HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Soeder, K. J. Articles by Collins, S. Search for Related Content PubMed PubMed Citation Articles by Soeder, K. J. Articles by Collins, S. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 17, 12017-12022, April 23, 1999

The ₃-Adrenergic Receptor Activates Mitogen-activated Protein Kinase in Adipocytes through a G_i-dependent Mechanism^*

Kurt J. Soeder, Sheridan K. Snedden, Wenhong Cao^§, Gregory J. Della Rocca^¶, Kiefer W. Daniel^§, Louis M. Luttrell, and Sheila Collins^§**

From the Departments of ^§ Psychiatry and Behavioral Sciences,  Pharmacology, ^¶ Biochemistry, and  Medicine, Duke University Medical Center, Durham, North Carolina 27710

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Promiscuous coupling between G protein-coupled receptors and multiple species of heterotrimeric G proteins provides a potential mechanism for expanding the diversity of G protein-coupled receptor signaling. We have examined the mechanism and functional consequences of dual G_s/G_i protein coupling of the ₃-adrenergic receptor (₃AR) in 3T3-F442A adipocytes. The ₃AR selective agonist disodium (R,R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL316,243) stimulated a dose-dependent increase in cAMP production in adipocyte plasma membrane preparations, and pretreatment of cells with pertussis toxin resulted in a further 2-fold increase in cAMP production by CL316,243. CL316,243 (5 µM) stimulated the incorporation of 8-azido-[³²P]GTP into G_s (1.57 ± 0.12; n = 3) and G_i (1.68 ± 0.13; n = 4) in adipocyte plasma membranes, directly demonstrating that ₃AR stimulation results in G_i-GTP exchange. The ₃AR-stimulated increase in 8-azido-[³²P]GTP labeling of G_i was equivalent to that obtained with the A₁-adenosine receptor agonist N⁶-cyclopentyladenosine (1.56 ± 0.07; n = 4), whereas inclusion of unlabeled GTP (100 µM) eliminated all binding. Stimulation of the ₃AR in 3T3-F442A adipocytes led to a 2-3-fold activation of mitogen-activated protein (MAP) kinase, as measured by extracellular signal-regulated kinase-1 and -2 (ERK1/2) phosphorylation. Pretreatment of cells with pertussis toxin (PTX) eliminated MAP kinase activation by ₃AR, demonstrating that this response required receptor coupling to G_i. Expression of the human ₃AR in HEK-293 cells reconstituted the PTX-sensitive stimulation of MAP kinase, demonstrating that this phenomenon is not exclusive to adipocytes or to the rodent ₃AR. ERK1/2 activation by the ₃AR was insensitive to the cAMP-dependent protein kinase inhibitor H-89 but was abolished by genistein and AG1478. These data indicate that constitutive ₃AR coupling to G_i proteins serves both to restrain G_s-mediated activation of adenylyl cyclase and to initiate additional signal transduction pathways, including the ERK1/2 MAP kinase cascade.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Long before the discovery of the ₃AR and its recognition as a unique, adipocyte-specific receptor controlling lipolysis and thermogenesis, Rodbell and colleagues (1) made the observation that there was an unusual, biphasic stimulation of cAMP production in adipocytes in response to the -adrenergic receptor agonist isoproterenol. Depending upon the concentration of GTP in the assay, isoproterenol could either stimulate or inhibit adenylyl cyclase activity in adipocyte plasma membranes. Murayama and Ui (2) showed that this inhibitory phase could be relieved by pretreatment of adipocytes with pertussis toxin (PTX).¹ This curious observation lay fallow until studied later in greater detail by Bégin-Heick (3-5). However, it was not until the cloning and characterization of the ₃AR gene and the development of selective ₃AR agonists (6, 7) that it was postulated that this novel adipocyte-specific AR may be responsible for the biphasic adenylyl cyclase response in adipocytes (8). We have previously noted that despite the relatively high level of expression of the ₃AR in adipocytes, the efficiency of coupling of the ₃AR to stimulation of adenylyl cyclase is low (9). However, there has been no clear biochemical demonstration of physical coupling of the ₃AR to G_i, other than comparative functional experiments in the presence or absence of PTX (10), nor has there been any indication of what additional second messenger pathway may be activated as a consequence of this putative coupling of ₃AR to G_i.

Recently, many G protein-coupled receptors have been shown to mediate cellular growth or differentiation responses through the activation of MAP kinase cascades (11). Receptors signaling via PTX-sensitive G_i/o proteins, as well as PTX-insensitive G_q/11 proteins may activate the ERK1/2 MAP kinase cascade through a mechanism involving tyrosine protein phosphorylation and the activation of the low molecular weight G protein p21^ras (12-14). Little is known about the potential role of ARs in the regulation of the MAP kinase pathway. Recently, we have found that in fibroblasts the ₂AR mediates Ras-dependent ERK1/2 activation through its ability to couple to a PTX-sensitive G_i protein (15). ₂AR coupling to G_i occurs as a result of PKA-dependent phosphorylation of the receptor, which effectively "switches" receptor coupling from G_s to G_i proteins. In contrast, ₂AR-mediated ERK1/2 activation in S49 lymphoma cells is an entirely G_s-dependent process (16). In this system, PKA-mediated phosphorylation of the low molecular weight GTPase, Rap1, promotes Ras-independent ERK1/2 activation; this process was shown to be independent of ₂AR interaction with G_i/o proteins. Therefore, it is not yet clear whether there is a common mechanism by which ARs activate MAP kinase.

Here, we demonstrate that stimulation of the ₃AR in adipocytes induces the direct activation of both G_s and G_i. In these cells, G_i activation results in both the attenuation of ₃AR-mediated stimulation of adenylyl cyclase and the activation of the ERK1/2 MAP kinase pathway. Unlike the ₂AR signal in fibroblasts, ₃AR activation of the ERK1/2 pathway is independent of cAMP and PKA. These data suggest that the promiscuous coupling of the ₃AR in adipocytes permits the simultaneous transduction of two independent signaling pathways. This property of the ₃AR may be responsible, in part, for the unique physiological effects of selective ₃AR agonists in vivo, such as their potency for stimulating lipolysis (17, 18) and their ability to prevent or reverse obesity (6, 19-22).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Chemicals-- CL316,243 was a gift from American Cyanamid Co. (Pearl River, NY). CGP20712A was a gift from CibaGeigy. Forskolin, bovine serum albumin (fraction V), insulin, (S)-()-propranolol, N⁶-2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, and salbutamol were purchased from Sigma. EGF was obtained from Upstate Biotechnology (Lake Placid, NY). 8-Azido-[³²P]GTP was purchased from Andotek Life Sciences (Irvine, CA). Pertussis toxin and genistein were purchased from Calbiochem. N⁶-Cyclopentyladenosine was obtained from RBI (Natick, MA). ICI 118551 was purchased from Cambridge Research Biochemicals (Wilmington, DE). Precast polyacrylamide gels were obtained from Novex (San Diego, CA).

Cell Culture and Transfections-- 3T3-F442A preadipocytes were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Inc.) at 37 °C in a humidified 5% CO₂ atmosphere. Upon reaching 90% confluence, the cells were stimulated to differentiate into adipocytes by culturing in a differentiation media composed of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5 µg/ml insulin (23). HEK-293 cells were grown in six-well dishes and transfected with 1.2 µg of pBC3 or pBC2 DNA (24) by calcium phosphate co-precipitation (25). Ten h later, cells were washed in 1 mM EGTA in phosphate-buffered saline and incubated in growth medium for 24 h. The cells were then serum-starved for 24 h and treated with PTX (100 ng/ml), propranolol (10 µM), SR59230A (25 µM), CL316,243 (10 µM), or isoproterenol (100 µM), and MAP kinase assays were performed as described below. Cells that were pretreated with PTX were incubated with the toxin for 16-20 h at a concentration of 100 ng/ml.

Adenylyl Cyclase Assays-- Plasma membranes were prepared from 3T3-F442A adipocytes or Sprague-Dawley epididymal white adipose tissue as follows. For cultured cells, the monolayers were gently rinsed with ice-cold phosphate-buffered saline. All subsequent steps were performed at 4 °C. Lysis buffer (5 mM Tris/2 mM EDTA, pH 7.2, containing 10 µg/ml soybean trypsin inhibitor, 10 µg/ml benzamidine, 2 µg/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride) was added, and the cells were released from the dish with a rubber policeman. Following gentle disruption with a glass/Teflon homogenizer, nuclei were removed by centrifuging for 5 min at 800 × g. The supernatant was collected and centrifuged at 40,000 × g for 20 min. The pellet was resuspended in fresh lysis buffer with a Teflon pestle, centrifuged again, and then resuspended in a small volume of lysis buffer. For rat adipocytes obtained from epididymal adipose tissue, the same procedure was followed except that first the tissue was finely minced, and adipocytes were isolated by incubating the tissue pieces with collagenase (Worthington) (26). Adenylyl cyclase activity in plasma membranes was measured as described previously (9). Control incubations in the absence or presence of isobutylmethylxanthine (0.25 mM) found no evidence of particulate cyclic nucleotide phosphodiesterase activity in these plasma membranes. In some experiments, cAMP production was measured in intact 3T3-F442A adipocytes. For these whole cell assays, growth media was replaced with serum-free medium 3 h prior to stimulation. The serum-free medium consisted of Dulbecco's modified Eagle's medium supplemented with 1 g/liter fraction V bovine serum albumin, 10 mM HEPES, pH 7.4, 100 units/ml penicillin, and 100 µg/ml streptomycin. Then cells were incubated with the indicated concentrations of -agonist in fresh serum-free medium for 10 min at 37 °C in the presence of 0.25 mM isobutylmethylxanthine. Reactions were stopped by the addition of ice-cold 5% trichloroacetic acid, and particulate material was removed by centrifugation. The cAMP concentrations from both assay methods were determined by radioimmunoassay using a polyclonal antiserum to iodinated cAMP (27). Protein concentrations were determined by the method of Bradford (28).

Western Blotting and Photolabeling of Heterotrimeric G Proteins-- Western blotting was performed with antibodies specific for individual G subunits. Adipocyte membranes (30 µg of protein) were solubilized in SDS-polyacrylamide gel electrophoresis sample buffer and resolved on 4-20% Novex gradient gels (San Diego, CA). A recombinant G protein standard mix containing G_i1, G_i2, G_i3, and G_o was included as a positive control (gift of Dr. Pat Casey). The proteins were then transferred to nitrocellulose membranes. The membranes were incubated for 16 h at 4 °C with rabbit anti-G_i1+2, anti-G_i3, or anti-G_s, 1:1000 dilution, followed by alkaline phosphatase-conjugated goat anti-rabbit antisera (1:10,000 dilution, Amersham Pharmacia Biotech) as secondary antibody. Immunoreactive bands were visualized by Storm PhosphorImager (Molecular Dynamics).

Synthesis and purification of the 8-azido-[³²P]GTP was performed as described (29, 30) with minor modifications (31). For some experiments, 8-azido-[³²P]GTP was purchased from Andotek Life Sciences. The photoaffinity labeling procedure of Offermanns (32) was followed with modifications (31). For labeling of the G_i subunits, adipocyte membranes (50 µg of protein) were incubated with 8-azido-[³²P]GTP (0.5 µCi) for 10 min at 30 °C in a volume of 60 µl containing 50 mM HEPES (pH 7.6), 1 mM EDTA, 50 mM NaCl, 500 µM MgCl₂, 50 µM GDP, 100 µM ATP, and 1 µg/ml adenosine deaminase in the presence or absence of the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine or the ₃AR-selective agonist CL316,243. Labeling of the G_s subunit was performed as above at a free Mg²⁺ concentration of 2-5 mM. The binding reactions were stopped by placing tubes on ice, and all subsequent steps were performed at 4 °C. The membranes were collected by centrifugation for 10 min at 14,000 × g and resuspended in a buffer containing 50 mM HEPES (pH 7.6), 1 mM EDTA, 50 mM NaCl, 500 µM MgCl₂, and 2 mM dithiothreitol. Following irradiation of the samples with a UV lamp (4 watts, 254 nm) at a distance of 3 cm for 5 min, the samples were collected by centrifugation as before and solubilized in Laemmli sample buffer. Proteins labeled with 8-azido-[³²P]GTP were resolved by SDS-polyacrylamide gel electrophoresis using a 10% polyacrylamide gel and visualized by a Storm PhosphorImager (Molecular Dynamics).

MAP Kinase Assays-- Activation of MAP kinase was determined by measuring the phosphorylation state of ERK 1 and ERK 2 (33). For this assay, 3T3-F442A cells were grown and differentiated in six-well culture plates and serum-starved for 24 h prior to stimulation with -adrenergic agonists or growth factors. The serum-free medium consisted of Dulbecco's modified Eagle's medium supplemented with 1 g/liter fraction V bovine serum albumin, 10 mM HEPES, pH 7.4, 100 units/ml penicillin, and 100 µg/ml streptomycin. Five min after the addition of -agonists or growth factors, the medium was removed, and the cells were solubilized by the direct addition of Laemmli sample buffer (34). These cell lysates were sonicated for 5 s, and aliquots (30 µg protein/lane) were resolved by SDS-polyacrylamide gel electrophoresis. ERK1/2 phosphorylation was detected by protein immunoblotting using a 1:3000 dilution of rabbit polyclonal phospho-MAP kinase-specific antisera (NEBiolabs) with a 1:10,000 dilution of alkaline phosphatase-conjugated goat anti-rabbit antisera (Amersham Pharmacia Biotech) as secondary antibody. Quantitation of ERK1/2 phosphorylation was performed using a Storm PhosphorImager (Molecular Dynamics). After quantitation of ERK1/2 phosphorylation, nitrocellulose membranes were stripped of immunoglobulin and reprobed using rabbit polyclonal anti-ERK 2 IgG (Upstate Biotechnology, Inc.) to confirm equal amounts of ERK 2 protein.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Stimulation of 3T3-F442A adipocyte plasma membranes with the selective ₃AR agonist CL316,243 resulted in a 3-4-fold increase in adenylyl cyclase activity Fig. 1A). Membranes prepared from cells pretreated with PTX exhibited a more robust increase in cAMP production, which was at least 2-fold above that observed in membranes from naive cells. Whereas basal levels of cAMP generation were elevated following PTX treatment, the effect of PTX on the -fold activation of adenylyl cyclase by forskolin was minimal (Fig. 1B). We obtained similar results from Chinese hamster ovary cells, which heterologously express the mouse ₃AR (data not shown) (35). These results suggest that the ₃AR is constitutively coupled to both G_s and G_i, because inhibition of G_i function leads to increased tonic production of cAMP.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   3AR agonist stimulation of cAMP production in 3T3-F442A adipocytes. A, dose-response curve for agonist stimulation of cAMP production in response to CL316,243. Membranes of untreated or PTX-pretreated adipocytes were prepared and incubated with increasing agonist concentrations. Cyclic AMP production was measured by radioimmunoassay using polyclonal antisera to iodinated cAMP. The data shown are representative of two dose-response experiments. B, basal (Bas) and forskolin (FSK)-stimulated cAMP production in untreated and PTX-pretreated membranes. The data shown are the average of four experiments.

Rat white adipocytes have been shown to contain the two splice variants of G_s and the three isoforms of G_i (_i1, _i2, _i3) (5, 36). As shown in Fig. 2, 3T3-F442A adipocytes express the same repertoire of G proteins as found in rat white adipocytes: two splice variants of G_s, G_i3, and G_i1/G_i2. Others have previously shown that adipocytes do not contain the PTX-sensitive G_o protein (36-39). To determine whether the PTX effects on cAMP production resulted from constitutive coupling of the ₃AR to both G_s and G_i, the photolabile GTP analog 8-azido-[³²P]GTP was used to measure ₃AR agonist-dependent GTP loading of G_s and G_i. This method has been used by several investigators to demonstrate physical coupling between G protein-coupled receptors and individual G protein  subunits, based upon several well defined criteria for receptor-G protein interaction (40-42). Because the binding of GTP analogs to G subunits is highly dependent upon free magnesium concentrations (31), we conducted our experiments in adipocyte plasma membranes under separate conditions favorable for 8-azido-[³²P]GTP labeling of each G family. Because the ₃AR is coupled to G_s and activation of adenylyl cyclase, Fig. 3A shows that under high magnesium conditions, which favor receptor-stimulated GTP loading of G_s, CL316,243 could stimulate specific 8-azido-[³²P]GTP labeling of G_s (1.57 ± 0.12; n = 3; p < 0.001). Under low magnesium conditions, which are optimal for determining receptor-stimulated GTP loading to G_i, there was a similar 1.68 ± 0.13-fold increase (n = 4; p < 0.001) in specific 8-azido-[³²P]GT G protein labeling of the 40-42-kDa G_i species (Fig. 3B). We obtained similar data from 3T3-F442A membranes (not shown). Fig. 3B also shows that the ability of CL316,243 to stimulate G_i-GTP exchange in adipocyte membranes was equivalent to that obtained with the A₁-adenosine receptor agonist N⁶-cyclopentyladenosine (1.56 ± 0.07; n = 4; p < 0.001). N⁶-Cyclopentyladenosine served as a positive control because the A₁-adenosine receptor is expressed in both primary adipocytes (43, 44) and differentiated 3T3-F442A adipocytes (45) and couples to all three G_i species (46, 47).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   G protein  subunit determination. Plasma membranes from 3T3-F442A adipocytes and rat epididymal white adipose tissue (EWAT) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The complement of G protein  subunits present was assessed by Western blotting as described under "Materials and Methods." Std, G protein standard.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Labeling of G subunits by 8-azido-[32P]GTP in adipocyte membranes in response to 3AR stimulation. A, 8-azido-[32P]GTP labeling of Gs in response to no stimulation (NS) or labeling with CL316,243 (CL) (5 µM) in rat white adipocyte membranes. B, 8-azido-[32P]GTP labeling of the 40-42-kDa Gi/o species in response to no stimulation, N6-cyclopentyladenosine (CPA) (50 nM), and CL316,243 (5 µM) in rat white adipocyte membranes. The effect of excess GTP is also shown. Data shown are the average of three experiments (for Gs) or four experiments (for Gi) and are expressed as -fold change over nonstimulated control. *, significantly different from unstimulated control, p < 0.001.

A number of G_i-coupled receptors have been shown capable of activating MAP kinase (11). Recently, Daaka and colleagues (15) reported that the ₂AR could couple to both G_s and G_i, with consequent activation of MAP kinase. We therefore determined whether one of the functional consequences of the coupling of the ₃AR to G_i in 3T3-F442A adipocytes was the stimulation of MAP kinase. Intact 3T3-F442A adipocytes treated with CL316,243 (5 µM) exhibited approximately a 2.5-fold stimulation of ERK1/2 phosphorylation (Fig. 4). Activation of the endogenous ₂AR in 3T3-F442A adipocytes with the ₂AR-selective agonist salbutamol also yielded a similar 2-fold stimulation of ERK1/2 phosphorylation. Both AR responses were less robust than that observed following activation of endogenous receptors for EGF, consistent with previous findings (13). Also shown in Fig. 4, pretreatment of 3T3-F442A adipocytes with PTX completely blocked ERK1/2 phosphorylation in response to either ₂AR or ₃AR stimulation, but it did not affect stimulation by EGF. The addition of propranolol (0.1 µM) eliminated ERK1/2 phosphorylation in response to salbutamol but had no effect on the activation of MAP kinase induced by CL316,243 (not shown), consistent with a CL-mediated effect exclusively through the ₃AR (7).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of -agonists and PTX on ERK1/2 phosphorylation in 3T3-F442A adipocytes. Quantitation of ERK1/2 phosphorylation subsequent to agonist stimulation was determined by Western blotting with phosphospecific anti-ERK1/2 rabbit antisera. Quantitation was performed using a Storm PhosphorImager (Molecular Dynamics). Data shown are the average of four experiments and are expressed as the -fold change over nonstimulated control for salbutamol (1 µM), CL316,243 (5 µM), and EGF (10 ng/ml). Pretreatment with PTX was as detailed under "Materials and Methods." *, significantly different from unstimulated control by one-way analysis of variance, p < 0.001.

The relative sensitivity of the ₃AR to activate MAP kinase versus cAMP accumulation in adipocytes was assessed by treating intact cells with increasing concentrations of CL316,243 (Fig. 5). Because the MAP kinase assays involve the stimulation of intact cells, we included cAMP dose-response data also from intact cells. The EC₅₀ for cAMP production in these experiments was 12 nM, whereas parallel measurements of MAP kinase activation (EC₅₀ = 280 nM) indicated that the PTX-sensitive coupling of the ₃AR to MAP kinase activation is less potent under these conditions. Nevertheless, the dose-response curves for both measurements yielded a unit slope, indicative of the high selectivity of CL316,243 for the ₃AR (7, 48). Note that the EC₅₀ for cAMP production in these whole cell experiments is significantly less than that found in plasma membranes (Fig. 1). However, it is generally recognized that dose-response curves as well as ligand binding data from intact cells are shifted "to the left," when compared with data obtained using isolated membrane preparations (49, 50). These comparative studies shown in Fig. 5 suggest that cAMP production is the favored pathway in response to activation of the ₃AR in adipocytes. It must be remembered, however, that the catalytic activity of the subsequent kinases and their juxtaposition to substrate targets will ultimately determine the relative importance of these two pathways. Future studies will address these issues.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Comparative dose-dependent stimulation of cAMP production () and MAP kinase activation () in 3T3-F442A adipocytes. Differentiated 3T3-F442A adipocytes were incubated with increasing concentrations of CL316,243 and processed for measurement of cAMP or ERK1/2 phosphorylation as detailed under "Materials and Methods." The data were analyzed by nonlinear curve-fitting routines (GraphPad Prism) (9). The results shown are from two independent experiments.

To determine whether the dual G_s/G_i coupling of the ₃AR and activation of MAP kinase was an adipocyte-specific event or a unique feature of the rodent ₃AR, we attempted to reconstruct these effects by transiently transfecting the human ₃AR into HEK-293 cells. As shown in Fig. 6A, transient transfection of the h₃AR resulted in a low basal level of ERK1/2 phosphorylation (lane 2), and stimulation with the ₃AR agonist increased ERK1/2 phosphorylation ~4-fold above basal level (lane 3), as compared with the 7-8-fold activation achieved by EGF (lane 7). There was no effect of the ₃AR agonist on mock-transfected cells (lane 1). This activation of MAP kinase by the h₃AR was completely blocked by the selective ₃AR antagonist, SR59230A (lane 4) (51). In addition, the nonselective AR agonist isoproterenol at a concentration capable of activating the ₃AR (100 µM), in the presence of propranolol, also led to an ~4-fold stimulation of ERK1/2 phosphorylation (lane 5). Finally, PTX completely eliminated MAP kinase activation (lane 6), as observed in 3T3-F442A adipocytes (Fig. 4). Together these data suggest that the dual coupling of the ₃AR to G_s/G_i, along with its activation of MAP kinase, is a general property of this receptor.

View larger version (41K): [in this window] [in a new window]   Fig. 6.   HEK-293 cells transiently expressing the human 3AR activate MAP kinase. A, HEK-293 cells were transiently transfected with the h3AR as described under "Materials and Methods." Cells were treated as shown, and ERK1/2 phosphorylation was determined. B, HEK-293 cells transiently expressing human 3AR or human 2AR were pretreated with H-89 (20 µM), genistein (5 µM), or AG1478 (3 µM) for 40 min before agonist stimulation. The results shown represent one of three experiments. CL, CL316,243; ISO, isoproterenol.

Mechanistically, ₂AR-mediated MAP kinase activation in fibroblasts requires sequential receptor coupling to G_s and G_i, because cAMP-dependent, PKA-mediated phosphorylation of the ₂AR is a prerequisite for receptor-G_i coupling (15). To compare the role of cAMP and PKA in AR-mediated MAP kinase activation in adipocytes, we compared the effects of the PKA inhibitor H-89 on ₂AR- and ₃AR-stimulated MAP kinase activation. As shown in Fig. 6B, H-89 blocked ₂AR-stimulated MAP kinase activation (lane 8), consistent with the results of Daaka et al. (15). In contrast, the ₃AR signal was insensitive to H-89 (lane 3). Fig. 6B also shows that MAP kinase activation by the ₃AR was sensitive to low micromolar concentrations of the tyrosine kinase inhibitor genistein (lane 4) and to the EGR receptor tyrphostin AG1478 (lane 5). These results indicate that, like other G_i-coupled receptors, the ₃AR employs a tyrosine kinase receptor in its mechanism of recruiting MAP kinase (52, 53). In other experiments, treatment with dibutyryl cAMP enhanced ₂AR-stimulated ERK1/2 phosphorylation by ~70%, consistent with the model of PKA-dependent ₂AR-G_i coupling. However, stimulation of MAP kinase by the ₃AR was unaffected by dibutyryl cAMP (data not shown). Together, these results indicate that MAP kinase activation via the ₃AR, unlike the ₂AR, is insensitive to modulation of the PKA pathway. Thus ₃AR-mediated MAP kinase activation can be distinguished in several ways from the ₂AR pathway. First, the ₃AR is not a substrate for PKA (24). Second, the ₃AR appears to be constitutively coupled to both G_s and G_i. Third, ₃AR-stimulated activation of cAMP is not required for activation of the MAP kinase cascade in adipocytes. In contrast to the finding of PKA-dependent ₂AR-mediated MAP kinase activation in S49 lymphoma cells (16), the ₃AR pathway in adipocytes is completely PTX-sensitive.

One of the remarkable features of the ₃AR is that treatment with ₃AR-selective agonists in vivo can prevent or reverse obesity due to either congenital or diet-induced etiology (6, 19-22, 54). These agents are also efficacious over prolonged periods of administration (21), which is quite distinct from the rapid desensitization and down-regulation that is characteristic of chronic treatment with ₁- and, particularly, ₂AR-selective drugs (55, 56). These unique properties of the ₃AR have been suggested to be related to the fact that ₃AR is resistant to desensitization (57), because it is not a substrate for either PKA or the G protein-coupled receptor kinases (24). Instead, our finding that the ₃AR is capable of activating both the PKA pathway and the MAP kinase cascade raises another possibility. The simultaneous recruitment of both signaling networks may result in a more potent stimulation of lipolysis and/or may promote the growth and differentiation of brown adipocytes, which is observed in all ₃AR agonist-treated animals (20, 21, 58). It will now be important for us to test the consequence of impairing one or the other of these signal transduction pathways to determine their contribution to these ₃AR-mediated responses.

	ACKNOWLEDGEMENTS

We thank Drs. Patrick Casey and Timothy Fields for advice and discussions on the synthesis and use of 8-azido-[³²P]GTP and the gift of recombinant G protein standard mix. We thank Dr. Suzie Mumby for the antisera to G_i3, Dr. Tom Gettys for the cAMP antisera, Dr. Neil Freedman for the gift of pBC₃ DNA, and Dr. Luciano Manara of SANOFI MIDI Research for the gift of SR59230A. We also acknowledge Dr. Robert J. Lefkowitz for helpful discussions during the development of this project.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants DK46793 and DK53092 (to S. C.) and DK02352 (to L. M. L.), National Institutes of Health Training Program Grants T32ES0731 (to K. J. S.) and T32GM07105 (to S. K. S.), and National Institutes of Health Medical Scientist Training Program Grant T32GM07171 (to G. J. D. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Duke University Medical Center, P. O. Box 3557, Durham, NC 27710. Tel.: 919-684-8991; Fax: 919-684-3071; E-mail: colli008{at}mc.duke.edu.

	ABBREVIATIONS

The abbreviations used are: PTX, pertussis toxin; AR, -adrenergic receptor; PKA, cAMP-dependent protein kinase; EGF, epidermal growth factor; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; CL316, 243, disodium (R,R)-5-[2[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate; SR59230A, 3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronapth-1-ylamino]-(2S)-2-propanol oxalate; AG1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; CGP20712A, 1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-O-methyl-4-trifluoromethyl-2-imidazoyl)phenoxy]-2-propanol methane sulfonate.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

1. Cooper, D., Schlegel, W., Lin, M., and Rodbell, M. (1979) J. Biol. Chem. 254, 8927-8931[Abstract/Free Full Text]
2. Murayama, T., and Ui, M. (1983) J. Biol. Chem. 258, 3319-3326[Abstract/Free Full Text]
3. Bégin-Heick, N. (1985) J. Biol. Chem. 260, 6187-6193[Abstract/Free Full Text]
4. Begin-Heick, N. (1987) 53, 1-8
5. Begin-Heick, N. (1990) Biochem. J. 268, 83-89[Medline] [Order article via Infotrieve]
6. Arch, J. R. S., Ainsworth, A. T., Cawthorne, M. A., Piercy, V., Sennitt, M. V., Thody, V. E., Wilson, C., and Wilson, S. (1984) Nature 309, 163-165[CrossRef][Medline] [Order article via Infotrieve]
7. Bloom, J. D., Dutia, M. D., Johnson, B. D., Wissner, A., Burns, M. G., Largis, E. E., Dolan, J. A., and Claus, T. H. (1992) J. Med. Chem. 35, 3081-3084[CrossRef][Medline] [Order article via Infotrieve]
8. Begin-Heick, N. (1995) J. Cell. Biochem. 58, 464-473[CrossRef][Medline] [Order article via Infotrieve]
9. Collins, S., Daniel, K. W., Rohlfs, E. M., Ramkumar, V., Taylor, I. L., and Gettys, T. W. (1994) Mol. Endocrinol. 8, 518-527[Abstract/Free Full Text]
10. Chaudhry, A., MacKenzie, R. G., Georgic, L. M., and Granneman, J. G. (1994) Cell. Signal. 6, 457-465[CrossRef][Medline] [Order article via Infotrieve]
11. Van Biesen, T., Luttrell, L. M., Hawes, B. E., and Lefkowitz, R. J. (1996) Endocr. Rev. 17, 698-714[Abstract/Free Full Text]
12. Koch, W., Hawes, B., Allen, L., and Lefkowitz, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12706-12710[Abstract/Free Full Text]
13. van Biesen, T., Hawes, B. E., Luttrell, D. K., Kreuger, K. M., Touhara, K., Porfiri, E., Sakaue, M., Luttrell, L. M., and Lefkowitz, R. J. (1995) Nature 376, 781-784[CrossRef][Medline] [Order article via Infotrieve]
14. Crespo, P., Xu, N., Simonds, W. F., and Gutkind, J. S. (1994) Nature 369, 418-420[CrossRef][Medline] [Order article via Infotrieve]
15. Daaka, Y., Luttrell, L. M., and Lefkowitz, R. J. (1997) Nature 390, 88-91[CrossRef][Medline] [Order article via Infotrieve]
16. Wan, Y., and Huang, X.-Y. (1998) J. Biol. Chem. 273, 14533-14537[Abstract/Free Full Text]
17. Hollenga, C., Brouwer, F., and Zaagsma, J. (1991) Eur. J. Pharmacol. 200, 325-330[CrossRef][Medline] [Order article via Infotrieve]
18. Hollenga, C., Brouwer, F., and Zaagsma, J. (1991) Br. J. Pharmacol. 102, 577-580[Medline] [Order article via Infotrieve]
19. Largis, E. E., Burns, M. G., Muenkel, H. A., Dolan, J. A., and Claus, T. H. (1994) Drug Dev. Res. 32, 69-76
20. Himms-Hagen, J., Cui, J., Danforth, E., Jr., Taatjes, D. J., Lang, S. S., Waters, B. L., and Claus, T. H. (1994) Am. J. Physiol. 266, R1371-R1382[Abstract/Free Full Text]
21. Collins, S., Daniel, K. W., Petro, A. E., and Surwit, R. S. (1997) Endocrinology 138, 405-413[Abstract/Free Full Text]
22. Sasaki, N., Uchida, E., Niiyama, M., Yoshida, T., and Saito, M. (1998) J. Vet. Med. Sci. 60, 465-469[CrossRef][Medline] [Order article via Infotrieve]
23. Djian, P., Phillips, M., and Green, H. (1985) J. Cell. Physiol. 124, 554-556[CrossRef][Medline] [Order article via Infotrieve]
24. Liggett, S., Freedman, N. J., Schwinn, D. A., and Lefkowitz, R. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 3665-3669[Abstract/Free Full Text]
25. Oppermann, M., Freedman, N. J., Alexander, R. W., and Lefkowitz, R. J. (1996) J. Biol. Chem. 271, 13266-13272[Abstract/Free Full Text]
26. Rodbell, M. (1964) J. Biol. Chem. 239, 375-380[Free Full Text]
27. Harper, J. F., and Brooker, G. (1975) J. Cyclic Nucleotide Res. 1, 207-218[Medline] [Order article via Infotrieve]
28. Bradford, M. (1976) Anal. Biochem. 72, 248-254[CrossRef][Medline] [Order article via Infotrieve]
29. Pfeuffer, T. (1977) J. Biol. Chem. 252, 7224-7234[Free Full Text]
30. Schafer, R., Christian, A. L., and Schulz, I. (1988) Biochem. Biophys. Res. Commun. 155, 1051-1059[CrossRef][Medline] [Order article via Infotrieve]
31. Fields, T. A., Linder, M. E., and Casey, P. J. (1994) Biochemistry 33, 6877-6883[CrossRef][Medline] [Order article via Infotrieve]
32. Offermanns, S., Schultz, G., and Rosenthal, W. (1991) Methods Enzymol. 195, 286-300[Medline] [Order article via Infotrieve]
33. Della Rocca, G. J., van Biesen, T., Daaka, Y., Luttrell, D. K., Luttrell, L. M., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 19125-19132[Abstract/Free Full Text]
34. Laemmli, U. (1970) Nature 227, 680-685[CrossRef][Medline] [Order article via Infotrieve]
35. Nahmias, C., Blin, N., Elalouf, J.-M., Mattei, M. G., Strosberg, A. D., and Emorine, L. J. (1991) EMBO J. 10, 3721-3727[Medline] [Order article via Infotrieve]
36. Gettys, T. W., Ramkumar, V., Uhing, R. J., Seger, L., and Taylor, I. L. (1991) J. Biol. Chem. 266, 15949-15955[Abstract/Free Full Text]
37. Chang, K., Pugh, W., Blanchard, S., McDermed, J., and Tam, J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4929-4933[Abstract/Free Full Text]
38. Rouot, B., Carrette, J., Lafontan, M., Lan Tran, P., Fehrentz, J., Bockaert, J., and Toutant, M. (1989) Biochem. J. 260, 307-310[Medline] [Order article via Infotrieve]
39. Hinsch, K. D., Rosenthal, W., Spicher, K., Binder, T., Gausepohl, H., Frank, R., Schultz, G., and Joost, H. G. (1988) FEBS Lett. 238, 191-196[CrossRef][Medline] [Order article via Infotrieve]
40. Wange, R. L., Smrcka, A. V., Sternweis, P. C., and Exton, J. H. (1991) J. Biol. Chem. 266, 11409-11412[Abstract/Free Full Text]
41. Gettys, T. W., Fields, T. A., and Raymond, J. R. (1994) Biochemistry 33, 4283-4290[CrossRef][Medline] [Order article via Infotrieve]
42. Herrlich, A., Kühn, B., Grosse, R., Schmid, A., Schultz, G., and Gudermann, T. (1996) J. Biol. Chem. 271, 16764-16772[Abstract/Free Full Text]
43. Vannucci, S. J., Klim, M., Martin, L. F., and LaNoue, K. F. (1989) Am. J. Physiol. 257, E871-E878[Abstract/Free Full Text]
44. Larrouy, D., Galitzky, J., and Lafontan, M. (1991) Eur. J. Pharmacol. 206, 139-147[CrossRef][Medline] [Order article via Infotrieve]
45. Ravid, K., and Lowenstein, J. M. (1988) Biochem. J. 249, 377-381[Medline] [Order article via Infotrieve]
46. Jockers, R., Linder, M. E., Hoheneger, M., Nanoff, C., Bertin, B., Strosberg, A. D., Marullo, S., and Freissmuth, M. (1994) J. Biol. Chem. 269, 32077-32084[Abstract/Free Full Text]
47. Munshi, R., Pang, I.-H., Sternweis, P. C., and Linden, J. (1991) J. Biol. Chem. 266, 22285-22289[Abstract/Free Full Text]
48. Rohlfs, E. M., Daniel, K. W., Premont, R. T., Kozak, L. P., and Collins, S. (1995) J. Biol. Chem. 270, 10723-10732[Abstract/Free Full Text]
49. Pittman, R. N., and Molinoff, P. B. (1983) Mol. Pharmacol. 24, 398-408[Abstract]
50. Clark, R. B. (1986) Adv. Cyclic Nucleotide Protein Phosphorylation Res. 20, 151-209[Medline] [Order article via Infotrieve]
51. Manara, L., Badone, D., Baroni, M., Boccardi, G., Cecchi, R., Croci, T., Giudice, A., Guzzi, U., Landi, M., and Le Fur, G. (1996) Br. J. Pharmacol. 117, 435-442[Medline] [Order article via Infotrieve]
52. Gutkind, J. (1998) J. Biol. Chem. 273, 1839-1842[Free Full Text]
53. Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 18677-18680[Free Full Text]
54. Yoshida, T., Sakane, N., Wakabayashi, Y., Umekawa, T., and Kondo, M. (1994) Life Sci. 54, 491-498[CrossRef][Medline] [Order article via Infotrieve]
55. Hausdorff, W., Caron, M., and Lefkowitz, R. (1990) FASEB J. 4, 2881-2889[Abstract]
56. Premont, R., Inglese, J., and Lefkowitz, R. (1995) FASEB J. 9, 175-182[Abstract]
57. Nantel, F., Bonin, H., Emorine, L. J., Ziberfarb, V., Strosberg, A. D., Bouvier, M., and Marullo, S. (1993) Mol. Pharmacol. 43, 548-555[Abstract]
58. Champigny, O., Ricquier, D., Blondel, O., Mayers, R. M., Briscoe, M. G., and Holloway, B. R. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10774-10777[Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Sato, D. S. Hutchinson, B. A. Evans, and R. J. Summers Mol. Pharmacol., November 1, 2008; 74(5): 1417 - 1428. [Abstract] [Full Text] [PDF]

				M. Sato, T. Horinouchi, D. S. Hutchinson, B. A. Evans, and R. J. Summers 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., November 1, 2007; 72(5): 1359 - 1368. [Abstract] [Full Text] [PDF]

				N. Kumar, J. Robidoux, K. W. Daniel, G. Guzman, L. M. Floering, and S. Collins Requirement of Vimentin Filament Assembly for beta3-Adrenergic Receptor Activation of ERK MAP Kinase and Lipolysis J. Biol. Chem., March 23, 2007; 282(12): 9244 - 9250. [Abstract] [Full Text] [PDF]

				J. Robidoux, N. Kumar, K. W. Daniel, F. Moukdar, M. Cyr, A. V. Medvedev, and S. Collins Maximal beta3-Adrenergic Regulation of Lipolysis Involves Src and Epidermal Growth Factor Receptor-dependent ERK1/2 Activation J. Biol. Chem., December 8, 2006; 281(49): 37794 - 37802. [Abstract] [Full Text] [PDF]

				O. S. Dallner, E. Chernogubova, K. A. Brolinson, and T. Bengtsson {beta}3-Adrenergic Receptors Stimulate Glucose Uptake in Brown Adipocytes by Two Mechanisms Independently of Glucose Transporter 4 Translocation Endocrinology, December 1, 2006; 147(12): 5730 - 5739. [Abstract] [Full Text] [PDF]

				N. R. Lenard, V. Prpic, A. W. Adamson, R. C. Rogers, and T. W. Gettys Differential coupling of beta3A- and beta3B-adrenergic receptors to endogenous and chimeric G{alpha}s and G{alpha}i Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E704 - E715. [Abstract] [Full Text] [PDF]

				M. Mongillo, C. G. Tocchetti, A. Terrin, V. Lissandron, Y.-F. Cheung, W. R. Dostmann, T. Pozzan, D. A. Kass, N. Paolocci, M. D. Houslay, et al. Compartmentalized Phosphodiesterase-2 Activity Blunts {beta}-Adrenergic Cardiac Inotropy via an NO/cGMP-Dependent Pathway Circ. Res., February 3, 2006; 98(2): 226 - 234. [Abstract] [Full Text] [PDF]

				R. Germack and J. M. Dickenson Induction of {beta}3-Adrenergic Receptor Functional Expression following Chronic Stimulation with Noradrenaline in Neonatal Rat Cardiomyocytes J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 392 - 402. [Abstract] [Full Text] [PDF]

				Z.-S. Zhang, H.-J. Cheng, K. Onishi, N. Ohte, T. Wannenburg, and C.-P. Cheng Enhanced Inhibition of L-type Ca2+ Current by {beta}3-Adrenergic Stimulation in Failing Rat Heart J. Pharmacol. Exp. Ther., December 1, 2005; 315(3): 1203 - 1211. [Abstract] [Full Text] [PDF]

				M. Sato, D. S. Hutchinson, T. Bengtsson, A. Floren, U. Langel, T. Horinouchi, B. A. Evans, and R. J. Summers Functional Domains of the Mouse {beta}3-Adrenoceptor Associated with Differential G Protein Coupling J. Pharmacol. Exp. Ther., December 1, 2005; 315(3): 1354 - 1361. [Abstract] [Full Text] [PDF]

				D. C. Adler-Wailes, H. Liu, F. Ahmad, N. Feng, C. Londos, V. Manganiello, and J. A. Yanovski Effects of the Human Immunodeficiency Virus-Protease Inhibitor, Ritonavir, on Basal and Catecholamine-Stimulated Lipolysis J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3251 - 3261. [Abstract] [Full Text] [PDF]

				C.-C. Juan, C.-L. Chang, Y.-H. Lai, and L.-T. Ho Endothelin-1 induces lipolysis in 3T3-L1 adipocytes Am J Physiol Endocrinol Metab, June 1, 2005; 288(6): E1146 - E1152. [Abstract] [Full Text] [PDF]

				D. S. Hutchinson, M. Sato, B. A. Evans, A. Christopoulos, and R. J. Summers Evidence for Pleiotropic Signaling at the Mouse {beta}3-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., March 1, 2005; 312(3): 1064 - 1074. [Abstract] [Full Text] [PDF]

				A. Robay, G. Toumaniantz, V. Leblais, and C. Gauthier Transfected {beta}3- but Not {beta}2-Adrenergic Receptors Regulate Cystic Fibrosis Transmembrane Conductance Regulator Activity via a New Pathway Involving the Mitogen-Activated Protein Kinases Extracellular Signal-Regulated Kinases Mol. Pharmacol., March 1, 2005; 67(3): 648 - 654. [Abstract] [Full Text] [PDF]

				S. Collins, W. Cao, and J. Robidoux Learning New Tricks from Old Dogs: {beta}-Adrenergic Receptors Teach New Lessons on Firing Up Adipose Tissue Metabolism Mol. Endocrinol., September 1, 2004; 18(9): 2123 - 2131. [Abstract] [Full Text] [PDF]

				A. Breit, M. Lagace, and M. Bouvier Hetero-oligomerization between {beta}2- and {beta}3-Adrenergic Receptors Generates a {beta}-Adrenergic Signaling Unit with Distinct Functional Properties J. Biol. Chem., July 2, 2004; 279(27): 28756 - 28765. [Abstract] [Full Text] [PDF]

				R. R. Bowers, W. T. L. Festuccia, C. K. Song, H. Shi, R. H. Migliorini, and T. J. Bartness Sympathetic innervation of white adipose tissue and its regulation of fat cell number Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2004; 286(6): R1167 - R1175. [Abstract] [Full Text] [PDF]

				B. CANNON and J. NEDERGAARD Brown Adipose Tissue: Function and Physiological Significance Physiol Rev, January 1, 2004; 84(1): 277 - 359. [Abstract] [Full Text] [PDF]

				E. Chernogubova, B. Cannon, and T. Bengtsson Norepinephrine Increases Glucose Transport in Brown Adipocytes via {beta}3-Adrenoceptors through a cAMP, PKA, and PI3-Kinase-Dependent Pathway Stimulating Conventional and Novel PKCs Endocrinology, January 1, 2004; 145(1): 269 - 280. [Abstract] [Full Text] [PDF]

				L. Akesson, B. Ahren, V. C. Manganiello, L. S. Holst, G. Edgren, and E. Degerman Dual Effects of Pituitary Adenylate Cyclase-Activating Polypeptide and Isoproterenol on Lipid Metabolism and Signaling in Primary Rat Adipocytes Endocrinology, December 1, 2003; 144(12): 5293 - 5299. [Abstract] [Full Text] [PDF]

				A. A. Coulter, C. M. Bearden, X. Liu, R. A. Koza, and L. P. Kozak Dietary fat interacts with QTLs controlling induction of Pgc-1{alpha} and Ucp1 during conversion of white to brown fat Physiol Genomics, July 7, 2003; 14(2): 139 - 147. [Abstract] [Full Text] [PDF]

				N. R. Lenard, T. W. Gettys, and A. J. Dunn Activation of beta 2- and beta 3-Adrenergic Receptors Increases Brain Tryptophan J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 653 - 659. [Abstract] [Full Text] [PDF]

				A. V. Medvedev, J. Robidoux, X. Bai, W. Cao, L. M. Floering, K. W. Daniel, and S. Collins Regulation of the Uncoupling Protein-2 Gene in INS-1 beta -Cells by Oleic Acid J. Biol. Chem., November 1, 2002; 277(45): 42639 - 42644. [Abstract] [Full Text] [PDF]

				S. Collins, W. Cao, K. W. Daniel, T. M. Dixon, A. V. Medvedev, H. Onuma, and R. Surwit Adrenoceptors, Uncoupling Proteins, and Energy Expenditure Experimental Biology and Medicine, December 1, 2001; 226(11): 982 - 990. [Abstract] [Full Text] [PDF]

				T. A. Kohout, H. Takaoka, P. H. McDonald, S. J. Perry, L. Mao, R. J. Lefkowitz, and H. A. Rockman Augmentation of Cardiac Contractility Mediated by the Human {beta}3-Adrenergic Receptor Overexpressed in the Hearts of Transgenic Mice Circulation, November 13, 2001; 104(20): 2485 - 2491. [Abstract] [Full Text] [PDF]

				F. Zalatan, J. A. Krause, and D. E. Blask Inhibition of Isoproterenol-Induced Lipolysis in Rat Inguinal Adipocytes in Vitro by Physiological Melatonin via a Receptor-Mediated Mechanism Endocrinology, September 1, 2001; 142(9): 3783 - 3790. [Abstract] [Full Text] [PDF]

				J. Klein, M. Fasshauer, M. Benito, and C. R. Kahn Insulin and the {beta}3-Adrenoceptor Differentially Regulate Uncoupling Protein-1 Expression Mol. Endocrinol., June 1, 2000; 14(6): 764 - 773. [Abstract] [Full Text]

				J. M. Fredriksson, J. M. Lindquist, G. E. Bronnikov, and J. Nedergaard Norepinephrine Induces Vascular Endothelial Growth Factor Gene Expression in Brown Adipocytes through a beta -Adrenoreceptor/cAMP/Protein Kinase A Pathway Involving Src but Independently of Erk1/2 J. Biol. Chem., April 28, 2000; 275(18): 13802 - 13811. [Abstract] [Full Text] [PDF]

				S. J. Keely, S. O. Calandrella, and K. E. Barrett Carbachol-stimulated Transactivation of Epidermal Growth Factor Receptor and Mitogen-activated Protein Kinase in T84 Cells Is Mediated by Intracellular Ca2+, PYK-2, and p60src J. Biol. Chem., April 21, 2000; 275(17): 12619 - 12625. [Abstract] [Full Text] [PDF]

				S. Maudsley, K. L. Pierce, A. M. Zamah, W. E. Miller, S. Ahn, Y. Daaka, R. J. Lefkowitz, and L. M. Luttrell The beta 2-Adrenergic Receptor Mediates Extracellular Signal-regulated Kinase Activation via Assembly of a Multi-receptor Complex with the Epidermal Growth Factor Receptor J. Biol. Chem., March 24, 2000; 275(13): 9572 - 9580. [Abstract] [Full Text] [PDF]

				K. L. Pierce, S. Maudsley, Y. Daaka, L. M. Luttrell, and R. J. Lefkowitz Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors PNAS, February 15, 2000; 97(4): 1489 - 1494. [Abstract] [Full Text] [PDF]

				W. Cao, L. M. Luttrell, A. V. Medvedev, K. L. Pierce, K. W. Daniel, T. M. Dixon, R. J. Lefkowitz, and S. Collins Direct Binding of Activated c-Src to the beta 3-Adrenergic Receptor Is Required for MAP Kinase Activation J. Biol. Chem., December 1, 2000; 275(49): 38131 - 38134. [Abstract] [Full Text] [PDF]

				W. Cao, A. V. Medvedev, K. W. Daniel, and S. Collins beta -Adrenergic Activation of p38 MAP Kinase in Adipocytes. cAMP INDUCTION OF THE UNCOUPLING PROTEIN 1 (UCP1) GENE REQUIRES p38 MAP KINASE J. Biol. Chem., July 13, 2001; 276(29): 27077 - 27082. [Abstract] [Full Text] [PDF]

				A. S. Greenberg, W.-J. Shen, K. Muliro, S. Patel, S. C. Souza, R. A. Roth, and F. B. Kraemer Stimulation of Lipolysis and Hormone-sensitive Lipase via the Extracellular Signal-regulated Kinase Pathway J. Biol. Chem., November 21, 2001; 276(48): 45456 - 45461. [Abstract] [Full Text] [PDF]

				J. M. Lindquist, J. M. Fredriksson, S. Rehnmark, B. Cannon, and J. Nedergaard beta 3- and alpha 1-Adrenergic Erk1/2 Activation Is Src- but Not Gi-mediated in Brown Adipocytes J. Biol. Chem., July 21, 2000; 275(30): 22670 - 22677. [Abstract] [Full Text] [PDF]

				H.-J. Cheng, Z.-S. Zhang, K. Onishi, T. Ukai, D. C. Sane, and C.-P. Cheng Upregulation of Functional {beta}3-Adrenergic Receptor in the Failing Canine Myocardium Circ. Res., September 28, 2001; 89(7): 599 - 606. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Soeder, K. J. Articles by Collins, S. Search for Related Content PubMed PubMed Citation Articles by Soeder, K. J. Articles by Collins, S. Social Bookmarking                      What's this?