the basal activity and controlling responsiveness of ␤ 2 AR signaling machinery within larger signaling platforms.

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“…For the ␣ 1 AR, PMA pretreatment results in phosphorylation of the receptor and decreased coupling to phosphoinositide hydrolysis . ␤ 2 AR function has also been shown to be decreased by phorbol esters, an effect associated with receptor phosphorylation (Bouvier et al 1991). One of the most well characterized G protein-coupled receptor signaling pathways that is altered by PKC-mediated receptor phosphorylation is that of the 5HT 1A receptor (Raymond, 1991), where the sites of this phosphorylation recently have been mapped (Lembo and Albert, 1995).…”

Section: Resultsmentioning

confidence: 99%

Phosphorylation and Functional Desensitization of the α_2A-Adrenergic Receptor by Protein Kinase C

et al. 1998

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We have investigated the potential for protein kinase C (PKC) to phosphorylate and desensitize the ␣ 2A -adrenergic receptor (␣ 2A AR). In whole-cell phosphorylation studies, recombinantly expressed human ␣ 2A AR displayed an increase in phosphorylation after short-term exposure to 100 nM phorbol 12-myristate-13-acetate (PMA) that was blocked by preincubation with a PKC inhibitor. This increase in receptor phosphorylation over basal amounted to 172 Ϯ 40% in COS-7 cells and 201 Ϯ 40% in Chinese hamster ovary cells. In permanently transfected Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of ␣ 2A AR function, amounting to a 68 Ϯ 4% decrease in the maximal agonist (UK14304)-stimulated intracellular calcium release. Such desensitization was blocked by the PKC inhibitor bisindolylmaleimide I and was not evoked by an inactive phorbol ester. The desensitization of this agonist response was not caused by PKC-mediated augmentation of G protein-coupled receptor kinase activity, because PMA-promoted desensitization of a mutated ␣ 2A AR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type ␣ 2A AR. To test whether PKC phosphorylation is a mechanism by which ␣ 2A AR can be regulated by other receptors, the ␣ 1b AR was co-expressed with the ␣ 2A AR in Chinese hamster ovary cells. Upon selective activation of ␣ 1b AR, the function of ␣ 2A AR underwent a 53 Ϯ 5% desensitization. Thus, cellular events that result in PKC activation promote phosphorylation of the ␣ 2A AR and lead to substantial desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the ␣ 2A AR and other receptors can occur.The ␣ 2 ARs regulate several effector systems including adenylyl cyclase, potassium channels, calcium channels, and inositol phosphate-mediated intracellular calcium release (Limbird, 1988;Liggett, 1996;Akerman et al. 1997;. Studies in intact organisms and cell culture systems have indicated that the function of G protein-coupled receptors, including ␣ 2 ARs, can be dynamically regulated under various physiologic and pathophysiologic conditions (Liggett and Lefkowitz, 1993;Liggett, 1997). We have recently delineated one pathway whereby persistent agonist activation results in a dampening of ␣ 2 AR signaling, termed desensitization, which is due to receptor phosphorylation by GRKs (Eason and Liggett, 1992;Liggett et al., 1992;Eason et al., 1995). GRK-mediated desensitization is evoked by agonist occupancy of the receptor, is independent of the generation of second messengers, and represents one mechanism of homologous desensitization of the ␣ 2 AR. The molecular basis of heterologous regulation of ␣ 2 AR has been largely unexplored. In the current study, we investigated the regulation of ␣ 2 AR signaling by PKC. This kinase was found to phosphorylate the ␣ 2A subtype, which resulted in a rapid desensitization of receptor function. These effect...

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Phorbol‐ester‐induced phosphorylation of the β₂‐adrenergic receptor decreases its coupling to G_s

Cited by 52 publications

References 34 publications

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β₂-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β₂-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

Cholesterol-dependent Separation of the β2-Adrenergic Receptor from Its Partners Determines Signaling Efficacy

Phosphorylation and Functional Desensitization of the α_2A-Adrenergic Receptor by Protein Kinase C

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Phorbol‐ester‐induced phosphorylation of the β2‐adrenergic receptor decreases its coupling to Gs

Cited by 52 publications

References 34 publications

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β2-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β2-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

Cholesterol-dependent Separation of the β2-Adrenergic Receptor from Its Partners Determines Signaling Efficacy

Phosphorylation and Functional Desensitization of the α2A-Adrenergic Receptor by Protein Kinase C

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Phorbol‐ester‐induced phosphorylation of the β₂‐adrenergic receptor decreases its coupling to G_s

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β₂-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β₂-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

Phosphorylation and Functional Desensitization of the α_2A-Adrenergic Receptor by Protein Kinase C