Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Woo, Anthony Yiu-Ho; Jóźwiak, Krzysztof; Toll, Lawrence; Tanga, Mary J.; Kozocas, Joseph A.; Jimenez, Lucita; Huang, Ying; Song, Ying; Płazińska, Anita; Pająk, Karolina; Paul, Rajib; Bernier, Michel; Wainer, Irving W.; Xiao, Rui‐Ping

doi:10.1074/jbc.m114.558882

Cited by 38 publications

(38 citation statements)

References 47 publications

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“…In support, (1) PTX treatment increased responses evoked by both the G s -selective (R,R)- and the dually coupled (R,S)-fenoterol isoforms (Fig. 1C & 2A); this should not occur if the PTX effect was dependent upon receptor activity, since β 2 ARs stimulated by (R,R)-fenoterol have been shown to only activate G s [29], [39]. (2) PTX treatment amplified forskolin-evoked cAMP accumulation and increased the potency of forskolin to evoke an inotropic response (Fig.…”

Section: Discussionmentioning

confidence: 69%

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

et al. 2014

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Background and purposeDespite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approachWe used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key resultsPTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implicationsTogether, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

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Section: Discussionmentioning

confidence: 69%

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

et al. 2014

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“…We have tested this hypothesis using the bitopic (R,S’)-MNF, a diastereoisomer of (R,R’)-MNF that has different β-AR selectivity and signaling. 13,59,60 The administration of (R,S’)-MNF to mice bearing a PANC-1 xenograft produced a >70% inhibition in tumor growth (data not shown). The data from the (R,S’)-MNF study will be reported elsewhere.…”

Section: Discussionmentioning

confidence: 94%

GPR55 receptor antagonist decreases glycolytic activity in PANC‐1 pancreatic cancer cell line and tumor xenografts

Bernier

Catazaro

Singh

et al. 2017

Intl Journal of Cancer

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The Warburg effect is a predominant metabolic pathway in cancer cells characterized by enhanced glucose uptake and its conversion to L-lactate and is associated with upregulated expression of HIF-1α and activation of the EGFR-MEK-ERK, Wnt-β-catenin and PI3K-AKT signaling pathways. (R,R’)-4’-methoxy-1-naphthylfenoterol ((R,R’)-MNF) significantly reduces proliferation, survival, and motility of PANC-1 pancreatic cancer cells through inhibition of the GPR55 receptor. We examined (R,R’)-MNF’s effect on glycolysis in PANC-1 cells and tumors. Global NMR metabolomics was used to elucidate differences in the metabolome between untreated and (R,R’)-MNF-treated cells. LC/MS analysis was used to quantify intracellular concentrations of β-hydroxybutyrate, carnitine and L-lactate. Changes in target protein expression were determined by Western blot analysis. Data was also obtained from mouse PANC-1 tumor xenografts after administration of (R,R’)-MNF. Metabolomics data indicate that (R,R’)-MNF altered fatty acid metabolism, energy metabolism, and amino acid metabolism and increased intracellular concentrations of β-hydroxybutyrate and carnitine while reducing L-lactate content. The cellular content of phosphoinositide-dependent kinase-1 and hexokinase 2 was reduced consistent with diminished PI3K-AKT signaling and glucose metabolism. The presence of the GLUT8 transporter was established and found to be attenuated by (R,R’)-MNF. Mice treated with (R,R’)-MNF had significant accumulation of L-lactate in tumor tissue relative to vehicle-treated mice, together with reduced levels of the selective L-lactate transporter MCT4. Lower intratumoral levels of EGFR, pyruvate kinase M2, β-catenin, hexokinase 2 and P-glycoprotein were also observed. The data suggest that (R,R’)-MNF reduces glycolysis in PANC-1 cells and tumors through reduced expression and function at multiple controlling sites in the glycolytic pathway.

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“…Another potential explanation for the differential effect of ICI-118,551 is based upon the coupling of the β 2 -AR to both G s and G i proteins. ( R,R ′)-MNF, like most synthetic β 2 -AR agonists, couples to both G s and G i proteins [50]. ICI-118,551 has been shown to reduces G s -dependent actions with either no effect on Gi coupling [51] or with some activation of G i -dependent signaling [52].…”

Section: Discussionmentioning

confidence: 99%

Concurrent activation of β 2 -adrenergic receptor and blockage of GPR55 disrupts pro-oncogenic signaling in glioma cells

Wnorowski

Such

Paul³

et al. 2017

Cellular Signalling

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Activation of β2-adrenergic receptor (β2AR) and deorphanized GPR55 has been shown to modulate cancer growth in diverse tumor types in vitro and in xenograft models in vivo. (R,R′)-4′-methoxy-1-naphthylfenoterol [(R,R′)-MNF] is a bivalent compound that agonizes β2AR but inhibits GPR55-mediated pro-oncogenic responses. Here, we investigated the molecular mechanisms underlying the anti-tumorigenic effects of concurrent β2AR activation and GPR55 blockade in C6 glioma cells using (R,R′)-MNF as a marker ligand. Our data show that (R,R′)-MNF elicited G1-phase cell cycle arrest and apoptosis, reduced serum-inducible cell motility, promoted the phosphorylation of PKA target proteins, and inhibited constitutive activation of ERK and AKT in the low nanomolar range, whereas high nanomolar levels of (R,R′)-MNF were required to block GPR55-mediated cell motility. siRNA knockdown and pharmacological inhibition of β2AR activity were accompanied by significant upregulation of AKT and ERK phosphorylation, and selective alteration in (R,R′)-MNF responsiveness. The effects of agonist stimulation of GPR55 on various readouts, including cell motility assays, were suppressed by (R,R′)-MNF. Lastly, a significant increase in phosphorylation-mediated inactivation of β-catenin occurred with (R,R′)-MNF, and we provided new evidence of (R,R′)-MNF-mediated inhibition of oncogenic β-catenin signaling in a C6 xenograft tumor model. Thus, simultaneous activation of β2AR and blockade of GPR55 may represent a novel therapeutic approach to combat the progression of glioblastoma cancer.

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Tyrosine 308 Is Necessary for Ligand-directed Gs Protein-biased Signaling of β2-Adrenoceptor

Cited by 38 publications

References 47 publications

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

GPR55 receptor antagonist decreases glycolytic activity in PANC‐1 pancreatic cancer cell line and tumor xenografts

Concurrent activation of β 2 -adrenergic receptor and blockage of GPR55 disrupts pro-oncogenic signaling in glioma cells

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