Publications by authors named "Bronwyn A Evans"

33 Publications

The metabolic effects of mirabegron are mediated primarily by β -adrenoceptors.

Pharmacol Res Perspect 2020 10;8(5):e00643

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Vic., Australia.

The β -adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity-related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at β-adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron-mediated effects were examined on cyclic AMP, UCP1 mRNA, [ H]-2-deoxyglucose uptake, cellular glycolysis, and O consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1-mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at β -adrenoceptors as they were largely absent in adipocytes derived from β -adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the β -adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the β -adrenoceptor.
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http://dx.doi.org/10.1002/prp2.643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437350PMC
October 2020

Upregulation of β-adrenoceptors-a general marker of and protective mechanism against hypoxia?

Naunyn Schmiedebergs Arch Pharmacol 2020 02 18;393(2):141-146. Epub 2019 Dec 18.

Department. of Pharmacology, Johannes Gutenberg University, Mainz, Germany.

β-Adrenoceptors exhibit a restricted expression pattern, particularly in humans. However, they have been found to be upregulated in various cancers and under several conditions associated with hypoperfusion such as congestive heart failure and diabetes for instance in the heart and other tissues. These conditions are frequently associated with hypoxia. Furthermore, direct induction of hypoxia has consistently been reported to cause upregulation of β-adrenoceptors across various tissues of multiple species including humans, rats, dogs, and fish. While a canonical hypoxia-response element in the promoter of the human β-adrenoceptor gene may play a role in this, no such sequence was found in rodent homologs. Moreover, not all upregulation of β-adrenoceptor protein is accompanied by increased expression of corresponding mRNA, indicating that the upregulation may involve factors other than transcriptional changes. We propose that upregulation of β-adrenoceptors at the mRNA and/or protein level is a general marker of hypoxic conditions. Moreover, it may be an additional pathway whereby cells and tissues adapt to reduced oxygen levels.
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http://dx.doi.org/10.1007/s00210-019-01780-6DOI Listing
February 2020

Adrenoceptors in white, brown, and brite adipocytes.

Br J Pharmacol 2019 07 7;176(14):2416-2432. Epub 2019 Apr 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Adrenoceptors play an important role in adipose tissue biology and physiology that includes regulating the synthesis and storage of triglycerides (lipogenesis), the breakdown of stored triglycerides (lipolysis), thermogenesis (heat production), glucose metabolism, and the secretion of adipocyte-derived hormones that can control whole-body energy homeostasis. These processes are regulated by the sympathetic nervous system through actions at different adrenoceptor subtypes expressed in adipose tissue depots. In this review, we have highlighted the role of adrenoceptor subtypes in white, brown, and brite adipocytes in both rodents and humans and have included detailed analysis of adrenoceptor expression in human adipose tissue and clonally derived adipocytes. We discuss important considerations when investigating adrenoceptor function in adipose tissue or adipocytes. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.
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http://dx.doi.org/10.1111/bph.14631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592855PMC
July 2019

Adrenoceptor regulation of the mechanistic target of rapamycin in muscle and adipose tissue.

Br J Pharmacol 2019 07 7;176(14):2433-2448. Epub 2019 Apr 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Victoria, Australia.

A vital role of adrenoceptors in metabolism and energy balance has been well documented in the heart, skeletal muscle, and adipose tissue. It has been only recently demonstrated, however, that activation of the mechanistic target of rapamycin (mTOR) makes a significant contribution to various metabolic and physiological responses to adrenoceptor agonists. mTOR exists as two distinct complexes named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) and has been shown to play a critical role in protein synthesis, cell proliferation, hypertrophy, mitochondrial function, and glucose uptake. This review will describe the physiological significance of mTORC1 and 2 as a novel paradigm of adrenoceptor signalling in the heart, skeletal muscle, and adipose tissue. Understanding the detailed signalling cascades of adrenoceptors and how they regulate physiological responses is important for identifying new therapeutic targets and identifying novel therapeutic interventions. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors-New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc.
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http://dx.doi.org/10.1111/bph.14616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592864PMC
July 2019

Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations.

Sci Signal 2018 10 9;11(551). Epub 2018 Oct 9.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

G protein-coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous β-adrenergic receptor (βAR) and muscarinic acetylcholine M receptor (MR) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal-regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations.
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http://dx.doi.org/10.1126/scisignal.aan1188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416780PMC
October 2018

Rosiglitazone and a β-Adrenoceptor Agonist Are Both Required for Functional Browning of White Adipocytes in Culture.

Front Endocrinol (Lausanne) 2018 30;9:249. Epub 2018 May 30.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

The recruitment of brite (or beige) adipocytes has been advocated as a means to combat obesity, due to their ability to phenotypically resemble brown adipocytes (BA). Lineage studies indicate that brite adipocytes are formed by differentiation of precursor cells or by direct conversion of existing white adipocytes, depending on the adipose depot examined. We have systematically compared the gene expression profile and a functional output (oxygen consumption) in mouse adipocytes cultured from two contrasting depots, namely interscapular brown adipose tissue, and inguinal white adipose tissue (iWAT), following treatment with a known browning agent, the peroxisome proliferator-activated receptor (PPARγ) activator rosiglitazone. Prototypical BA readily express uncoupling protein (UCP)1, and upstream regulators including the β-adrenoceptor and transcription factors involved in energy homeostasis. Adipocytes from inguinal WAT display maximal UCP1 expression and mitochondrial uncoupling only when treated with a combination of the PPARγ activator rosiglitazone and a β-adrenoceptor agonist. In conclusion, brite adipocytes are fully activated only when a browning agent (rosiglitazone) and a thermogenic agent (β-adrenoceptor agonist) are added in combination. The presence of rosiglitazone throughout the 7-day culture period partially masks the effects of β-adrenoceptor signaling in inguinal white adipocyte cultures, whereas including rosiglitazone only for the first 3 days promotes robust β-adrenoceptor expression and provides an improved window for detection of β-adrenoceptor responses.
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http://dx.doi.org/10.3389/fendo.2018.00249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992408PMC
May 2018

INSL5 activates multiple signalling pathways and regulates GLP-1 secretion in NCI-H716 cells.

J Mol Endocrinol 2018 04;60(3):213-224

Drug Discovery BiologyMonash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Insulin-like peptide 5 (INSL5) is a newly discovered gut hormone expressed in colonic enteroendocrine L-cells but little is known about its biological function. Here, we show using RT-qPCR and hybridisation that mRNA is highly expressed in the mouse colonic mucosa, colocalised with proglucagon immunoreactivity. In comparison, mRNA for RXFP4 (the cognate receptor for INSL5) is expressed in various mouse tissues, including the intestinal tract. We show that the human enteroendocrine L-cell model NCI-H716 cell line, and goblet-like colorectal cell lines SW1463 and LS513 endogenously express Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr/Tyr), AKT (Thr and Ser) and S6RP (Ser) and inhibited cAMP production but did not stimulate Ca release. Acute INSL5 treatment had no effect on GLP-1 secretion mediated by carbachol or insulin, but modestly inhibited forskolin-stimulated GLP-1 secretion in NCI-H716 cells. However, chronic INSL5 pre-treatment (18 h) increased basal GLP-1 secretion and prevented the inhibitory effect of acute INSL5 administration. LS513 cells were found to be unresponsive to INSL5 despite expressing Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.
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http://dx.doi.org/10.1530/JME-17-0152DOI Listing
April 2018

α-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes.

Biochem Pharmacol 2018 02 24;148:27-40. Epub 2017 Nov 24.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address:

The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the α-adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α-AR signalling in CHO-K1 cells co-expressing the human α-AR and GLUT4 (CHOαGLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α-AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α-AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHOαGLUT4myc cells, siRNA directed against rictor but not raptor suppressed α-AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α-AR agonists. Our findings identify a novel link between the α-AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α-AR selective agonists as tools in the treatment of cardiac dysfunction.
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http://dx.doi.org/10.1016/j.bcp.2017.11.016DOI Listing
February 2018

The PPARγ agonist rosiglitazone promotes the induction of brite adipocytes, increasing β-adrenoceptor-mediated mitochondrial function and glucose uptake.

Cell Signal 2018 Jan 29;42:54-66. Epub 2017 Sep 29.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Monash University, Parkville, Victoria 3052, Australia; Department of Pharmacology, 9 Ancora Imparo Way, Monash University, Clayton, Victoria 3800, Australia. Electronic address:

Recruitment and activation of brite (or beige) adipocytes has been advocated as a potential avenue for manipulating whole-body energy expenditure. Despite numerous studies illustrating the differences in gene and protein markers between brown, brite and white adipocytes, there is very little information on the adrenergic regulation and function of these brite adipocytes. We have compared the functional (cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, extracellular acidification rates, calcium influx) profiles of mouse adipocytes cultured from three contrasting depots, namely interscapular brown adipose tissue, and inguinal or epididymal white adipose tissues, following chronic treatment with the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone. Prototypical brown adipocytes readily express β-adrenoceptors, and β-adrenoceptor stimulation increases cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, and extracellular acidification rates. Treatment of brown adipocytes with rosiglitazone increases uncoupling protein 1 (UCP1) levels, and increases β-adrenoceptor mitochondrial function but does not affect glucose uptake responses. In contrast, inguinal white adipocytes only express UCP1 and β-adrenoceptors following rosiglitazone treatment, which results in an increase in all β-adrenoceptor-mediated functions. The effect of rosiglitazone in epididymal white adipocytes, was much lower compared to inguinal white adipocytes. Rosiglitazone also increased α-adrenoceptor mediated increases in calcium influx and glucose uptake (but not mitochondrial function) in inguinal and epididymal white adipocytes. In conclusion, the PPARγ agonist rosiglitazone promotes the induction and function of brite adipocytes cultured from inguinal and epididymal white adipose depots.
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http://dx.doi.org/10.1016/j.cellsig.2017.09.023DOI Listing
January 2018

Factors influencing biased agonism in recombinant cells expressing the human α -adrenoceptor.

Br J Pharmacol 2017 Jul 10;174(14):2318-2333. Epub 2017 Jun 10.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Background And Purpose: Agonists acting at GPCRs promote biased signalling via Gα or Gβγ subunits, GPCR kinases and β-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α -adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline.

Experimental Approach: Intracellular Ca mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism.

Key Results: Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation.

Conclusion And Implications: We have shown that while adrenergic agonists display bias at human α -adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.
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http://dx.doi.org/10.1111/bph.13837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481649PMC
July 2017

Adrenoceptors promote glucose uptake into adipocytes and muscle by an insulin-independent signaling pathway involving mechanistic target of rapamycin complex 2.

Pharmacol Res 2017 02 23;116:87-92. Epub 2016 Dec 23.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia. Electronic address:

Uptake of glucose into skeletal muscle and adipose tissue plays a vital role in metabolism and energy balance. Insulin released from β-islet cells of the pancreas promotes glucose uptake in these target tissues by stimulating translocation of GLUT4 transporters to the cell surface. This process is complex, involving signaling proteins including the mechanistic (or mammalian) target of rapamycin (mTOR) and Akt that intersect with multiple pathways controlling cell survival, growth and proliferation. mTOR exists in two forms, mTOR complex 1 (mTORC1), and mTOR complex 2 (mTORC2). mTORC1 has been intensively studied, acting as a key regulator of protein and lipid synthesis that integrates cellular nutrient availability and energy balance. Studies on mTORC2 have focused largely on its capacity to activate Akt by phosphorylation at Ser473, however recent findings demonstrate a novel role for mTORC2 in cellular glucose uptake. For example, agonists acting at β-adrenoceptors (ARs) in skeletal muscle or β-ARs in brown adipose tissue increase glucose uptake in vitro and in vivo via mechanisms dependent on mTORC2 but not Akt. In this review, we will focus on the signaling pathways downstream of β-ARs that promote glucose uptake in skeletal muscle and brown adipocytes, and will highlight how the insulin and adrenergic pathways converge and interact in these cells. The identification of insulin-independent mechanisms that promote glucose uptake should facilitate novel treatment strategies for metabolic disease.
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http://dx.doi.org/10.1016/j.phrs.2016.12.022DOI Listing
February 2017

The actions of relaxin family peptides on signal transduction pathways activated by the relaxin family peptide receptor RXFP4.

Naunyn Schmiedebergs Arch Pharmacol 2017 Jan 26;390(1):105-111. Epub 2016 Nov 26.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052, Australia.

The relaxin family peptide receptor 4 (RXFP4) is a G protein-coupled receptor (GPCR) expressed in the colorectum with emerging roles in metabolism and appetite regulation. It is activated by its cognate ligand insulin-like peptide 5 (INSL5) that is expressed in enteroendocrine L cells in the gut. Whether other evolutionarily related peptides such as relaxin-2, relaxin-3, or INSL3 activate RXFP4 signal transduction mechanisms with a pattern similar to or distinct from INSL5 is still unclear. In this study, we compare the signaling pathways activated by various relaxin family peptides to INSL5. We found that, like INSL5, relaxin-3 activated ERK1/2, p38MAPK, Akt, and S6RP phosphorylations leading to increased cell proliferation and also caused GRK and β-arrestin-mediated receptor internalization. Interestingly, relaxin-3 was slightly more potent than INSL5 in ERK1/2 and Akt phosphorylations, but both peptides were almost equipotent in adenylyl cyclase inhibition, S6RP phosphorylation, and cell proliferation. In addition, relaxin-3 showed greater efficacy only in Akt phosphorylation but not in the other pathways investigated. In contrast, no signaling activity or receptor internalization mechanisms were observed following relaxin-2 and INSL3. In conclusion, relaxin-3 is a high-efficacy agonist at RXFP4 with a comparable signal transduction profile to INSL5.
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http://dx.doi.org/10.1007/s00210-016-1321-8DOI Listing
January 2017

Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor.

Br J Pharmacol 2017 05 13;174(10):1077-1089. Epub 2016 Jul 13.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Background And Purpose: Insulin-like peptide 5 (INSL5) is a two-chain, three-disulfide-bonded peptide of the insulin/relaxin superfamily, uniquely expressed in enteroendocrine L-cells of the colon. It is the cognate ligand of relaxin family peptide RXFP4 receptor that is mainly expressed in the colorectum and enteric nervous system. This study identifies new signalling pathways activated by INSL5 acting on RXFP4 receptors.

Experimental Approach: INSL5/RXFP4 receptor signalling was investigated using AlphaScreen® proximity assays. Recruitment of Gα proteins by RXFP4 receptors was determined by rescue of Pertussis toxin (PTX)-inhibited cAMP and ERK1/2 responses following transient transfection of PTX-insensitive Gα C351I mutants. Cell proliferation was studied with bromodeoxyuridine. RXFP4 receptor interactions with β-arrestins, GPCR kinase 2 (GRK2), KRas and Rab5a was assessed with real-time BRET. Gene expression was investigated using real-time quantitative PCR. Insulin release was measured using HTRF and intracellular Ca flux monitored in a Flexstation® using Fluo-4-AM.

Key Results: INSL5 inhibited forskolin-stimulated cAMP accumulation and increased phosphorylation of ERK1/2, p38MAPK, Akt Ser , Akt Thr and S6 ribosomal protein. cAMP and ERK1/2 responses were abolished by PTX and rescued by mGα , mGα and mGα and to a lesser extent mGα and mGα . RXFP4 receptors interacted with GRK2 and β-arrestins, moved towards Rab5a and away from KRas, indicating internalisation following receptor activation. INSL5 inhibited glucose-stimulated insulin secretion and Ca mobilisation in MIN6 insulinoma cells and forskolin-stimulated cAMP accumulation in NCI-H716 enteroendocrine cells.

Conclusions And Implications: Knowledge of signalling pathways activated by INSL5 at RXFP4 receptors is essential for understanding the biological roles of this novel gut hormone.

Linked Articles: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.
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http://dx.doi.org/10.1111/bph.13522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406383PMC
May 2017

Could burning fat start with a brite spark? Pharmacological and nutritional ways to promote thermogenesis.

Mol Nutr Food Res 2016 Jan 27;60(1):18-42. Epub 2015 Aug 27.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia.

There are two types of adipose tissue with distinct functions-white adipose tissue stores chemical energy as triglycerides, whereas brown adipose tissue consumes energy and releases heat (thermogenesis) in response to sympathetic nerve activity. In humans, treatments that promote greater brown adipose tissue deposition and/or activity would be highly beneficial in regimes aimed at reducing obesity. Adult humans have restricted populations of prototypical brown adipocytes in the neck and chest areas, but recent advances have established that adipocytes with similar properties, termed "brite" adipocytes, can be recruited in subcutaneous depots thought to be primarily white adipose tissue. These brite adipocytes express the protein machinery required for thermogenesis, but to assess brite adipocytes as viable therapeutic targets we need to understand how to promote conversion of white adipocytes to brite adipocytes and ways to increase optimal energy consumption and thermogenesis in these brite adipocytes. This can be accomplished by pharmacological and nutritional therapies to differing degrees, as reviewed in detail here.
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http://dx.doi.org/10.1002/mnfr.201500251DOI Listing
January 2016

Does the autonomic nervous system contribute to the initiation and progression of prostate cancer?

Asian J Androl 2013 Nov 21;15(6):715-6. Epub 2013 Oct 21.

In the July 12 issue of Science magazine, researchers from the Albert Einstein College of Medicine, the Mount Sinai School of Medicine, the Durham VA Medical Centre and Duke University published an elegant study demonstrating that the sympathetic nervous system, acting through β2 and β3-adrenoceptors in the prostate, plays an important role in the initiation of prostate cancer, while the parasympathetic nervous system plays a role in the dissemination of tumour metastases via M1 muscarinic receptors. These findings are significant because they indicate that receptors associated with the autonomic nervous system may be viable targets for prostate cancer therapy.
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http://dx.doi.org/10.1038/aja.2013.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3854038PMC
November 2013

Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

PLoS One 2013 25;8(7):e68841. Epub 2013 Jul 25.

Commissariat à l'Énergie Atomique Et Aux Énergies Alternatives, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, Gif sur Yvette, France.

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068841PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723878PMC
March 2014

β2-Adrenoceptor-mediated regulation of glucose uptake in skeletal muscle--ligand-directed signalling or a reflection of system complexity?

Naunyn Schmiedebergs Arch Pharmacol 2013 Sep 9;386(9):757-60. Epub 2013 May 9.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, 399 Royal Parade, Parkville, Melbourne, VIC, 3052, Australia.

The capacity of G protein-coupled receptors (GPCRs) to activate multiple G protein isoforms and additional effectors such as β-arrestins has become a well-established paradigm and provides the basis for developing drugs that preferentially activate beneficial signalling pathways. There are many published examples of ligand-directed signalling, and recent studies have provided direct evidence that different agonists stabilise distinct GPCR conformations. This field is rapidly evolving, but a key question is whether signalling bias observed in heterologous cell expression systems can be translated to physiological systems of therapeutic relevance. The paper by Ngala et al. in this issue of the journal addresses the capacity of agonists acting at the β2-adrenoceptor to engender signalling bias in relation to glucose uptake in isolated skeletal muscle, an area of considerable potential interest in targeting insulin-independent pathways for the treatment of type 2 diabetes. The authors show that clenbuterol and BRL37344 have opposite effects on glucose uptake, despite both having agonist actions at β2-adrenoceptors. This study underlines some of the obstacles associated with studies in a complex physiological system but nonetheless highlights the need to consider signalling bias in the relevant target tissue when developing novel drugs.
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http://dx.doi.org/10.1007/s00210-013-0879-7DOI Listing
September 2013

Interaction with caveolin-1 modulates G protein coupling of mouse β3-adrenoceptor.

J Biol Chem 2012 Jun 25;287(24):20674-88. Epub 2012 Apr 25.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and the Department of Pharmacology, Monash University, Parkville, Victoria 3052, Australia.

Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knock-out mice suggest that β(3)-adrenoceptor (β(3)-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the β(3)-AR or solely with downstream signaling proteins. We have addressed this question by examining the impact of membrane rafts and caveolin-1 on the differential signaling of mouse β(3a)- and β(3b)-AR isoforms that diverge at the distal C terminus. Only the β(3b)-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the β(3a)-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the β(3)-AR agonist CL316243 became PTX-sensitive, suggesting Gα(i/o) coupling. The β(3a)-AR C terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant β(3a)-ARs (F389A/Y392A/F398A or P384S/F389A) promoted PTX-sensitive cAMP responses, and in situ proximity assays demonstrated an association between caveolin-1 and the wild type β(3a)-AR but not the mutant receptors. In membrane preparations, the β(3b)-AR activated Gα(o) and mediated PTX-sensitive cAMP responses, whereas the β(3a)-AR did not activate Gα(i/o) proteins. The endogenous β(3a)-AR displayed Gα(i/o) coupling in brown adipocytes from caveolin-1 knock-out mice or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the β(3a)-AR with caveolin inhibits coupling to Gα(i/o) proteins and suggest that signaling is modulated by a raft-enriched complex containing the β(3a)-AR, caveolin-1, Gα(s), and adenylyl cyclase.
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http://dx.doi.org/10.1074/jbc.M111.280651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370250PMC
June 2012

β(2)-Adrenoceptors increase translocation of GLUT4 via GPCR kinase sites in the receptor C-terminal tail.

Br J Pharmacol 2012 Mar;165(5):1442-56

Department of Physiology, The Wenner-Gren Institute, Arrhenius Laboratories F3, Stockholm University, Stockholm, Sweden.

Background And Purpose: β-Adrenoceptor stimulation induces glucose uptake in several insulin-sensitive tissues by poorly understood mechanisms.

Experimental Approach: We used a model system in CHO-K1 cells expressing the human β(2)-adrenoceptor and glucose transporter 4 (GLUT4) to investigate the signalling mechanisms involved.

Key Results: In CHO-K1 cells, there was no response to β-adrenoceptor agonists. The introduction of β(2)-adrenoceptors and GLUT4 into these cells caused increased glucose uptake in response to β-adrenoceptor agonists. GLUT4 translocation occurred in response to insulin and β(2)-adrenoceptor stimulation, although the key insulin signalling intermediate PKB was not phosphorylated in response to β(2)-adrenoceptor stimulation. Truncation of the C-terminus of the β(2)-adrenoceptor at position 349 to remove known phosphorylation sites for GPCR kinases (GRKs) or at position 344 to remove an additional PKA site together with the GRK phosphorylation sites did not significantly affect cAMP accumulation but decreased β(2)-adrenoceptor-stimulated glucose uptake. Furthermore, inhibition of GRK by transfection of the βARKct construct inhibited β(2)-adrenoceptor-mediated glucose uptake and GLUT4 translocation, and overexpression of a kinase-dead GRK2 mutant (GRK2 K220R) also inhibited GLUT4 translocation. Introducing β(2)-adrenoceptors lacking phosphorylation sites for GRK or PKA demonstrated that the GRK sites, but not the PKA sites, were necessary for GLUT4 translocation.

Conclusions And Implications: Glucose uptake in response to activation of β(2)-adrenoceptors involves translocation of GLUT4 in this model system. The mechanism is dependent on the C-terminus of the β(2)-adrenoceptor, requires GRK phosphorylation sites, and involves a signalling pathway distinct from that stimulated by insulin.
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http://dx.doi.org/10.1111/j.1476-5381.2011.01647.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372728PMC
March 2012

Quantification of functional selectivity at the human α(1A)-adrenoceptor.

Mol Pharmacol 2011 Feb 26;79(2):298-307. Epub 2010 Oct 26.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, 399 Royal Parade, Parkville, Victoria 3052, Australia.

Although G protein-coupled receptors are often categorized in terms of their primary coupling to a given type of Gα protein subunit, it is now well established that many show promiscuous coupling and activate multiple signaling pathways. Furthermore, some agonists selectively activate signaling pathways by promoting interaction between distinct receptor conformational states and particular Gα subunits or alternative signaling proteins. We have tested the capacity of agonists to stimulate Ca(2+) release, cAMP accumulation, and changes in extracellular acidification rate (ECAR) at the human α(1A)-adrenoceptor. Signaling bias factors were determined by novel application of an operational model of agonism and compared with the reference endogenous agonist norepinephrine; values significantly different from 1.0 indicated an agonist that promoted receptor conformations distinct from that favored by norepinephrine. Oxymetazoline was a full agonist for ECAR and a partial agonist for Ca(2+) release (bias factor 8.2) but failed to stimulate cAMP production. Phenylephrine showed substantial bias toward ECAR versus Ca(2+) release or cAMP accumulation (bias factors 21 and 33, respectively) but did not display bias between Ca(2+) and cAMP pathways. Cirazoline and N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl]methanesulfonamide (A61603) displayed bias toward cAMP relative to Ca(2+) release (bias factors of 7.4 and 8.6). It is noteworthy that epinephrine, a second endogenous adrenoceptor agonist, did not display bias relative to norepinephrine. Our finding that phenylephrine displayed significant signaling bias, despite being highly similar in structure to epinephrine, indicates that subtle differences in agonist-receptor interaction can affect conformational changes in cytoplasmic domains and thereby modulate the repertoire of effector proteins that are activated.
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http://dx.doi.org/10.1124/mol.110.067454DOI Listing
February 2011

The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK-AMPK-dependent mechanism.

Cell Signal 2010 Jul 4;22(7):1104-13. Epub 2010 Mar 4.

Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, 3800, Australia.

The role of muscarinic acetylcholine receptors (mAChRs) in regulating glucose uptake in L6 skeletal muscle cells was investigated. [(3)H]-2-Deoxyglucose uptake was increased in differentiated L6 cells by insulin, acetylcholine, oxotremorine-M and carbachol. mAChR-mediated glucose uptake was inhibited by the AMPK inhibitor Compound C. Whole cell radioligand binding using [(3)H]-N-methyl scopolamine chloride identified mAChRs in differentiated but not undifferentiated L6 cells and M(3) mAChR mRNA was detected only in differentiated cells. M(3) mAChRs are Gq-coupled, and cholinergic stimulation by the mAChR agonists acetylcholine, oxotremorine-M and carbachol increased Ca(2+) in differentiated but not undifferentiated L6 cells. This was due to muscarinic but not nicotinic activation as responses were antagonised by the muscarinic antagonist atropine but not the nicotinic antagonist tubocurarine. Western blotting showed that both carbachol and the AMPK activator AICAR increased phosphorylation of the AMPKalpha subunit at Thr172, with responses to carbachol blocked by Compound C and the CaMKK inhibitor STO609 but not by the PI3K inhibitor wortmannin. AICAR-stimulated AMPK phosphorylation was not sensitive to STO-609, confirming that this compound inhibits CaMKK but not the classical AMPK kinase LKB1. The TAK1 inhibitor (5Z)-7-oxozeaenol and the G(i) inhibitor pertussis toxin both failed to block AMPK phosphorylation in response to carbachol. Using CHO-K1 cells stably expressing each of the mAChR subtypes (M(1)-M(4)), it was determined that only the M(1) and M(3) mAChRs phosphorylate AMPK, confirming a G(q)-dependent mechanism. This study demonstrates that activation of M(3) mAChRs in L6 skeletal muscle cells stimulates glucose uptake via a CaMKK-AMPK-dependent mechanism, independent of the insulin-stimulated pathway.
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http://dx.doi.org/10.1016/j.cellsig.2010.03.004DOI Listing
July 2010

Ligand-directed signalling at beta-adrenoceptors.

Br J Pharmacol 2010 Mar 2;159(5):1022-38. Epub 2010 Feb 2.

Monash Institute of Pharmaceutical Sciences & Department of Pharmacology, Parkville, Vic, Australia.

beta-Adrenoceptors (ARs) classically mediate responses to the endogenous ligands adrenaline and noradrenaline by coupling to Gsalpha and stimulating cAMP production; however, drugs designed as beta-AR agonists or antagonists can activate alternative cell signalling pathways, with the potential to influence clinical efficacy. Furthermore, drugs acting at beta-ARs have differential capacity for pathway activation, described as stimulus trafficking, biased agonism, functional selectivity or ligand-directed signalling. These terms refer to responses where drug A has higher efficacy than drug B for one signalling pathway, but a lower efficacy than drug B for a second pathway. The accepted explanation for such responses is that drugs A and B have the capacity to induce or stabilize distinct active conformations of the receptor that in turn display altered coupling efficiency to different effectors. This is consistent with biophysical studies showing that drugs can indeed promote distinct conformational states. Agonists acting at beta-ARs display ligand-directed signalling, but many drugs acting as cAMP antagonists are also able to activate signalling pathways central to cell survival and proliferation or cell death. The observed complexity of drug activity at beta-ARs, prototypical G protein-coupled receptors, necessitates rethinking of the approaches used for screening and characterization of novel therapeutic agents. Most studies of ligand-directed signalling employ recombinant cell systems with high receptor abundance. While such systems are valid for examining upstream signalling events, such as receptor conformational changes and G protein activation, they are less robust when comparing downstream signalling outputs as these are likely to be affected by complex pathway interactions.
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http://dx.doi.org/10.1111/j.1476-5381.2009.00602.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2839261PMC
March 2010

RXFP1 couples to the Galpha-Gbetagamma-PI3K-PKCzeta pathway via the final 10 amino acids of the receptor C-terminal tail.

Ann N Y Acad Sci 2009 Apr;1160:117-20

Department of Pharmacology, Monash University, Clayton, Victoria 3800, Australia.

The relaxin family peptide receptors RXFP1 and RXFP2 are highly similar receptors that share approximately 80% amino acid sequence homology. Constitutively active receptors couple to increased cAMP accumulation, which is important for relaxin-mediated decidualization and myometrial inhibition. Despite the high homology, the receptors couple to different G-proteins to affect cAMP accumulation. This study aimed to determine the region of RXFP1 that directs coupling to the delayed Galpha(i3) pathway by using receptor mutagenesis. Receptor chimeras suggested that activation of this pathway by RXFP1 was dependent upon the membrane-anchored domain of the receptor. Further receptor mutagenesis showed that activation of the Galpha(i3)-Gbetagamma-PI3K-PKCzeta cAMP pathway by RXFP1 is dependent upon the C-terminal 10 amino acids of the receptor and absolutely requires Arg(752).
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http://dx.doi.org/10.1111/j.1749-6632.2008.03813.xDOI Listing
April 2009

Relaxin family peptide receptor (RXFP1) coupling to G(alpha)i3 involves the C-terminal Arg752 and localization within membrane Raft Microdomains.

Mol Pharmacol 2009 Feb 24;75(2):415-28. Epub 2008 Nov 24.

Monash Institute for Pharmaceutical Sciences, Parkville, Australia.

The relaxin family peptide receptors (RXFP) 1 and 2 are targets for the relaxin family peptides relaxin and insulin-like peptide 3 (INSL3), respectively. Although both receptors and peptides share a high degree of sequence identity, the cAMP signaling pathways activated by the two systems are quite distinct. Relaxin activation of RXFP1 initially results in accumulation of cAMP via G(alpha)(s), but this is modulated by inhibition of cAMP through G(alpha)(oB). Over time, RXFP1 recruits coupling to G(alpha)(i3), causing additional cAMP accumulation via a G(alpha)(i3)-Gbetagamma-phosphoinositide 3-kinase (PI3K)-protein kinase C (PKC)zeta pathway. In contrast, INSL3 activation of RXFP2 results in accumulation of cAMP only via G(alpha)(s), modulated by cAMP inhibition through G(alpha)(oB). Thus, the aim of this study was to identify the cause of differential G-protein coupling between these highly similar receptors. Construction of chimeric receptors revealed that G(alpha)(i3) coupling is dependent upon the transmembrane region of RXFP1 and independent of the receptor ectodomain or ligand bound. Generation of C-terminal truncated receptors identified the terminal 10 amino acids of the RXFP1 C terminus as essential for G(alpha)(i3) signaling, and point mutations revealed an obligatory role for Arg(752). RXFP1-mediated G(alpha)(i3), but not G(alpha)(s) or G(alpha)(oB), signaling was also found to be dependent upon membrane rafts, and RXFP1 coupled to G(alpha)(i3) after only 3 min of receptor stimulation. Therefore, RXFP1 coupling to the G(alpha)(i3)-Gbetagamma-PI3K-PKCzeta pathway requires the terminal 10 amino acids of the RXFP1 C terminus and membrane raft localization, and the observed delay in this pathway occurs downstream of G(alpha)(i3).
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http://dx.doi.org/10.1124/mol.108.051227DOI Listing
February 2009

The beta3-adrenoceptor agonist 4-[[(Hexylamino)carbonyl]amino]-N-[4-[2-[[(2S)-2-hydroxy-3-(4-hydroxyphenoxy)propyl]amino]ethyl]-phenyl]-benzenesulfonamide (L755507) and antagonist (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]-ethyl]phenyl]benzenesulfonamide (L748337) activate different signaling pathways in Chinese hamster ovary-K1 cells stably expressing the human beta3-adrenoceptor.

Mol Pharmacol 2008 Nov 6;74(5):1417-28. Epub 2008 Aug 6.

Department of Pharmacology, P.O. Box 13E, Monash University VIC 3800, Australia.

This study identifies signaling pathways activated by the beta(2)-/beta(3)-adrenoceptor (AR) agonist zinterol, the selective beta(3)-AR agonist L755507, and the selective beta(3)-AR antagonist L748337 in CHO-K1 cells expressing human beta(3)-adrenoceptors. Zinterol and L755507 caused a robust concentration-dependent increase in cAMP accumulation (pEC(50) values of 8.5 and 12.3, respectively), whereas L748337 had low efficacy. Maximal cAMP accumulation with zinterol and L755507 was increased after pretreatment with pertussis toxin, indicating that the human beta(3)-AR couples to G(i) and to G(s). In contrast to cAMP, zinterol, L755507 and L748337 increased phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) with very high potency (pEC(50) values of 10.9, 11.7, and 11.6). These compounds also stimulated phosphorylation of p38 mitogen-activated protein kinase (MAPK) but with much lower potency than Erk1/2 (pEC(50) values of 5.9, 5.5, and 5.7, respectively). Pertussis toxin completely blocked Erk1/2 and p38 MAPK phosphorylation in response to L748337, demonstrating a requirement for G(i/o) coupling, whereas L755507-stimulated p38 MAPK phosphorylation was not inhibited by pertussis toxin, and Erk1/2 phosphorylation was inhibited by only 30%. We found that high levels of cAMP interfered with agonist-activated p38 MAPK phosphorylation. L748337 increased extracellular acidification rate (ECAR) in the cytosensor microphysiometer with efficacy similar to zinterol and L755507, albeit with lower potency (pEC(50) value of 7.2 compared with zinterol, 8.1, and L755507, 8.6). The ECAR response to L748337 was largely via activation of p38 MAPK, demonstrated by 65% inhibition with 4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol (RWJ67657). We conclude that the beta(3)-AR agonist L755507 couples to both G(s) and G(i) to activate adenylate cyclase and MAPK signaling, whereas the beta(3)-AR antagonist L748337 couples predominantly to G(i) to activate MAPK signaling.
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http://dx.doi.org/10.1124/mol.108.046979DOI Listing
November 2008

Ligand-directed signaling at the beta3-adrenoceptor produced by 3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate (SR59230A) relative to receptor agonists.

Mol Pharmacol 2007 Nov 23;72(5):1359-68. Epub 2007 Aug 23.

Department of Pharmacology, PO Box 13E, Monash University VIC 3800, Australia.

This study examines signaling pathways activated by the mouse beta(3)-adrenoceptor (AR) expressed in Chinese hamster ovary cells at high (CHObeta(3)H) or low (CHObeta(3)L) levels. Functional responses included extracellular acidification rate (ECAR), cAMP accumulation, and p38 mitogen-activated protein kinase (MAPK) or extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation. (-)-Isoproterenol and the beta(3)-AR agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-decarboxylate (CL316243) caused concentration-dependent increases in cAMP accumulation and ECAR in CHObeta(3)H and CHObeta(3)L cells. For cAMP accumulation, the beta(3)-AR ligand SR59230A was a partial agonist in CHObeta(3)H and an antagonist in CHObeta(3)L cells but for ECAR was an agonist at both expression levels. This suggested that SR59230A, which is normally regarded as an antagonist, can selectively activate pathways leading to ECAR. Examination of the pathways stimulated by (-)-isoproterenol, CL316243, and SR59230A for both ECAR and cAMP accumulation suggested that the cAMP pathway predominates in CHObeta(3)H cells, whereas p38 MAPK is a major contributor to ECAR in CHObeta(3)L cells and was the sole contributor to responses to SR59230A. Western blots of p38 MAPK and Erk1/2 phosphorylation confirmed that MAPKs are activated in CHObeta(3)H and CHObeta(3)L cells by CL316243 and SR59230A but that SR59230A has much higher efficacy. In addition, p38 MAPK phosphorylation displayed differences in drug potency and efficacy between CHObeta(3)H and CHObeta(3)L cells related to inhibition of the response by cAMP. Thus, CL316243 and SR59230A display reversed orders of efficacy for cAMP accumulation compared with Erk1/2 and p38 MAPK phosphorylation, providing a strong indication of ligand-directed signaling.
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http://dx.doi.org/10.1124/mol.107.035337DOI Listing
November 2007

Multiple signalling pathways involved in beta2-adrenoceptor-mediated glucose uptake in rat skeletal muscle cells.

Br J Pharmacol 2006 Feb;147(4):446-54

Department of Pharmacology, PO Box 13E, Monash University, Victoria 3800, Australia.

1. Beta-adrenoceptor (AR) agonists increase 2-deoxy-[3H]-D-glucose uptake (GU) via beta2-AR in rat L6 cells. The beta-AR agonists, zinterol (beta2-AR) and (-)-isoprenaline, increased cAMP accumulation in a concentration-dependent manner (pEC50=9.1+/-0.02 and 7.8+/-0.02). Cholera toxin (% max increase 141.8+/-2.5) and the cAMP analogues, 8-bromo-cAMP (8Br-cAMP) and dibutyryl cAMP (dbcAMP), also increased GU (196.8+/-13.5 and 196.4+/-17.3%). 2. The adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (50 microM), significantly reduced cAMP accumulation to zinterol (100 nM) (109.7+35.0 to 21.6+4.5 pmol well(-1)), or forskolin (10 microM) (230.1+/-58.0 to 107.2+/-26.3 pmol well(-1)), and partially inhibited zinterol-stimulated GU (217+/-26.3 to 176.1+/-20.4%). The protein kinase A (PKA) inhibitor, 4-cyano-3-methylisoquinoline (100 nM), did not inhibit zinterol-stimulated GU. The PDE4 inhibitor, rolipram (10 microM), increased cAMP accumulation to zinterol or forskolin, and sensitised the GU response to zinterol, indicating a stimulatory role of cAMP in GU. 3. cAMP accumulation studies indicated that the beta2-AR was desensitised by prolonged stimulation with zinterol, but not forskolin, whereas GU responses to zinterol increased with time, suggesting that receptor desensitisation may be involved in GU. Receptor desensitisation was not reversed by inhibition of PKA or Gi. 4. PTX pretreatment (100 ng ml(-1)) inhibited insulin or zinterol-stimulated but not 8Br-cAMP or dbcAMP-stimulated GU. The PI3K inhibitor, LY294002 (1 microM), inhibited insulin- (174.9+/-5.9 to 142.7+/-2.7%) and zinterol- (166.9+/-7.6 to 141.1+/-8.1%) but not 8 Br-cAMP-stimulated GU. In contrast to insulin, zinterol did not cause phosphorylation of Akt. 5. The results suggest that GU in L6 cells involves three mechanisms: (1) an insulin-dependent pathway involving PI3K, (2) a beta2-AR-mediated pathway involving both cAMP and PI3K, and (3) a receptor-independent pathway suggested by cAMP analogues that increase GU independently of PI3K. PKA appears to negatively regulate beta2-AR-mediated GU.
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http://dx.doi.org/10.1038/sj.bjp.0706626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1616992PMC
February 2006

Functional domains of the mouse beta3-adrenoceptor associated with differential G protein coupling.

J Pharmacol Exp Ther 2005 Dec 6;315(3):1354-61. Epub 2005 Sep 6.

Department of Pharmacology, P.O. Box 13E, Monash University, Victoria 3800, Australia.

Alternative splicing of mouse beta3-adrenoceptor transcripts produces an additional receptor isoform (beta3b-adrenoceptor) with a C terminus comprising 17 amino acids distinct from the 13 in the known receptor (beta3a-adrenoceptor). We have shown that the beta3b-adrenoceptor couples to both Gs and Gi, whereas the beta3a-adrenoceptor couples only to Gs. To define the regions involved in this differential G protein coupling, we have compared wild-type, truncated, and mutant beta3-adrenoceptors. In Chinese hamster ovary cells expressing beta3-adrenoceptors truncated at the splicing point, cAMP accumulation with CL316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] increased by 59% following pretreatment with pertussis toxin, suggesting that the C-terminal region of the beta3a-adrenoceptor inhibits coupling to Gi. We next utilized the cell-penetrating peptide Transportan 10 (Tp10) to introduce peptides comprising the different C-terminal tail fragments into cells expressing beta3a-adrenoceptor, beta3b-adrenoceptor, and the truncated beta3-adrenoceptor. Treatment with beta3a-Tp10 (1 microM) caused cAMP responses to CL316243 in the beta3a-adrenoceptor to become pertussis toxin-sensitive and display a 30% increase over control, whereas the other peptides did not affect any receptor. Mutation at a potential tyrosine phosphorylation site (Tyr392Ala beta3a-adrenoceptor) did not alter responses or pertussis toxin sensitivity relative to the parent receptor. Surprisingly, a Ser388Ala/Ser389Ala mutant beta3b-adrenoceptor became unresponsive to CL316243 while retaining an extracellular acidification rate response to SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate]. Our findings suggest that the beta3a-adrenoceptor cannot couple to Gi because of conformational changes induced by a protein(s) that interacts with residues in the C-terminal tail or because this protein(s) affects the intracellular localization of the beta3a-adrenoceptor.
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http://dx.doi.org/10.1124/jpet.105.091736DOI Listing
December 2005

Evidence for pleiotropic signaling at the mouse beta3-adrenoceptor revealed by SR59230A [3-(2-Ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanol oxalate].

J Pharmacol Exp Ther 2005 Mar 1;312(3):1064-74. Epub 2004 Dec 1.

Department of Pharmacology, P.O. Box 13E, Monash University, Victoria 3800, Australia.

This study examines the action of the beta(3)-adrenoceptor antagonist SR59230A [3-(2-ethylphenoxy)-1-[(1,S)-1,2,3,4-tetrahydronapth-1-ylamino]-2S-2-propanoloxalate] at cloned mouse beta(3)-adrenoceptors expressed in Chinese hamster ovary cells (CHO-K1-beta(3)) or endogenously expressed in 3T3-F442A adipocytes or ileum. SR59230A displayed partial agonist properties compared with the beta(3)-adrenoceptor agonist CL316243 [(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate] in CHO-K1-beta(3) with the intrinsic activity increasing with the level of receptor expression. Functional affinity values for SR59230A at each level of receptor expression were in agreement with pK(I) values determined by binding. In cytosensor microphysiometer studies, SR59230A was a full agonist for increases in extracellular acidification rates (ECARs) at all levels of receptor expression, and antagonist actions were measurable only in medium- or low-expressing cells. In 3T3-F442A adipocytes, SR59230A antagonized CL316243-mediated increases of cAMP and had no agonist actions. However, in the cytosensor micro-physiometer, SR59230A (acting via beta(3)-adrenoceptors) was an agonist with an intrinsic activity greater than CL316243. In mouse ileum, SR59230A relaxed smooth muscle, although concentration-response curves were biphasic. Relaxant effects were produced by concentrations that did not affect cAMP levels. Differences in tissue responses to SR59230A were not caused by major differences in expression of Galphas. ECAR responses were not affected by pretreatment of cells with pertussis toxin, indicating that signaling did not involve Gi. Therefore, SR59230A displays agonist and antagonist actions at the mouse beta(3)-adrenoceptor. Because SR59230A only antagonized accumulation of cAMP in 3T3-F442A adipocytes yet in the same cells was an agonist for ECAR, cAMP-independent signaling pathways must mediate part of the agonist actions in the microphysiometer.
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http://dx.doi.org/10.1124/jpet.104.076901DOI Listing
March 2005

The Janus faces of adrenoceptors: factors controlling the coupling of adrenoceptors to multiple signal transduction pathways.

Clin Exp Pharmacol Physiol 2004 Nov;31(11):822-7

Molecular Pharmacology Laboratory, Department of Pharmacology, Monash University, Clayton, Victoria, Australia.

1. The adrenoceptors (AR) are an important subfamily of rhodopsin-like G-protein-coupled receptors that couple to an increasingly large number of signalling mechanisms. Two important factors that determine the pathways that are used are the C-terminal region of the receptor and the agonist used to activate the receptor. 2. Studies of splice variants of the mouse beta3-AR showed that the C-terminus is a factor controlling the signalling characteristics. Although these receptors differ only at the C-terminus, the beta3b-AR coupled to both Gs and Gi, whereas the beta3a-AR coupled solely to Gs. 3. Examination of four splice variants of the human alpha1A-AR showed that all were able to couple to pertussis toxin-sensitive G-proteins, even though they have radically different C-terminal regions. 4. Comparison of the effects of the beta3-AR ligands CL316243 and SR59230A showed that both can activate the mouse beta3-AR but that SR59230A uses pathways other than cAMP accumulation in 3T3-F442A cells. 5. Examination of a series of alpha1-AR agonists for their ability to activate a number of signalling pathways revealed that A61603 acted as a full agonist in all assays, whereas oxymetazoline was unable to cause cAMP accumulation, suggesting agonist-selective signalling at the human alpha1A-AR.
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http://dx.doi.org/10.1111/j.1440-1681.2004.04094.xDOI Listing
November 2004