Role of G protein-coupled receptor kinases (GRKs) in ß2 -adrenoceptor-mediated glucose uptake.

Truncation of the C-terminal tail of the ß2 -AR, transfection of ßARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the ß2 -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant ß2 -ARs were generated and receptor affinity for [3 H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by ß2 -AR agonists, cAMP accumulation, GLUT4 translocation, [3 H]-2-deoxyglucose uptake, and ß2 -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between ß2 -AR and ß-arrestin2 or between ß2 -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to ß2 -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to ß2 -AR agonists occurred in CHO-GLUT4myc cells expressing ß2 -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type ß2 -AR. However, ß2 -ARs lacking phosphorylation sites failed to recruit ß-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the ß2 -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

The metabolic effects of mirabegron are mediated primarily by ß3 -adrenoceptors.

The ß3 -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 ß-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, [3 H]-2-deoxyglucose uptake, cellular glycolysis, and O2 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 ß3 -adrenoceptors as they were largely absent in adipocytes derived from ß3 -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 ß3 -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 ß3 -adrenoceptor.

Upregulation of ß3-adrenoceptors-a general marker of and protective mechanism against hypoxia?

ß3-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 ß3-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 ß3-adrenoceptor gene may play a role in this, no such sequence was found in rodent homologs. Moreover, not all upregulation of ß3-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 ß3-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.

Adrenoceptors in white, brown, and brite adipocytes.

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.

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

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.

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

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 ß2-adrenergic receptor (ß2AR) and muscarinic acetylcholine M3 receptor (M3R) 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.

Rosiglitazone and a ß3-Adrenoceptor Agonist Are Both Required for Functional Browning of White Adipocytes in Culture.

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 ß3-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 ß3-adrenoceptor agonist. In conclusion, brite adipocytes are fully activated only when a browning agent (rosiglitazone) and a thermogenic agent (ß3-adrenoceptor agonist) are added in combination. The presence of rosiglitazone throughout the 7-day culture period partially masks the effects of ß3-adrenoceptor signaling in inguinal white adipocyte cultures, whereas including rosiglitazone only for the first 3 days promotes robust ß3-adrenoceptor expression and provides an improved window for detection of ß3-adrenoceptor responses.

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

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 in situ hybridisation that Insl5 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 RXFP4. Stimulation of NCI-H716 cells with INSL5 produced phosphorylation of ERK1/2 (Thr202/Tyr204), AKT (Thr308 and Ser473) and S6RP (Ser235/236) and inhibited cAMP production but did not stimulate Ca2+ 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 RXFP4 Another enteroendocrine L-cell model, mouse GLUTag cells did not express detectable levels of Rxfp4 and were unresponsive to INSL5. This study provides novel insights into possible autocrine/paracrine roles of INSL5 in the intestinal tract.

α1A-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes.

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 α1A-adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α1A-AR signalling in CHO-K1 cells co-expressing the human α1A-AR and GLUT4 (CHOα1AGLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca2+ mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α1A-AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α1-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 CHOα1AGLUT4myc cells, siRNA directed against rictor but not raptor suppressed α1A-AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α1A-AR agonists. Our findings identify a novel link between the α1A-AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α1A-AR selective agonists as tools in the treatment of cardiac dysfunction.

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

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 ß3-adrenoceptors, and ß3-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 ß3-adrenoceptor mitochondrial function but does not affect glucose uptake responses. In contrast, inguinal white adipocytes only express UCP1 and ß3-adrenoceptors following rosiglitazone treatment, which results in an increase in all ß3-adrenoceptor-mediated functions. The effect of rosiglitazone in epididymal white adipocytes, was much lower compared to inguinal white adipocytes. Rosiglitazone also increased α1-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.

Factors influencing biased agonism in recombinant cells expressing the human α1A -adrenoceptor.

BACKGROUND AND PURPOSE: Agonists acting at GPCRs promote biased signalling via Gα or Gßγ subunits, GPCR kinases and ß-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 α1A -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 Ca2+ 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 Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca+2 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-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ 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 α1A -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.

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

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 Gαi/o proteins by RXFP4 receptors was determined by rescue of Pertussis toxin (PTX)-inhibited cAMP and ERK1/2 responses following transient transfection of PTX-insensitive Gαi/o C351I mutants. Cell proliferation was studied with bromodeoxyuridine. RXFP4 receptor interactions with ß-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 Ca2+ 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 Ser473 , Akt Thr308 and S6 ribosomal protein. cAMP and ERK1/2 responses were abolished by PTX and rescued by mGαoA , mGαoB and mGαi2 and to a lesser extent mGαi1 and mGαi3 . RXFP4 receptors interacted with GRK2 and ß-arrestins, moved towards Rab5a and away from KRas, indicating internalisation following receptor activation. INSL5 inhibited glucose-stimulated insulin secretion and Ca2+ 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.

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

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 ß-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.

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

Uptake of glucose into skeletal muscle and adipose tissue plays a vital role in metabolism and energy balance. Insulin released from ß-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 ß2-adrenoceptors (ARs) in skeletal muscle or ß3-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 ß-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.

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

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.

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

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 ß2 and ß3-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.

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

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.

ß2-Adrenoceptor-mediated regulation of glucose uptake in skeletal muscle--ligand-directed signalling or a reflection of system complexity?

The capacity of G protein-coupled receptors (GPCRs) to activate multiple G protein isoforms and additional effectors such as ß-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 ß2-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 ß2-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.

Interaction with caveolin-1 modulates G protein coupling of mouse ß3-adrenoceptor.

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 ß(3)-adrenoceptor (ß(3)-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the ß(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 ß(3a)- and ß(3b)-AR isoforms that diverge at the distal C terminus. Only the ß(3b)-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the ß(3a)-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the ß(3)-AR agonist CL316243 became PTX-sensitive, suggesting Gα(i/o) coupling. The ß(3a)-AR C terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant ß(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 ß(3a)-AR but not the mutant receptors. In membrane preparations, the ß(3b)-AR activated Gα(o) and mediated PTX-sensitive cAMP responses, whereas the ß(3a)-AR did not activate Gα(i/o) proteins. The endogenous ß(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 ß(3a)-AR with caveolin inhibits coupling to Gα(i/o) proteins and suggest that signaling is modulated by a raft-enriched complex containing the ß(3a)-AR, caveolin-1, Gα(s), and adenylyl cyclase.

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

BACKGROUND AND PURPOSE: ß-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 ß(2)-adrenoceptor and glucose transporter 4 (GLUT4) to investigate the signalling mechanisms involved. KEY RESULTS: In CHO-K1 cells, there was no response to ß-adrenoceptor agonists. The introduction of ß(2)-adrenoceptors and GLUT4 into these cells caused increased glucose uptake in response to ß-adrenoceptor agonists. GLUT4 translocation occurred in response to insulin and ß(2)-adrenoceptor stimulation, although the key insulin signalling intermediate PKB was not phosphorylated in response to ß(2)-adrenoceptor stimulation. Truncation of the C-terminus of the ß(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 ß(2)-adrenoceptor-stimulated glucose uptake. Furthermore, inhibition of GRK by transfection of the ßARKct construct inhibited ß(2)-adrenoceptor-mediated glucose uptake and GLUT4 translocation, and overexpression of a kinase-dead GRK2 mutant (GRK2 K220R) also inhibited GLUT4 translocation. Introducing ß(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 ß(2)-adrenoceptors involves translocation of GLUT4 in this model system. The mechanism is dependent on the C-terminus of the ß(2)-adrenoceptor, requires GRK phosphorylation sites, and involves a signalling pathway distinct from that stimulated by insulin.