α<sub>2A</sub>- and α<sub>2C</sub>-Adrenergic Receptors Form Homo- and Heterodimers:  The Heterodimeric State Impairs Agonist-Promoted GRK Phosphorylation and β-Arrestin Recruitment

Small, Kersten M.; Schwarb, Mary Rose; Glinka, Clare B.; Theiss, Cheryl T.; Brown, Kari; Seman, Carrie A.; Liggett, Stephen B.

doi:10.1021/bi052074z

Cited by 62 publications

(50 citation statements)

References 28 publications

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“…A band of ϳ67 kDa was detected from the lysates of the EC by Western blot (Fig. 2G), consistent with the reported molecular mass of ␣ 2A ARs (43). The specificity of ␣ 2A AR antibody was further confirmed by the result showing that the immunofluorescence of ␣ 2A ARs was detected only in HEK293 cells co-expressing ␣ 2A ARs and GFP but not in HEK293 cells expressing GFP alone (Fig.…”

Section: Ne Inhibits the Neuronal Excitability In The Superficial Laysupporting

confidence: 86%

Noradrenergic Depression of Neuronal Excitability in the Entorhinal Cortex via Activation of TREK-2 K+ Channels

Xiao

Deng

Rojanathammanee

et al. 2009

Journal of Biological Chemistry

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The entorhinal cortex is closely associated with the consolidation and recall of memories, Alzheimer disease, schizophrenia, and temporal lobe epilepsy. Norepinephrine is a neurotransmitter that plays a significant role in these physiological functions and neurological diseases. Whereas the entorhinal cortex receives profuse noradrenergic innervations from the locus coeruleus of the pons and expresses high densities of adrenergic receptors, the function of norepinephrine in the entorhinal cortex is still elusive. Accordingly, we examined the effects of norepinephrine on neuronal excitability in the entorhinal cortex and explored the underlying cellular and molecular mechanisms. Application of norepinephrine-generated hyperpolarization and decreased the excitability of the neurons in the superficial layers with no effects on neuronal excitability in the deep layers of the entorhinal cortex. Norepinephrine-induced hyperpolarization was mediated by ␣ 2A adrenergic receptors and required the functions of G␣ i proteins, adenylyl cyclase, and protein kinase A. Norepinephrine-mediated depression on neuronal excitability was mediated by activation of TREK-2, a type of two-pore domain K ؉ channel, and mutation of the protein kinase A phosphorylation site on TREK-2 channels annulled the effects of norepinephrine. Our results indicate a novel action mode in which norepinephrine depresses neuronal excitability in the entorhinal cortex by disinhibiting protein kinase A-mediated tonic inhibition of TREK-2 channels. The entorhinal cortex (EC)2 is an essential structure in the limbic system that is closely related to emotional control (1), consolidation and recall of memories (2, 3), Alzheimer disease (4, 5), schizophrenia (6, 7), and temporal lobe epilepsy (8, 9). The physiological and pathological roles of the EC are likely to be determined by its unique position in the brain; the EC serves as the interface to control the flow of information into and out of the hippocampus. Afferents from the olfactory structures, parasubiculum, perirhinal cortex, claustrum, amygdala, and neurons in the deep layers of the EC (layers V-VI) (10, 11) converge onto the superficial layers (layer II/III) of the EC, whereas the axons of principal neurons in layer II form the major component of perforant path that innervates the dentate gyrus and CA3 (12), and those of the pyramidal neurons in layer III form the temporoammonic pathway and synapse onto the distal dendrites of pyramidal neurons in the CA1 and subiculum (12)(13)(14). The output from the hippocampus is then projected to the deep layers of the EC that relay information back to the superficial layers (15-18) and to other cortical areas (10).The EC receives abundant noradrenergic projections from the locus coeruleus in the brain stem (19 -21) and expresses ␣ 1 (22), ␣ 2 (23-25), and ␤ (26) adrenergic receptors (ARs), although the identities of cells expressing these ARs in the EC remain to be determined. Accordingly, application of norepinephrine (NE) in the EC has been shown to inhibit glu...

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Section: Ne Inhibits the Neuronal Excitability In The Superficial Laysupporting

confidence: 86%

Noradrenergic Depression of Neuronal Excitability in the Entorhinal Cortex via Activation of TREK-2 K+ Channels

Xiao

Deng

Rojanathammanee

et al. 2009

Journal of Biological Chemistry

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“…The recent observation that P2Y12 receptors form homo-oligomers in platelets (19) suggested to us that P2Y12 may help to recruit arrestin-2 to PAR4 by forming a heterodimer or oligomer that facilitates arrestin binding. The idea that GPCR dimers may be required for arrestin-dependent signaling has precedent in both the muscarinic and ␣-adrenergic receptor systems (33,34). In fact, we have detected the agonist-dependent association of PAR4 with P2Y12 in human platelets using an immunoprecipitation approach (Fig.…”

Section: Figure 8 Arterial Thrombus Formation In Wt and Arrestin-2mentioning

confidence: 78%

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

D'Angelo

Chavez

et al. 2011

Journal of Biological Chemistry

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Arrestins can facilitate desensitization or signaling by G protein-coupled receptors (GPCR) in many cells, but their roles in platelets remain uncharacterized. Because of recent reports that arrestins can serve as scaffolds to recruit phosphatidylinositol-3 kinases (PI3K)s to GPCRs, we sought to determine whether arrestins regulate PI3K-dependent Akt signaling in platelets, with consequences for thrombosis. Co-immunoprecipitation experiments demonstrate that arrestin-2 associates with p85 PI3K␣/␤ subunits in thrombin-stimulated platelets, but not resting cells. The association is inhibited by inhibitors of P2Y12 and Src family kinases (SFKs). The function of arrestin-2 in platelets is agonist-specific, as PAR4-dependent Akt phosphorylation and fibrinogen binding were reduced in arrestin-2 knock-out platelets compared with WT controls, but ADP-stimulated signaling to Akt and fibrinogen binding were unaffected. ADP receptors regulate arrestin recruitment to PAR4, because co-immunoprecipitates of arrestin-2 with PAR4 are disrupted by inhibitors of P2Y1 or P2Y12. P2Y1 may regulate arrestin-2 recruitment to PAR4 through protein kinase C (PKC) activation, whereas P2Y12 directly interacts with PAR4 and therefore, may help to recruit arrestin-2 to PAR4. Finally, arrestin2؊/؊ mice are less sensitive to ferric chloride-induced thrombosis than WT mice, suggesting that arrestin-2 can regulate thrombus formation in vivo. In conclusion, arrestin-2 regulates PAR4-dependent signaling pathways, but not responses to ADP alone, and contributes to thrombus formation in vivo.Arrestins are cytoplasmic proteins that were originally characterized by their ability to associate with agonist-activated G protein-coupled receptors (GPCRs), 2 mediating their internalization and desensitization (1). More recent studies suggest that arrestins play additional roles in GPCR signaling, by serving as scaffolds to recruit signaling complexes to the receptor, thereby facilitating activation of G protein-dependent and -independent pathways (2, 3). One such arrestinmediated pathway is the PI3K-dependent activation of the Ser-Thr kinase, Akt (4, 5). In fibroblasts, colorectal, and gastric carcinoma cells, arrestins have been found to play a critical role in localizing PI3K to GPCR complexes through an interaction with Src family kinases (SFKs) (6 -8). Perhaps most relevant for platelet agonists, thrombin-stimulated Akt phosphorylation involved activation of both G i and G q : G i -dependent signaling to Akt required ras activation, while G q -dependent Akt activation required arrestin-2 (9).Previous work from our laboratory and others has demonstrated that Akt-dependent pathways contribute to platelet activation by G protein-coupled receptors (10, 11). Yet, the mechanisms leading to Akt activation in platelets remain incompletely defined. Multiple laboratories have demonstrated that thrombin-dependent Akt phosphorylation in platelets is reduced by about 90% in the presence of inhibitors for the G i -coupled ADP receptor, P2Y12, and is blocked by inhibi...

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“…Equal amounts of protein (typically 15 μg) were loaded in all lanes. Western blots were performed using standard enhanced chemiluminescence techniques as previously described in detail (8,27), with signals from the membranes acquired with a Fuji LAS-3000 charged-coupled acquisition system. Quantification of the immunoreactive bands was performed with software from the manufacturer (ImageGauge version 4.22; Fuji).…”

Section: Methodsmentioning

confidence: 99%

Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation

McGraw

Elwing²,

Fogel³

et al. 2007

J. Clin. Invest.

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Receptor-mediated airway smooth muscle (ASM) contraction via G αq , and relaxation via G αs , underlie the bronchospastic features of asthma and its treatment. Asthma models show increased ASM G αi expression, considered the basis for the proasthmatic phenotypes of enhanced bronchial hyperreactivity to contraction mediated by M 3 -muscarinic receptors and diminished relaxation mediated by β 2 -adrenergic receptors (β 2 ARs). A causal effect between G i expression and phenotype has not been established, nor have mechanisms whereby G i modulates G q /G s signaling. To delineate isolated effects of altered G i , transgenic mice were generated overexpressing G αi2 or a G αi2 peptide inhibitor in ASM. Unexpectedly, G αi2 overexpression decreased contractility to methacholine, while G αi2 inhibition enhanced contraction. These opposite phenotypes resulted from different crosstalk loci within the G q signaling network: decreased phospholipase C and increased PKCα, respectively. G αi2 overexpression decreased β 2 AR-mediated airway relaxation, while G αi2 inhibition increased this response, consistent with physiologically relevant coupling of this receptor to both G s and G i . IL-13 transgenic mice (a model of asthma), which developed increased ASM G αi , displayed marked increases in airway hyperresponsiveness when G αi function was inhibited. Increased G αi in asthma is therefore a double-edged sword: a compensatory event mitigating against bronchial hyperreactivity, but a mechanism that evokes β-agonist resistance. By selective intervention within these multipronged signaling modules, advantageous G s /G q activities could provide new asthma therapies. IntroductionAirway smooth muscle (ASM) contraction and relaxation are primarily regulated by G protein-coupled receptors, the former mediated by receptors signaling to G q and the latter by those that couple to G s (1, 2). Many inflammatory cascades in asthma evoke bronchoconstriction by promoting local increases of G q receptor agonists such as acetylcholine, cysteinyl leukotrienes, prostaglandins, and histamine, which activate their cognate receptors expressed on ASM. There appear to be fewer G s -coupled receptors that act via endogenous agonists to counteract bronchoconstriction, but the β 2 -adrenergic receptor (β 2 AR) of ASM is the target of pharmacologically administered β-agonists and is typically highly effective in relaxing constricted airways. The molecular events and critical transduction elements for these 2 classes of receptors are well recognized. Agonist binding to receptors such as the M 3 -muscarinic receptor promote disassociation of heterotrimeric G q into G α and G βγ subunits, with the α subunit activating phospholipase C (PLC; which promotes inositol-3 phosphate and diacylglycerol production) and the latter activating PKC. Receptors such as the β 2 AR act via G αs to stimulate the effector adenylyl cyclase, resulting in cAMP production and activation of PKA. Substantial interest has

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α_2A- and α_2C-Adrenergic Receptors Form Homo- and Heterodimers: The Heterodimeric State Impairs Agonist-Promoted GRK Phosphorylation and β-Arrestin Recruitment

Cited by 62 publications

References 28 publications

Noradrenergic Depression of Neuronal Excitability in the Entorhinal Cortex via Activation of TREK-2 K+ Channels

Noradrenergic Depression of Neuronal Excitability in the Entorhinal Cortex via Activation of TREK-2 K+ Channels

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation

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α2A- and α2C-Adrenergic Receptors Form Homo- and Heterodimers: The Heterodimeric State Impairs Agonist-Promoted GRK Phosphorylation and β-Arrestin Recruitment

Cited by 62 publications

References 28 publications

Noradrenergic Depression of Neuronal Excitability in the Entorhinal Cortex via Activation of TREK-2 K+ Channels

Noradrenergic Depression of Neuronal Excitability in the Entorhinal Cortex via Activation of TREK-2 K+ Channels

Arrestin-2 Differentially Regulates PAR4 and ADP Receptor Signaling in Platelets

Crosstalk between Gi and Gq/Gs pathways in airway smooth muscle regulates bronchial contractility and relaxation

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Product

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α_2A- and α_2C-Adrenergic Receptors Form Homo- and Heterodimers: The Heterodimeric State Impairs Agonist-Promoted GRK Phosphorylation and β-Arrestin Recruitment