Characterization of G Protein-Coupled Receptor Regulation in Antisense mRNA-Expressing Cells with Reduced Arrestin Levels

Mundell, Stuart J.; Loudon, Robert P.; Benovic, Jeffrey L.

doi:10.1021/bi990361v

Cited by 59 publications

(57 citation statements)

References 36 publications

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“…Previous reports suggest that the EP4-R, but not the EP2-R, can display rapid agonist-induced desensitization due to multiple phosphorylation sites within its C-terminal sequence (42,43). Furthermore, it is suggested that this effect is attenuated in ␤-arrestin antisense-expressing cells (44). It is conceivable that the desensitization of EP2/4-Rs seen here is also due to PKA-mediated receptor phosphorylation.…”

Section: Discussionmentioning

confidence: 53%

Dynamic Regulation, Desensitization, and Cross-talk in Discrete Subcellular Microdomains during β2-Adrenoceptor and Prostanoid Receptor cAMP Signaling

Willoughby

Baillie

Lynch

et al. 2007

Journal of Biological Chemistry

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Dynamic and localized actions of cAMP are central to the generation of discrete cellular events in response to a range of G s -coupled receptor agonists. In the present study we have employed a cyclic nucleotide-gated channel sensor to report acute changes in cAMP in the restricted cellular microdomains adjacent to two different G s -coupled receptor pathways, ␤ 2 -adrenoceptors and prostanoid receptors that are expressed endogenously in HEK293 cells. We probed by either selective small interference RNA-mediated knockdown or dominant negative overexpression the contribution of key signaling components in the rapid attenuation of the local cAMP signaling and subsequent desensitization of each of these G-protein-coupled receptor signaling pathways immediately following receptor activation. Direct measurements of cAMP changes just beneath the plasma membrane of single HEK293 cells reveal novel insights into key regulatory roles provided by protein kinase A-RII, ␤-arrestin2, cAMP phosphodiesterase-4D3, and cAMP phosphodiesterase-4D5. We provide new evidence for distinct modes of cAMP down-regulation in these two G s -linked pathways and show that these distinct G-protein-coupled receptor signaling systems are subject to unidirectional, heterologous desensitization that allows for limited cross-talk between distinct, dynamically regulated pools of cAMP.The ␤ 2 -adrenergic receptor (␤ 2 AR) 2 and prostanoid receptors (EP2R and EP4R) are G s -linked receptors that, upon agonist occupancy, activate adenylyl cyclases (ACs) to produce a diverse number of cAMP-dependent biological actions. Many tissues types, including airway epithelium and cardiac muscle, endogenously express both classes of G-protein-coupled receptor to generate cellular increases in cAMP following appropriate stimuli (1, 2). Differential agonist actions are thus conjectured to be permitted via compartmentalization of cAMP signals to regions close to individual G-protein-coupled receptor subtypes (3, 4). These local cAMP signals can then mediate specific downstream events through the regulation of neighboring effector molecules such as protein kinase A (PKA), exchange protein directly activated by cAMP, and cyclic nucleotide-gated channels (CNGCs). Temporally distinct, localized cAMP signals can be produced by two main factors: firstly, rapid cAMP hydrolysis by spatially organized cAMP phosphodiesterases (PDEs) (3-9) and, secondly, rapid receptor uncoupling or "desensitization" causing reduced G s -coupled receptor responsiveness to subsequent or prolonged agonist application (10, 11).The study of separate pools of cAMP deriving from different receptors in the same cell is most directly examined at the single cell level. Recent real-time, single cell approaches have provided some insight into the composition of signaling complexes whose tethered PDEs serve to shape a localized cAMP signal (9, 12, 13). However, the characteristics of the other key regulators comprising rapid attenuation of cAMP signal and receptor desensitization have not been addressed a...

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Section: Discussionmentioning

confidence: 53%

Dynamic Regulation, Desensitization, and Cross-talk in Discrete Subcellular Microdomains during β2-Adrenoceptor and Prostanoid Receptor cAMP Signaling

Willoughby

Baillie

Lynch

et al. 2007

Journal of Biological Chemistry

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“…Adenovirus Construction-Recombinant adenoviruses that encode ␤-galactosidase or ␤-arrestin2 or ␤-arrestin2 antisense were prepared as described previously (30,31). Briefly, ␤-galactosidase, ␤-arrestin2, or ␤-arrestin2 antisense was cloned into shuttle vector pAdTrack-CMV by using standard cloning protocols, and the shuttle vectors were recombined with pAdEasy-1, which harbors a CMV-driven green fluorescent protein, to form the viral constructs.…”

Section: Methodsmentioning

confidence: 99%

β-Arrestin2 Is Critically Involved in CXCR4-mediated Chemotaxis, and This Is Mediated by Its Enhancement of p38 MAPK Activation

Sun¹,

Cheng²,

Ma³

et al. 2002

Journal of Biological Chemistry

339

295

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Chemotaxis mediated by chemokine receptors such as CXCR4 plays a key role in lymphocyte homing and hematopoiesis as well as in breast cancer metastasis. We have demonstrated previously that ␤-arrestin2 functions to attenuate CXCR4-mediated G protein activation and to enhance CXCR4 internalization. Here we show further that the expression of ␤-arrestin2 in both HeLa and human embryonic kidney 293 cells significantly enhances the chemotactic efficacy of stromal cell-derived factor 1␣, the specific agonist of CXCR4, whereas the suppression of ␤-arrestin2 endogenous expression by antisense or RNA-mediated interference technology considerably attenuates stromal cell-derived factor 1␣-induced cell migration. Expression of ␤-arrestin2 also augmented chemokine receptor CCR5-mediated but not epidermal growth factor receptor-mediated chemotaxis, indicating the specific effect of ␤-arrestin2. Further analysis reveals that expression of ␤-arrestin2 strengthened CXCR4-mediated activation of both p38 MAPK and ERK, and the suppression of ␤-arrestin2 expression blocked the activation of two kinases. Interestingly, inhibition of p38 MAPK activation (but not ERK activation) by its inhibitors or by expression of a dominantnegative mutant of p38 MAPK effectively blocked the chemotactic effect of ␤-arrestin2. Expression of a dominant-negative mutant of ASK1 also exerted the similar blocking effect. The results of our study suggest that ␤-arrestin2 can function not only as a regulator of CXCR4 signaling but also as a mediator of stromal cellderived factor 1␣-induced chemotaxis and that this activity probably occurs via the ASK1/p38 MAPK pathway.

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“…Arrestin2 and GRK2 were detected using the equivalent of 8.9 μg tissue per lane, arrestin3, GRK3, and GRK5 -35.7, and GRK6 -120 μg tissue per lane. Arrestins were detected with arrestin2- [75] (1:6,000) or arrestin3-specific [79] (1:700) affinity-purified rabbit polyclonal antibodies. We used rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) to quantify GRK2 (catalog # sc-562; 1:500), GRK3 (catalog # sc-563; 1:300), and GRK5 (catalog # sc-565; 1:500).…”

Section: Western Blottingmentioning

confidence: 99%

“…If the compensation for DA depletion was aimed at restoring a normal level of G protein-mediated signaling via DA receptors, a down-regulation of arrestins and GRKs in the PD brain should be expected. It is well established that reduction of the arrestin and/or GRK concentration invariably reduces GPCR desensitization and internalization and enhances G protein-mediated signaling [3,14,27,41,58,75,103]. Therefore, decreased concentrations of arrestin/GRKs in the DA-depleted basal ganglia would impede desensitization of DA receptors and help sustain the G protein-mediated signaling.…”

Section: Functional Significance Of Changes In Arrestin and Grk Exprementioning

confidence: 99%

Arrestins and two receptor kinases are upregulated in Parkinson's disease with dementia

Bychkov

Joyce

et al. 2008

Neurobiology of Aging

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Arrestins and G proteins-coupled receptor kinases (GRKs) regulate signaling and trafficking of G protein-coupled receptors. We investigated changes in the expression of arrestins and GRKs in the striatum of patients with Parkinson's disease without (PD) or with dementia (PDD) at post mortem using Western blotting and ribonuclease protection assay. Both PD and PDD groups had similar degree of dopamine depletion in all striatal regions. Arrestin proteins and mRNAs were increased in the PDD group throughout striatum. Protein and mRNA of GRK5, the major subtype in the human striatum, and GRK3 were also upregulated, whereas GRK2 and 6 were mostly unchanged. The PD group had lower concentration of arrestins and GRKs than the PDD group. There was no statistical link between the load of Alzheimer's pathology and the expression of these signaling proteins. Upregulation of arrestins and GRK in PDD may confer resistance to the therapeutic effects of levodopa often observed in these patients. In addition, increased arrestin and GRK concentrations may lead to dementia via perturbation of multiple signaling mechanisms.

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Characterization of G Protein-Coupled Receptor Regulation in Antisense mRNA-Expressing Cells with Reduced Arrestin Levels

Cited by 59 publications

References 36 publications

Dynamic Regulation, Desensitization, and Cross-talk in Discrete Subcellular Microdomains during β2-Adrenoceptor and Prostanoid Receptor cAMP Signaling

Dynamic Regulation, Desensitization, and Cross-talk in Discrete Subcellular Microdomains during β2-Adrenoceptor and Prostanoid Receptor cAMP Signaling

β-Arrestin2 Is Critically Involved in CXCR4-mediated Chemotaxis, and This Is Mediated by Its Enhancement of p38 MAPK Activation

Arrestins and two receptor kinases are upregulated in Parkinson's disease with dementia

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