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Originally published In Press as doi:10.1074/jbc.M701998200 on May 10, 2007

J. Biol. Chem., Vol. 282, Issue 29, 21529-21541, July 20, 2007
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Lysophosphatidic Acid Regulates Trafficking of beta2-Adrenergic Receptors

THE G{alpha}13/p115RhoGEF/JNK PATHWAY STIMULATES RECEPTOR INTERNALIZATION*

Elena Shumay{ddagger}1, Jiangchuan Tao{ddagger}1, Hsien-yu Wang§, and Craig C. Malbon{ddagger}2

From the Departments of {ddagger}Pharmacology and §Physiology and Biophysics, Diabetes and Metabolic Diseases Research Program, School of Medicine, State University of New York, Stony Brook, New York 11794-8661

Received for publication, March 7, 2007 , and in revised form, May 9, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Lysophosphatidic acid is an important lipid ligand regulating many aspects of cell function, including proliferation and migration. Operating via heterotrimeric G proteins to downstream effectors, lysophosphatidic acid was shown to regulate the function and trafficking of the G protein-coupled beta2-adrenergic receptor. C3 exotoxin, expression of dominant negative RhoA, and inhibition of c-Jun N-terminal kinase blocked the ability of lysophosphatidic acid to sequester the beta2-adrenergic receptor, whereas expression of constitutively active G{alpha}13, p115RhoGEF, or RhoA mimicked lysophosphatidic acid (LPA) action, stimulating the internalization of the G{alpha}s-coupled beta2-adrenergic receptor. This study revealed a novel cross-talk exerted from the LPA/G{alpha}13/p115RhoGEF/RhoA pathway to the beta2-adrenergic receptor/G{alpha}s/adenylyl cyclase pathway, attenuating the ability of beta-adrenergic agonists to act following stimulation of cells by LPA as may occur during beta-adrenergic therapy of an inflammatory response.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A major focus of research in cell signaling is the study of the integration or "cross-talk" among signaling pathways (1, 2). For more than a decade it has been observed in G protein-coupled signaling paradigms that pathways cross-talk at several levels. Chronic activation of the inhibitory adenylyl cyclase pathway leads to a compensatory change in the stimulatory adenylyl cyclase pathway at the level of gene expression (3). Chronic activation of the stimulatory adenylyl cyclase pathway likewise influences the expression of the elements of the opposing inhibitory adenylyl cyclase pathway (4). Many examples of such cross-talk have been reported for pathways regulated by G protein-coupled receptors (GPCRs)3 and their cognate G proteins. Similarly cross-talk has been observed operating between receptor tyrosine kinases and GPCR-mediated signaling (5, 6). The regulation of catecholamine action at the level of the beta2-adrenergic receptor (beta2AR) by insulin is prototypic for this aspect of cross-talk (7). The function of the beta2AR is impaired by insulin action with the beta2AR acting as a substrate for the activated insulin receptor itself (8) as well as for the downstream protein kinase of the insulin receptor, Akt (9).

Although cross-talk among G protein-mediated pathways controlling a common second messenger, cyclic AMP, has been detailed, much less is known about whether or not cross-talk occurs among signaling pathways with divergent second messenger readouts. In an effort to explore cross-talk in this context, we sought to investigate whether signaling via a G{alpha}13-mediated pathway influenced signaling of the beta2AR/G{alpha}s/adenylyl cyclase pathway. Lysophosphatidic acid (LPA) is a lipid mediator that acts upon members of the GPCR subfamily of LPA receptors that can couple to G{alpha}13 and in some cases other heterotrimeric G proteins (10). Using human carcinoma A431 as well as human embryonic kidney (HEK) 293 cells as models (11), we tested the ability of LPA to regulate beta2AR action, elucidating a novel cross-talk from a G{alpha}13-mediated pathway in which G{alpha}13, p115RhoGEF, RhoA, and ultimately JNK regulated the trafficking of beta2ARs in the beta2AR/G{alpha}s/adenylyl cyclase pathway. Activation of this LPA-sensitive pathway led to a reduction in the cell surface complement of beta2AR and attenuated beta2AR-mediated signaling events as well as recovery from agonist-induced desensitization.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Key reagents used in these studies were obtained from the following sources: Clostridium botulinum exotoxin (C3) and LPA from Sigma, SP600125 from A. G. Scientific, Inc. (San Diego, CA), SB203580 and Ro-20-1724 from Calbiochem, and ProLong® anti-fade reagent from Invitrogen.


Figure 1
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  		FIGURE 1.
LPA attenuates cyclic AMP accumulation in response to beta-adrenergic agonist. A, human epidermoid carcinoma A431 cells were treated in the absence or presence of LPA (5 µM) for 30 min followed by a 0-15-min treatment with either Iso (10 µM) or the plant diterpene forskolin (FK;30 µM). Cyclic AMP accumulation was measured at the times indicated. The data presented are mean values ± S.E. for cyclic AMP accumulation (-fold over basal) from three or more independent experiments. Basal cyclic AMP accumulation was 0.31 ± 0.025 pmol/105 cells. * denotes p ≤ 0.05 for the difference between without and with LPA in the presence of Iso. B, A431 cells were treated with and without Iso (10 µM) for 5 min and then treated without and with LPA (5 µM) concurrently for the times indicated. Cyclic AMP was measured at the time points indicated. The data presented are mean values ± S.E. for cyclic AMP accumulation (-fold over basal) from three or more independent experiments. Cyclic AMP accumulation was 0.41 ± 0.021 and 18.01 ± 0.63 pmol/105 cells for -Iso and +Iso at time = 0, respectively. * denotes p ≤ 0.05 for the difference between without and with LPA in the presence of Iso. C, A431 cells were treated with LPA (5 µM) for 0-90 min. At each of the time points indicated for LPA treatment, Iso (10 µM) was added, and the cyclic AMP accumulation was then measured over a 5-min period. The data shown are the mean values ± S.E. from at least three separate experiments. * denotes p ≤ 0.05 for the difference from the zero time control, having no prior LPA treatment. D and E, A431 cells were treated with and without Iso (10 µM; D as shown in B) or bradykinin (BK;10nM; E) for 5 min and then treated without and with LPA (5 µM) concurrently for the times indicated. Cyclic AMP was measured at the time points indicated. F, A431 cells were treated without and with C3 exotoxin (20 µg/ml) for 30 min and then concurrently without or with either isoproterenol (10 µM), LPA (5 µM), or forskolin (30 µM). Cyclic AMP accumulation was measured at 15 min following the addition of either Iso or forskolin. * denotes p ≤ 0.05 for the difference between without and with treatment with C3 exotoxin in the presence of Iso. G, A431 cells were transiently transfected with an expression vector harboring either QLG{alpha}13, QLG{alpha}11, CA-p115RhoGEF, or DN-RhoA and seeded in 96-well plates for 24 h. On the day of experiment, where indicated, cells were pretreated with LPA (5 µM) or C3 exotoxin (20 µg/ml) for 1 h prior to stimulation with isoproterenol (+Iso;10 µM for 5 min). The data presented are mean values ± S.E. for cyclic AMP accumulation (pmol/105 cells) from three or more independent experiments. * denotes p ≤ 0.05 for the difference from control + Iso group. H, A431 cells were transiently transfected without and with an expression vector harboring either CA-p115RhoGEF or DN-RhoA and seeded in 96-well plates for 24 h. On the day of experiment, where indicated, cells were pretreated or not with LPA (5 µM) for 1 h prior to stimulation without or with isoproterenol (+Iso;10 µM for 5 min). The data presented are mean values ± S.E. for cyclic AMP accumulation (pmol/104 cells) from three or more independent experiments. * denotes p ≤ 0.05 for the difference from control + Iso group. I, A431 cells were treated without (time = 0) or with LPA (5 µM) for 10-60 min. The time course of the activation of JNK in response to LPA was measured by immunoblotting (IB) of whole-cell lysates in which the blots were stained with antibodies to phosphospecific (activated) JNK (p-JNK) or with antibodies to JNK; the second antibody staining provided both a measure of JNK abundance and a loading control for the SDS-PAGE. The image is representative of at least three similar experiments. Densitometric scanning of the autoradiograph revealed a 3.4-fold increase in the relative amount of phospho-JNK in response to LPA treatment for 60 min.

 
Cell Culture—Human epidermoid carcinoma A431 and HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) in a humidified atmosphere of 5% CO2. For all experimental trials herein, stable transfectant clones, transiently transfected cells, or untreated, wild-type cells were cultured on tissue culture plates (6- and 96-well plates) or on chamber slides and kept in serum-free growth medium for 18 h prior to assays.

Plasmids and Transfections—The expression vector pCMV5 plasmid harboring the constitutively activated, GTPase-deficient Q226L mutant form of G{alpha}13 (QLG{alpha}13) was obtained from Dr. Alfred Gilman (Pharmacology, University of Texas South-western Medical School, Dallas, TX). For empty vector controls the pCDNA3 plasmid was used. Expressed proteins were epitope-tagged either with the hemagglutinin antigen (HA-tagged) or with c-Myc (Myc-tagged). The HA-tagged versions of constitutively active p115-RhoGEF (pCMV5-HA-p115-{Delta}N{Delta}C-RhoGEF, residues 249-802) and of the dominant negative p115RhoGEF (pCMV5-{Delta}DH-RhoGEF with excision of residues 466-547) were gifts from Dr. Gideon Bollag (ONYX Pharmaceuticals, Richmond, CA). The HA-tagged versions of the constitutively active Cdc42 (pCDNA3-HA-Cdc42 (Q61L)) and dominant negative Cdc42 (pCDNA3-HA-Cdc42 (T17N)) as well as the constitutively active Rac1 (pCDNA3-HA-Rac1 (Q61L)) and dominant negative Rac1 (pCDNA3-HA-Rac1 (T17N)) plasmids were gifts from Dr. Richard A. Cerione (Department of Molecular Medicine, Cornell University, Ithaca, NY). The c-Myc-tagged version of the dominant negative RhoA (pCDNA3-Myc-RhoA (T19N)) plasmid was a gift from Dr. Dafna Bar-Sagi (Department of Biochemistry, New York University, NY) and subsequently was re-engineered to incorporate an HA tag. The constitutively active version of RhoA (pCDNA3-myc-RhoA (Q63L)) plasmid was a gift from Dr. Alan Hall (CRC Oncogene and Signal Transduction Group, Medical Research Council Laboratory for Molecular Cell Biology, University College, London, UK). A431 cells were transfected with one or more plasmids using Lipofectamine according to manufacturer's instructions (Invitrogen). The A431 cells stably expressing a green fluorescent protein (GFP)-tagged version of beta2AR (fusion of enhanced GFP moiety to the C terminus of the receptor) described previously (12, 13) were utilized in experiments seeking to image the beta2AR.

Suppression of G Protein {alpha} Subunits—Knockdown of G protein subunits was achieved by treating the cells for 48-72 h with siRNA targeting the subunit. siRNA targeting G{alpha}q and G{alpha}13 were synthesized (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and introduced into A431 cells according to the manufacturer's instructions. Briefly cells growing in a 6-well plate in antibiotic-free medium were treated with siRNA duplex and incubated for 8 h. Thereafter the medium was supplemented with standard growth medium, and the incubation was maintained for an additional 40-64 h. siRNA-treated cells were analyzed for the efficiency of the knockdown of targeted G protein subunits by immunoblotting and thereafter used in the imaging experiments.

Assay of Intracellular Accumulation of Cyclic AMP—Cells transiently transfected with the QLG{alpha}13, QLG{alpha}11, CA-p115, CA-RhoA, CA-Cdc42, and/or CA-Rac1 were seeded in a 96-well plate for 24 h. On the day of experiment, cells were treated with either LPA (5 µM) or C3 (20 µg/ml) for 1 h before the challenge with isoproterenol (10 µM). Cell culture medium then was replenished with Krebs-Ringer phosphate buffer containing the phosphodiesterase inhibitor Ro-20-1724 (10 µM), and the cells were challenged with the isoproterenol for the indicated times. The reaction was terminated by the addition of ethanol to 10% (v/v), and the cyclic AMP content was measured by the competitive binding assay as described previously (14). To assay receptor resensitization, cells were challenged with isoproterenol (10 µM) for 5 min to provoke full desensitization and then washed free of beta-adrenergic agonist to allow recovery over the time period indicated (0, 30, or 60 min) before a second challenge with the beta-adrenergic agonist for 5 min. Each sampling was performed in triplicates. The results displayed throughout are mean values ± S.E. from three or more separate experiments.

[3H]CGP-12177 Assay of Surface beta2AR—The cell surface complement of beta2AR was quantified using the hydrophilic, cell-impermeable radiolabeled beta-adrenergic antagonist [3H]CGP-12177. Briefly cells were treated with or without isoproterenol (10 µM for 30 min) or LPA (5 µM) at 37 °C. The cells were washed and placed on ice for 6 h in the presence of 70 nM [3H]CGP-12177 with or without propranolol (10 µM) to define the amount of nonspecific binding. At the end of the incubation period, the cell masses (in triplicate) were rapidly washed free of unbound ligand and collected on Whatman GF/C filters by vacuum filtration. The amount of [3H]CGP-12177 that was bound to cell surface beta2AR was quantified by liquid scintillation spectrometry. Radioligand binding that is insensitive to the presence of propranolol (typically <10%) is defined as "nonspecific."

Confocal Microscopy—Cells stably expressing GFP-tagged beta2AR were used as such or transiently transfected with an expression vector harboring the cDNA of a signaling element (where indicated) and grown on glass slides. Prior to confluence, the cell cultures were either untreated or stimulated with isoproterenol (10 µM) or LPA (5 µM). At the end of the treatments, cells were washed twice with Hanks' balanced salt solution, fixed (100% methanol at -20 °C for 15 min), and returned to Hanks' balanced salt solution. Fixed objects were embedded in ProLong anti-fade reagent. Images were acquired on the Zeiss LCM510 microscope fitted with an argon laser (oil immersion, 63x objective). Serial sections were acquired as a z-stack. z-stacks of images were processed using Bitplane software (Imaris 4.2) and exported as TIFF files.

Statistical Analysis—The results in the figures are presented as the mean ± S.E. The data were compiled from assays sampled in triplicate from three or more separate experiments. * denotes significance of the difference from the "control" value of interest with p ≤ 0.05. For the microscopic studies, images were acquired from five to seven different fields for each condition; the fields were obtained from one of three or more duplicate experiments. Representative images are shown.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
LPA Suppresses beta2AR-stimulated Cyclic AMP Accumulation—We explored the hypothesis that LPA action might include cross-talk to the beta2AR/Gs/adenylyl cyclase signaling pathway (Fig. 1). Human epidermoid carcinoma A431 cells first were treated without and with LPA (5 µM) for 30 min followed by stimulation with the beta-adrenergic agonist isoproterenol (Iso; 10 µM) for 2-15 min, and cyclic AMP accumulation was measured over a 5-min interval (Fig. 1A). LPA treatment blunted the response of the cells to stimulation with isoproterenol. The ability of the plant diterpene forskolin to stimulated cyclic AMP accumulation under these same conditions, in contrast, was unaffected by LPA. Next a time course for the action of LPA was investigated (Fig. 1B). Attenuation of beta-adrenergic action by LPA was measured in the same manner over a 90-min exposure to the lipid mediator. Suppression of beta-adrenergic action occurred within 30-60 min and continued to increase until the 90-min post-LPA treatment sampling, the last time point measured. The effect of LPA was examined on A431 cells with no prior stimulation by isoproterenol (Fig. 1C). When naïve cells are treated with LPA and their ability to accumulate cyclic AMP in response to isoproterenol was measured over a 90-min period (with Iso treatment and cyclic AMP accumulation measured only over a 5-min interval at the time points indicated), the time course of LPA-induced suppression of the beta-adrenergic cyclic AMP response was similar (Fig. 1, compare C with A). We tested whether LPA treatment would impact the ability of a G{alpha}s protein-coupled receptor other than the beta2-adrenergic receptor to stimulate cyclic AMP accumulation (Fig. 1, D and E). Cells were treated without and with LPA for 30 min and then stimulated with either isoproterenol (Fig. 1D) or bradykinin (Fig. 1E, BK). The cyclic AMP response of A431 cells to stimulation by bradykinin was attenuated by treating the cells with LPA as was observed for the response to stimulation of beta2-adrenergic receptors with Iso (Fig. 1, A-D).

The C. botulinum ADP-ribosyltransferase "C3" exotoxin targets a major LPA downstream element, the small GTPase RhoA (15), and has been shown to inactivate RhoA in A431 cells (16). As LPA appears to negatively modulate beta-adrenergic signaling, treatment with the C3 exotoxin, which blocks some LPA signaling, was tested to ascertain its influence on isoproterenol-stimulated cyclic AMP accumulation. Treatment with C3 exotoxin (20 µg/ml) potentiated the ability of isoproterenol to stimulate cyclic AMP accumulation (Fig. 1F). No significant change in cyclic AMP accumulation was observed in response to LPA as compared with Iso (Fig. 1F).

To probe whether the response to LPA was mediated by G{alpha}13, a G protein to which LPA receptors can couple, we compared the effects of LPA with that of expression of the constitutively active Q226L mutant of G{alpha}13 (QLG{alpha}13) on the cyclic AMP response to isoproterenol stimulation (Fig. 1G). Transient expression of QLG{alpha}13 mimicked LPA action, reducing the beta-adrenergic stimulation of cyclic AMP accumulation by more than 60%. The Q209L mutant of G{alpha}11 also is constitutively active (17) and was used as a control. Expression of Q209L mutant of G{alpha}11 (QLG{alpha}11), in contrast to expression of QLG{alpha}13, had no influence on the ability of the beta-adrenergic pathway to stimulate cyclic AMP accumulation (Fig. 1G). A major downstream effector for G{alpha}13 is the guanine nucleotide exchange factor p115RhoGEF (18, 19), so we investigated whether the expression of the constitutively active p115RhoGEF (CA-p115), a mutant lacking the N- and C-regulatory domains (residues 249-802) of this GEF (20), would influence the cyclic AMP response to isoproterenol (Fig. 1G). Transient expression of CA-p115RhoGEF mimicked LPA action and proved to be a most potent attenuator of the beta-adrenergic receptor-mediated cyclic AMP response, reducing the accumulation of cyclic AMP by more than 80%. If our hypothesis that LPA was cross-talking to the beta2AR/Gs/adenylyl cyclase signaling pathway through the G{alpha}13/p115RhoGEF/RhoA triad was correct, transient transfection of the A431 cells with an expression vector harboring the dominant negative mutant ({Delta}DH-RhoGEF (20)) of RhoA (DN-RhoA) might be expected to block the attenuation by LPA. Expression of DN-RhoA indeed provoked a potentiation of the cyclic AMP response to beta-adrenergic stimulation as did the treatment with the C3 exotoxin (Fig. 1, F and G). We tested the ability of the DN-RhoA to impact LPA signaling at the levels of LPA and/or p115RhoGEF (Fig. 1H). Expression of DN-RhoA abolished the ability of LPA to suppress beta2AR/Gs/adenylyl cyclase signaling in response to isoproterenol. Expression of DN-RhoA likewise abolished the ability of CA-p115RhoGEF to signal to this same level (Fig. 1H).

The mitogen-activated protein kinase JNK has been shown to operate downstream of the G{alpha}13/p115RhoGEF/RhoA pathway (21, 22). We examined the ability of LPA to stimulate JNK activation in A431 cells (Fig. 1I). Stimulating the cells with LPA provoked a robust activation of JNK (Fig. 1I) that was detected within 10 min of LPA treatment and maintained to 60 min. Taken together, these data demonstrate at a functional level that LPA, operating via G{alpha}13, p115RhoA, and RhoA activation, suppresses catecholamine action mediated by beta2ARs.

LPA Regulates Cellular Trafficking of the beta2AR—The ability of LPA to attenuate the beta-adrenergic regulation of cyclic AMP accumulation provoked us to ask whether this lipid mediator also regulated the trafficking of the beta2AR. RhoA action includes changes in cellular trafficking (23), and it seemed possible that the influence of LPA via G{alpha}13 and the ability of DN-RhoA expression to block the LPA effect (Fig. 1) was a reflection of RhoA activation. The trafficking of the beta2AR was explored using a fusion protein of the human beta2AR in which the enhanced GFP is engineered into the C terminus of the receptor. This GFP-tagged beta2AR displays full functional capability (12, 13, 24, 25). By confocal microscopy we probed the receptor localization in both the unstimulated (-) cells as well as in cells stimulated for 30 min with the beta-agonist isoproterenol (10 µM; +Iso), which caused desensitization and internalization of beta2ARs (Fig. 2A). In the absence of treatment with beta-adrenergic agonist, beta2ARs were observed largely confined to the cell membrane (Fig. 2A, top left-hand panel, control; white arrows highlight cell membrane-associated receptor). In cells treated with isoproterenol (+Iso), the complement of cell membrane-localized beta2AR was depleted and an accumulation of internalized beta2AR occurred (Fig. 2A, top right-hand panel; yellow arrowheads highlight cytoplasmic, internalized receptor).

Treating the cells with LPA mimicked treatment with beta-adrenergic agonist, stimulating the internalization of beta2AR and accumulation of the receptor in cytoplasmic locales in the absence of beta-adrenergic agonist (Fig. 2A). For the cells treated with the beta-adrenergic agonist or with LPA, the sequestered beta2ARs were observed as large "punctate" aggregates localized to the cytoplasmic regions of the cells. Treatment with LPA and beta-agonist in combination largely appeared to result in little further internalization, although some discrete, larger punctate aggregates of internalized beta2AR were observed in some fields (Fig. 2A).

To test further the hypothesis that LPA drives the sequestration of beta2AR via LPA activation of G{alpha}13 and of its downstream effector p115RhoGEF (26), cells were transiently transfected with constitutively active forms of either G{alpha}13 (QLG{alpha}13) or p115RhoGEF (CA-p115), and the localization of the beta2AR was examined by microscopy (Fig. 2A). Mimicking LPA treatment, expression of the QLG{alpha}13 alone provoked internalization of the beta2AR. Treating the cells expressing the QLG{alpha}13 with the beta-agonist isoproterenol resulted in little additional effect. Expression of QLG{alpha}11, in contrast, had no effect on beta2AR localization; the beta2ARs appeared as observed in the control, untreated cells (data not shown). Expression of CA-p115RhoGEF, like the expression of QLG{alpha}13, also mimicked LPA action and promoted receptor internalization (Fig. 2A).

The data obtained by confocal analysis of the GFP-tagged beta2AR were tested further and quantified independently by equilibrium binding studies of whole-cell preparations with the hydrophilic beta-adrenergic antagonist [3H]CGP-12177. This CGP-12177 ligand does permeate the cell membrane and reports back from only those beta2AR that are accessible on the cell surface and to the bulk solution. [3H]CGP-12177 binding data confirmed the results obtained by confocal microscopy, i.e. expression of either QLG{alpha}13 or CA-p115RhoGEF mimicked the effects of LPA itself on beta2AR internalization (Fig. 2B). We also analyzed the time course for internalization of beta2AR in cells treated with either LPA or Iso (Fig. 2C). The time course for Iso displayed internalization of receptors within 5 min of challenge and a plateau of maximal internalization at 30-60 min. LPA-stimulated beta2AR sequestration displayed a time course similar to that observed for Iso as measured by CGP-12177 binding.

If G{alpha}13 mediates the ability of LPA to stimulate internalization of beta2ARs, then suppression of this G protein {alpha} subunit would be expected to attenuate the effect of LPA on receptor trafficking. We tested this hypothesis directly using siRNA to knock down the expression of G{alpha}13 (Fig. 2D, inset). The siRNAs effectively suppressed the expression of their cognate G{alpha} subunit targets (Fig. 2D, inset), whereas treatment with the commercially designed control siRNA (Cont) did not. Treatment of A431 cells with siRNA targeting G{alpha}13 (G{alpha}13-KD) abolished the ability of LPA to internalize beta2ARs (Fig. 2D), again suggesting that G{alpha}13 specifically is coupling the LPA response to the trafficking of beta2AR. Suppressing the expression of G{alpha}13 with siRNA likewise abolished the ability of LPA to activate JNK (data not shown). Treating cells with siRNA either targeting G{alpha}q (G{alpha}q-KD) to suppress the expression of this G protein {alpha} subunit (Fig. 2D, inset) or with the control siRNA, in sharp contrast, had no apparent effect on the ability of LPA to internalize beta2ARs (Fig. 2D). We conducted CGP-12177 binding studies in parallel to the confocal studies to quantify amounts of beta2AR internalized in response to LPA versus Iso in cells treated with siRNA to knock down either G{alpha}13 or G{alpha}q (Fig. 2E). The data obtained from the independent CGP-12177 binding studies (Fig. 2E) agree well with results from the confocal images (Fig. 2D), i.e. G{alpha}13 mediates LPA action on beta2AR internalization.


Figure 2
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  		FIGURE 2.
Activation of the LPA/G{alpha}13/p115RhoA pathway stimulates internalization of beta2AR. A, A431 clones stably expressing GFP-tagged beta2AR (expressed at 5% of wild-type levels) were subjected to either pretreatment with LPA (5 µM) for 1 h or transiently transfected to express either QLG{alpha}13 or CA-p115RhoGEF and then either untreated (-) or treated for 30 min with the beta-adrenergic agonist isoproterenol (+Iso;10 µM). Confocal microscopy was performed, and representative fields of GFP-beta2AR are displayed as accumulated "z-stacks." White arrows highlight beta2AR localized to the cell membrane; yellow arrowheads highlight internalized beta2AR. The images displayed are representative of three or more separate experiments performed on separate occasions. Bar, 10 µm. B, A431 cells were transiently transfected with or without an expression vector harboring either QLG{alpha}13 or CA-p115RhoGEF and seeded in 96-well plates for 24 h. On the day of experiment, where indicated, cells were pretreated without or with LPA (5 µM) for 1 h prior to subsequent stimulation with isoproterenol (+Iso;10 µM for 30 min). The cell surface complement of beta2AR was probed by equilibrium radioligand binding studies using a hydrophilic, cell-impermeable, radiolabeled beta-adrenergic antagonist, [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells is set at "100%," which is 3.7 x 104 receptors/cell. The amount of beta2AR internalized in response to these treatments was calculated, and the results are displayed as "percentage of receptor internalized." These data are mean values ± S.E. of at least three separate experiments. * denotes p ≤ 0.05 for the difference from the control -Iso group. C, A431 cells were treated with and without either Iso (10 µM) or LPA (5 µM) for the time periods indicated. The cell surface complement of beta2AR was probed by equilibrium radioligand binding studies using [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells is set at 100%, the amount of receptor internalized in response to these treatments was calculated, and the results displayed are percentage of receptor internalized. These data are mean values ± S.E. of at least three separate experiments. * denotes p ≤ 0.05 for the difference from the time = 0 group. D, knockdown of G{alpha}13 (G{alpha}13-KD), but not G{alpha}q (G{alpha}q-KD), by siRNA in A431 cells blocks the ability of LPA (5 µM for 60 min) to internalize GFP-tagged beta2AR as determined by confocal microscopy. Inset, suppression of G protein {alpha} subunit expression by treatment with siRNA targeting either G{alpha}13 or G{alpha}q. Cont, non-targeting siRNA reagents provided as a control by the commercial supplier. The immunoblotting (IB) data are from a single experiment and representative of at least three separate experiments. The blots were stained with antibodies against either G{alpha}13 or G{alpha}q subunits. The extent of subunit suppression was >90% for those subunits targeted by their specific siRNAs. Panels marked Control were treated with a non-targeting, control siRNA supplied by the commercial source. The cells were treated without or with LPA (+LPA). Immunoblotting of beta-actin is provided as a loading control for the SDS-PAGE. E, knockdown of G{alpha}13 (G{alpha}13-KD), but not G{alpha}q (G{alpha}q-KD), by siRNA in A431 cells blocks the ability of LPA (5 µM for 60 min), but not Iso (10 µM for 30 min), to internalize beta2AR as determined by equilibrium radioligand binding studies using [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells is set at 100%. The amount of beta2AR internalized in response to siRNA treatment was calculated, and the results displayed as "percentage of beta2AR internalized." These data are mean values ± S.E. of at least three separate experiments. * denotes p ≤ 0.05 for the difference from the control + LPA group. F, prospective projection and sectional views of images of GFP-beta2ARs expressed in A431 cells. Images processed using advanced Bitplane image analysis software are displayed for untreated (Control) cells and cells transiently transfected to express CA-p115RhoGEF (CA-p115). F, inset, immunoblotting of HA-tagged CA-p115RhoGEF expressed in A431 cells. Immunoblots of whole-cell lysates were stained with anti-HA antibodies to identify immune complexes of the expressed CA-p115RhoGEF. Bar, 20 µm.

 
A prospective projection and sectional view of the localization of beta2AR in untreated cells (control) as compared with cells expressing the constitutively active mutant of p115RhoGEF (CA-p115) was created in an attempt to obtain a more detailed depiction of the disposition of the internalized beta2AR in untreated cells and cells expressing CA-p115RhoGEF (Fig. 2F). The prospective view of the native cells (control) clearly shows the localization of GFP-tagged beta2AR predominantly in the cell membrane, whereas in the cells transiently transfected to express the CA-p115RhoGEF (CA-p115) translocation and internalization of the beta2AR to the cytoplasm in the absence of LPA is obvious (Fig. 2F, upper panels). The cross-sectional views highlight the differences between beta2AR localization in control cells as compared with cells expressing CA-p115RhoGEF. In the untreated cells, beta2ARs were densely localized to the plane of the cell membrane. For the cells expressing the CA-p115RhoGEF, beta2ARs localized to the cytoplasm and in close proximity to the inner leaflet of the cell membrane (Fig. 2F, lower panels). Immunoblots stained with anti-HA antibodies confirmed the expression of the HA-tagged CA-p115RhoGEF mutant in the cells (Fig. 2F, inset).

LPA Regulates Internalization of the beta2AR via Small Molecular Weight GTPases—We explored the effects of both C3 exotoxin and expression of DN-RhoA on the cellular localization of GFP-tagged beta2AR (Fig. 3A). The patterns of localization of beta2AR in the control, untreated cells (-, Fig. 3A) compared with the cells treated with either C3 exotoxin (C3) or transiently transfected to express DN-RhoA are all similar (Fig. 3A, +Iso). The internalization of the beta2AR in response to isoproterenol treatment likewise was largely unaffected by treatment with C3 exotoxin or by expression of DN-RhoA. The observations from the confocal analysis were tested using the CGP-12177 radioligand binding assay (Fig. 3B). The results of both assays are in agreement; there was little change in the amount of cell membrane-localized beta2AR in cells treated with either C3 exotoxin or those expressing the DN-RhoA (Fig. 3, A and B). These data suggest that the effects of LPA on beta2AR trafficking may be operating via a pathway with little commonality with the Iso/beta2AR/Gs/AC/cyclic AMP signaling pathway.

The imaging studies revealed that activation of the LPA/G{alpha}13/p115RhoGEF pathway leads to a sequestration of beta2AR from the cell membrane in the absence of beta-adrenergic agonist. The results from studies of the cyclic AMP response suggest that signaling downstream of p115RhoGEF involves Rho family GTPases. We tested the role of RhoA, Rac1, and Cdc42 in beta2AR trafficking by examining the effects of expression of the constitutively active mutants of each of these small molecular weight GTPases on the complement of cell surface-localized beta2AR (Fig. 3C). Cells were treated either with LPA or transiently transfected to express CA mutants of various elements in the G{alpha}13/p115RhoGEF/RhoA pathway. Equilibrium binding studies of cell surface beta2AR were conducted using [3H]CGP-12177. Treating cells with LPA reduced the complement of beta2AR on the cell membrane (Fig. 3C), as expected, as did expression of either QLG{alpha}13 or CA-p115 (Fig. 3C). Expression of the CA-RhoA alone, like that of either QLG{alpha}13 or CA-p115RhoGEF alone, stimulated a reduction in the amount of cell membrane-associated beta2AR (Fig. 3C). Expression of CA-RhoA, in contrast, stimulated a reduction in surface beta2AR substantially greater than that observed in those cells expressing either QLG{alpha}13 or CA-p115. Expression of the constitutively active mutants of either Rac1 (CA-Rac1) or Cdc42 (CA-Cdc42) alone, in contrast to the expression of CA-RhoA, had no effect on the relative amount of beta2AR available in these cells to bind the cell-impermeant CGP-12177 ligand. Immunoblots stained with the appropriate antibodies demonstrated comparable levels of expression of the CA-RhoA, CA-Rac1, and CA-Cdc42 (Fig. 3C, inset).

We also investigated whether activation of isoproterenol-stimulated beta2AR sequestration was influenced by treatment of the cells with LPA as compared with transient expression of constitutively active mutants of G{alpha}13 (QLG{alpha}13), p115RhoGEF (CA-p115), RhoA (CA-RhoA), Rac1 (CA-Rac1), or Cdc42 (CA-Cdc42) for 24 h prior (Fig. 3D). Isoproterenol, which stimulated a homologous, beta-adrenergic-induced internalization that was more robust than the heterologous effects of LPA (Fig. 2), stimulated a 40-50% internalization, which was unaffected by the addition of LPA. Expression of either QLG{alpha}13 or CA-p115RhoGEF likewise had no effect on Iso-stimulated sequestration of beta2AR (Fig. 3D). As noted above, expression of CA-RhoA itself induced beta2AR internalization. Curiously expression of either Rac-1 or CA-Cdc42 attenuated the ability of isoproterenol to sequester beta2AR (Fig. 3D).


Figure 3
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  		FIGURE 3.
C3 exotoxin and expression of DN-RhoA block whereas expression of QLG{alpha}13, CA-p115RhoGEF, and CA-RhoA mimics the ability of LPA to internalize beta2AR. A, A431 clones expressing GFP-beta2AR were subjected to either pretreatment with C3 exotoxin (20 µg/ml) for 1 h or transiently transfected for 24 h with an expression vector harboring DN-RhoA. Then the cells were either untreated (-) or treated for 30 min with isoproterenol (+Iso;10 µM). Confocal analysis of the autofluorescent GFP-beta2AR was conducted. The images displayed are representative of those obtained from three or more separate experiments performed on separate occasions. Bar, 10 µm. Cell surface-localized beta2ARs are decorated with white arrows; cytoplasmic, internalized beta2ARs are decorated with yellow arrowheads. B, A431 cells were pretreated or not with C3 exotoxin (20 µg/ml) for 1 h or transiently transfected for 24 h with an expression vector harboring DN-RhoA. Then the cells were either untreated (-Iso/-LPA) or treated with either isoproterenol (+Iso;10 µM) or LPA (+LPA;5 µM). The cell surface complement of beta2AR was probed by equilibrium radioligand binding studies using [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells is set at 100%. The amount of beta2AR internalized in response to these treatments was calculated, and the results displayed as percentage of beta2AR internalized. These data are mean values ± S.E. from three or more separate experiments. * denotes p ≤ 0.05 for the difference from control + LPA group. C, A431 cells were transiently transfected with or without an expression vector harboring either QLG{alpha}13, CA-p115RhoGEF, CA-RhoA, CA-Rac1, or CA-Cdc42 and seeded in 96-well plates for 24 h. On the day of experiment, where indicated, cells were treated without or with LPA (5 µM) for 1 h. The cell surface complement of beta2AR was probed by equilibrium radioligand binding studies using [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells (~4.2 x 104 receptors/cell) is set at 100%, and the results are displayed as "cell surface beta2AR (percentage of control)." These data are mean values ± S.E. of at least three separate experiments. * denotes p ≤ 0.05 for the difference from the control cells. The expression of the small molecular weight GTPases was established by immunoblotting (IB) of whole-cell lysates stained with antibodies against the HA-tagged versions of the small molecular weight GTPases being expressed exogenously. beta-Actin staining provides an index of loading controls for the immunoblotting. D, A431 cells were transiently transfected with or without an expression vector harboring either QLG{alpha}13, CA-p115RhoGEF, CA-RhoA, CA-Rac1, or CA-Cdc42 and seeded in 96-well plates for 24 h. On the day of experiment, where indicated, cells were untreated (Control) or treated with isoproterenol (+Iso;10 µM). One group was concurrently treated with isoproterenol and also LPA (5 µM) for 1 h. The cell surface complement of beta2AR was probed by equilibrium radioligand binding studies using [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells is set at 100%, and the results are displayed as cell surface beta2AR (percentage of control). These data are mean values ± S.E. of at least three separate experiments. * denotes p ≤ 0.05 for the difference from control + Iso group. The expression of the HA-tagged mutant signaling proteins was established by immunoblotting of whole-cell lysates stained with anti-HA antibodies (not shown). E, A431and HEK293 cells were untreated (no stimulation (no stim.)) or treated with either Iso (10 µM for 30 min) or LPA (5 µM for 60 min). The cell surface complement of beta2AR was probed by equilibrium radioligand binding studies using [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells is set at 100%, and the results are displayed as cell surface beta2AR (percentage of control). These data are mean values ± S.E. of at least three separate experiments. * denotes p ≤ 0.05 for the difference from the untreated (no stimulation) control cell group.

 
We tested LPA action on the complement of surface beta2AR in a second cell line, HEK293 cells. HEK293 cells, like the A431 cells, displayed LPA-induced reduction in surface beta2ARs (Fig. 3E). Activation of the LPA/G{alpha}13/p115RhoGEF/RhoA pathway in HEK293 cells appeared to operate in a manner similar to that observed in A431 cells, i.e. LPA stimulated internalization of beta2AR (Fig. 3E).


Figure 4
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  		FIGURE 4.
Activation of the LPA/G{alpha}13/p115RhoGEF pathway blocks the resensitization of beta2AR following agonist-induced desensitization. A431 cells were transiently transfected for 24 h with an expression vector harboring either QLG{alpha}13 or CA-p115RhoGEF. These cells and those stimulated with LPA (5 µM for 1 h) were tested to ascertain whether the activation of the LPA/G{alpha}13/p115RhoGEF pathway alters the resensitization of the beta2AR. Receptor desensitization was achieved by a challenge (30 min) with Iso (10 µM). To assay resensitization, the isoproterenol-treated, desensitized cell groups were subjected to a washout followed by a 0-, 30-, or 60-min recovery period. The cells were then challenged with a second stimulation (5 min, 10 µM) by isoproterenol (control). The time-dependent progressive increase of cyclic AMP accumulation after the second challenge with Iso reflects the resensitization process, which is complete within 60 min. The data displayed are mean values ± S.E. from four separate experiments. * denotes p ≤ 0.05 for the difference from the cyclic AMP accumulation observed in the zero time groups. W, washout of agonist for 30 (w30) or 60 (w60) min.

 
LPA Suppresses Recovery of Desensitized beta2AR—Activation of the LPA pathway leads to attenuation of beta-adrenergic agonist activation of the stimulatory adenylyl cyclase pathway and later to increased internalization of beta2AR. We investigated the effects of activating the LPA/G{alpha}13/p115RhoGEF pathway at several points on resensitization/recovery of desensitized beta2AR (Fig. 4). To desensitize beta2AR, cells were treated with beta-adrenergic agonist (10 µM isoproterenol for 30 min) to provoke full desensitization (i.e. recovery period time = 0) and then washed free of agonist. The cells were allowed to recover for 0, 30, or 60 min and then restimulated with isoproterenol at each of these times for a 5-min interval designed to measure the extent of resensitization (Fig. 4). The cyclic AMP response to a second challenge with isoproterenol administered at washout (recovery period = 0 min) was determined and set as the control. In the control situation, recovery of the cyclic AMP response (i.e."resensitization") is complete within 60 min of washout of the beta-adrenergic agonist from the first challenge (25). In the LPA-treated cells, in contrast, the cells displayed a persistent state of desensitization following the washout of beta-adrenergic agonist. No resensitization, even at 60 min postwashout of the beta-adrenergic agonist, was observed in LPA-treated cells. Thus, in the absence of treatment with beta-adrenergic agonist, LPA stimulated internalization of beta2AR (Fig. 2). For cells desensitized in response to stimulation by the beta-adrenergic agonist isoproterenol, LPA blocked the resensitization process for cyclic AMP accumulation once the beta-adrenergic agonist had been washed out (Fig. 4).

We also tested the effects of activation of downstream signaling elements in the pathway to ascertain whether G{alpha}13 and/or the p115RhoGEF effector were mediating the ability of LPA to block the resensitization process. In cells transiently transfected to express the constitutively active QLG{alpha}13 mutant, the effects on the recovery process were the same as those following treatment with LPA, i.e. the resensitization of the beta2AR-mediated cyclic AMP response was absent. Likewise expression of the constitutively active p115RhoGEF mutant (CA-p115) was observed to abolish the resensitization of the beta2AR-mediated response. These data provide a partial explanation for the ability of LPA to regulate the beta2AR-stimulated activation of cyclic AMP accumulation. LPA activation of the G{alpha}13/p115RhoGEF/RhoA pathway at any point reduces the cell surface complement of beta2AR and also suppresses the characteristic ability of the desensitized beta2AR to undergo resensitization. Resensitization is a process observed for most members of the GPCR superfamily of receptors following agonist-induced desensitization/receptor internalization (1, 27).

JNK Mediates LPA Regulation of beta2AR—Activation of the LPA/G{alpha}13/p115RhoGEF pathway leads to activation of downstream signaling to the mitogen-activated protein kinase cascade (28, 29) with activation of JNK acting prominently as a bridge to gene expression and other responses (Fig. 1B). We investigated the signaling downstream of the G{alpha}13/p115RhoGEF/RhoA triad to the mitogen-activated protein (MAP) kinase cascade (30) by the use of selective inhibitors of the three terminal MAP kinase branches: p38, JNK, and Erk1,2 MAPKs (2). The following MAPK-selective inhibitors were used in these experiments: SB203580 for p38 kinase (31), SP600125 for JNK (32), and PD98059 for MEK (33), which operates upstream of Erk1,2 (Fig. 5A). Control cells not treated with any MAP kinase inhibitor displayed the characteristic sequestration of GFP-tagged beta2AR in response to LPA (5 µM for 30 min). Cell surface-localized beta2ARs are highlighted with white arrows in the absence of LPA; cytoplasmic, internalized receptors are highlighted with yellow arrowheads in the LPA-treated (+LPA) control cells that were not treated with inhibitors. Treating the cells for 60 min with inhibitors of MAP kinases alone had little effect on the apparent cellular distribution of beta2AR in the absence of treatment with LPA (Fig. 5A). Pretreating cells with the p38 kinase inhibitor SB203580 (1.0 µM) had little effect on the ability of LPA to provoke beta2AR internalization. Inhibition of the upstream Erk1,2 kinase MEK with PD98059 (5 µM) also had no effect on LPA-induced beta2AR internalization (not shown). In sharp contrast to the lack of effects of inhibitors of p38 and Erk1,2 branches of the MAPK cascade, treating the cells with the JNK-selective inhibitor SB203580 effectively blocked the ability of LPA to sequester beta2AR internally (Fig. 5A). The confocal studies were complemented by equilibrium binding studies using the CGP-12177 (Fig. 5B). LPA stimulated the internalization of beta2AR in cells not treated with MAPK inhibitors (control). In the presence of the JNK inhibitor SP600125, the ability of LPA to stimulate beta2AR sequestration was abolished. Treating the cells with the p38 inhibitor SB203580, in contrast, had no effect on the ability of LPA to stimulate internalization of beta2AR as measured by radioligand binding. We next compared the ability of LPA and CA downstream signaling elements to provoke beta2AR internalization in the absence and presence of SP600125 versus SB203580 (Fig. 5C). Either LPA alone or expression of QLG{alpha}13, of CA-p115RhoGEF, or of CA-RhoA individually demonstrated the ability to stimulate beta2AR sequestration; the JNK inhibitor SP600125 effectively blocked the ability of each of these agents to internalize beta2AR (Fig. 5C). Similar treatment with the p38 inhibitor had no effects on receptor internalization. The ability of isoproterenol to stimulate beta2AR sequestration was insensitive to either the JNK or the p38 inhibitor, again suggesting that the sequestration of beta2AR by LPA and by beta-adrenergic agonists operates via different pathways.

We examined the effects of the JNK inhibition on the ability of LPA to attenuate signaling by the beta2AR/G{alpha}s/adenylyl cyclase/cyclic AMP pathway (Fig. 5D). The cyclic AMP response to stimulation by isoproterenol was assayed in cells treated with or without LPA, demonstrating the ability of LPA to attenuate the response. When the same experiment was performed in the presence of the JNK inhibitor SP600125, the ability of LPA to suppress the cyclic AMP response to Iso was lost (Fig. 5D). Treating the cells with the p38 inhibitor, in contrast, had no effect on LPA action to the level of cyclic AMP accumulation. Finally we investigated at the level of cyclic AMP accumulation the ability of DN-RhoA to block the downstream signaling of LPA itself or expression of CA-p115RhoGEF to suppress the beta-adrenergic action of isoproterenol (Fig. 5E). Expression of DN-RhoA and CA-p115RhoGEF was established by immunoblotting (not shown). Either LPA alone or expression of CA-p115RhoGEF attenuated beta-adrenergic stimulated accumulation of intracellular cyclic AMP; co-expression of DN-RhoA abolished the action of LPA or of expression of CA-p115RhoGEF in this assay. These data suggest that the MAP kinase cascade regulated by the G{alpha}13/p115RhoGEF/RhoA cascade terminates with the activation of JNK (Fig. 6) in response to LPA and is obligate for LPA cross-talk to the beta2AR/G{alpha}s/adenylyl cyclase pathway. Chemical inhibition of JNK abolished that ability of the G{alpha}13/p115RhoGEF/RhoA pathway to mediate the ability of LPA to cross-talk to the beta2AR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Cross-talk in cell signaling is essential for integration of signals from a variety of receptors; prominent among them are members of the superfamily of GPCRs (1, 27). Cross-talk was discovered first upon chronic activation of the stimulatory or the inhibitory adenylyl cyclase pathways, which were shown to influence the opposing pathway at the level of gene expression, protein expression, and/or post-translational modification of the elements that compose the two pathways (34). Cross-talk between GPCR-mediated pathways and those regulated by receptor and non-receptor tyrosine kinases also has emerged as a paradigm in cell signaling (35). In the current work, the existence of cross-talk from pathways other than those controlling cyclic AMP was investigated, notably seeking to probe for the possible cross-talk from the G{alpha}13-mediated pathway to that of the G{alpha}s-mediated stimulatory adenylyl cyclase pathway. Cross-talk from the G{alpha}13/p115RhoGEF/RhoA pathway to other G-protein-mediated pathways has not been reported, and our initial observations on possible LPA regulation of beta2AR signaling fostered this study. To explore this possibility, we examined the ability of LPA operating through G{alpha}13 to influence the prototypic G protein coupled to the stimulatory adenylyl cyclase pathway, the beta2AR.


Figure 5
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  		FIGURE 5.
LPA-stimulated sequestration of beta2AR is blocked by JNK inhibitor SP600125. A, the effects of selective inhibitors of MAPKs on the ability of LPA to sequester beta2AR were tested directly. The inhibitors used were as follows: p38 inhibitor SB203580 (1 µM), JNK inhibitor SP600125 (0.4 µM), and Erk1, 2 via MEK inhibitor PD980572 (1 µM; not shown). A431 cells expressing GFP-tagged beta2AR were treated with a MAPK inhibitor for 60 min prior to stimulation without (-) or with LPA (+LPA;5 µM for 1 h). Images of the beta2AR were acquired by confocal microscopy. The images displayed are representative of those obtained from at least three separate experiments. Bar, 10 µm. Cell surface-localized beta2ARs are decorated with white arrows; cytoplasmic, internalized beta2ARs are decorated with yellow arrowheads. B, A431 cells were treated with and without a MAPK inhibitor (SP600125 or SB203580) for 30 min prior to being treated concurrently without (-LPA) and with LPA (+LPA;5 µM for 1 h). The cell surface complement of beta2AR was probed by equilibrium radioligand binding studies using [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells is set at 100%. The amount of beta2AR internalized in response to these treatments was calculated, and the results are displayed as percentage of receptor internalized. These data are mean values ± S.E. of at least three separate experiments. * denotes p ≤ 0.05 for the difference from -LPA group. C, A431 cells were transiently transfected with or without an expression vector harboring either QLG{alpha}13, CA-p115RhoGEF, or CA-RhoA and seeded in 96-well plates for 24 h. On the day of experiment, where indicated, cells were untreated (-) or treated for 30 min with a MAPK inhibitor (+SP600125 or +SB203580). Then the cells were treated without or with LPA (5 µM for 1 h) or Iso (10 µM for 30 min). The cell surface complement of beta2AR was probed by equilibrium radioligand binding studies using [3H]CGP-12177. The cell surface complement of beta2AR in untreated cells is set at 100%. The amount of beta2AR internalized in response to these treatments was calculated, and the results are displayed as percentage of beta2AR internalized. These data are mean values ± S.E. of at least three separate experiments. * denotes p ≤ 0.05 for the difference from the groups not treated with a MAPK inhibitor. D, A431 cells were untreated (Control) or treated with and without a MAPK inhibitor (+SP600125 or +SB203580) for 30 min prior to being treated concurrently without (-LPA) and with LPA (+LPA;5 µM for 1 h). The cells were treated with LPA (5 µM for 1 h) and then either without or with isoproterenol (+Iso;10 µM for 5 min). The data presented are mean values ± S.E. for cyclic AMP accumulation (pmol/104 cells) from three or more independent experiments. * denotes p ≤ 0.05 for the difference from the groups treated with LPA and no MAPK inhibitor. E, A431 cells were transiently transfected with empty vector or an expression vector harboring either CA-p115RhoGEF, DN-RhoA, or both CA-p115RhoGEF and DN-RhoA and seeded in 96-well plates for 24 h. On the day of experiment, where indicated, cells were treated with LPA (5 µM for 1 h) and then either without (-Iso) or with isoproterenol (+Iso;10 µM for 5 min). The data presented are mean values ± S.E. for cyclic AMP accumulation (pmol/104 cells) from three or more independent experiments. * denotes p ≤ 0.05 for the difference from the +Iso-treated control group. Expression of DN-RhoA and CA-p115RhoGEF was established by immunoblotting.

 
LPA is a lipid mediator with a diverse set of roles in cell signaling, including in inflammation in which LPA stimulates cell proliferation, survival, migration, and platelet aggregation (10, 30). Activation of beta2ARs, in contrast, can inhibit release of proinflammatory mediators, regulate eosinophil survival and action, regulate T-cell maturation and action, and inhibit tumor necrosis factor-{alpha} release (36, 37). This ability of beta2ARs to suppress inflammatory responses, however, is known to attenuate and to offer little suppression of chronic inflammation suggesting the possibility that agents that promote inflammation might compromise beta-adrenergic action at some level (38, 39). These earlier observations fostered our study of LPA effects, mediated largely via the G{alpha}13/p115RhoGEF/RhoA pathway, on cross-talking to the signaling of the beta2AR/G{alpha}s/stimulatory adenylyl cyclase pathway.


Figure 6
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  		FIGURE 6.
Schematic of LPA/G{alpha}13/p115RhoGEF pathway "cross-talking" to the beta2AR/G{alpha}s/AC pathway. The distribution of beta2AR from the cell surface (exofacial) to the cytoplasm (internalized) is depicted. Signaling from beta-adrenergic receptor activates its cognate G protein, Gs, which stimulates the activity of adenylyl cyclase (AC) and generates cyclic AMP accumulation. A homologous desensitization and internalization of beta2ARs in response to a beta-adrenergic agonist are catalyzed by the combined activation of the G protein-coupled receptor kinase (GRK) and of protein kinase A (PKA). Resensitization and recycling of the beta2AR occurs in the absence of beta-adrenergic agonist. Recycling provokes the movement of the sequestered, internalized receptor back to the cell membrane. LPA provokes heterologous desensitization and increased accumulation of internalized beta2AR, in the absence of beta-adrenergic agonist, by signaling through its cell surface receptor (LPAR). The activated LPAR, in turn, activates its cognate G protein, G13, and its downstream effector p115RhoGEF and thereby small molecular weight G proteins, especially RhoA. RhoA signals via the mitogen-activated protein kinase cascade from the level of MEKK to MKK to JNK. Expression of a constitutively active version of G{alpha}13, p115RhoGEF, or RhoA itself mimics LPA action on beta2AR internalization. Treatment with C3 exotoxin, expression of dominant negative RhoA, or treatment with the JNK inhibitor SP600125 abolishes or sharply attenuates LPA action on beta2AR trafficking. Thus, activation of the LPAR, as occurs in an inflammatory response, can attenuate the actions mediated by beta2AR through increasing the amount of internalized beta2AR absent from the cell membrane, a novel form of cross-talk among G protein-mediated pathways.

 
The current study revealed an important crossover point for the LPA and beta2-adrenergic responses, i.e. beta2AR trafficking (Fig. 6). Treating cells with LPA suppressed the ability of Iso to stimulate cyclic AMP accumulation and ultimately also led to increased amounts of internalized beta2ARs lost from the cell membrane. This reduction in the cell surface complement of beta2AR was deduced by the results of three independent readouts: analysis of functional consequences on the beta2AR/G{alpha}s/AC/cyclic AMP pathway, image analysis using autofluorescent beta2AR-enhanced GFP fusion protein, and radioligand binding analysis with a unique, cell-impermeant beta-adrenergic ligand that can quantify the relative amounts of cell surface receptor. All three of these readouts provide a compelling demonstration of the ability of LPA to alter beta2AR function and localization. The next question to answer was the details of the pathway responsible for downstream regulation of beta2AR by this lipid mediator.

The downstream signaling pathway of G{alpha}13 provides a number of experimental approaches designed to ascertain the level of involvement of key elements in the LPA effects (Fig. 6). LPA, operating via an LPA receptor (LPAR), can couple to several heterotrimeric G proteins, prominently G{alpha}i2 and G{alpha}13 (10). G{alpha}i2 was eliminated as a candidate on the basis of its well known ability to inhibit adenylyl cyclase. A G{alpha}i-induced decline in cyclic AMP in response to LPA was not observed in A431 cells. Such an LPA-stimulated, G{alpha}i-mediated decline in intracellular cyclic AMP would act to inhibit rather than to potentiate beta2AR internalization. Our initial hypothesis that G{alpha}13 was mediating the LPA effect on beta2AR trafficking was tested at several downstream loci, each confirming and extending the central hypothesis. LPA, operating via one or more of its three receptors (LPARs) all found in human epidermoid A431 cells (40), has been shown to signal via G{alpha}13 via two linked, but largely independent, signaling cascades. Activation of the LPA receptor, its cognate G protein G{alpha}13, the p115RhoGEF effector, and ultimately the small molecular weight GTPase RhoA constitutes the elements of the first cascade (29, 41). The ability of C3 exotoxin and expression of the dominant negative RhoA to potentiate beta2AR-stimulated cyclic AMP accumulation provided the first insight. Expression of the constitutively active G{alpha}13 (QLG{alpha}13), p115RhoGEF (CA-p115), or RhoA (CA-RhoA) provoked suppression of beta2AR function through internalization of the beta2AR, mimicking LPA action in its absence. The ability of LPA to activate phospholipase C-{epsilon} via G{alpha}12/13 in simian kidney COS-7 cells is likewise linked to activation of Rho (42).

The MAP kinase cascade links small molecular weight GTPases to downstream protein kinases (21). We made use of selective inhibitors of the MAP kinases p38, JNK, and Erk1,2 (via MEK inhibition) to dissect the elements of the obligate pathway from LPA to regulation of beta2AR trafficking (43). The ability of SP600125 (inhibitor of JNK) to selectively block the LPA response completed the more distal aspects of the signaling map, as elucidated for G{alpha}13 action in mouse F9 teratocarcinoma and P19 embryonal carcinoma cells (28, 29, 41), i.e. from MEKK1/4, via MKK4/MKK7, to JNK (Fig. 6). The A431 cells were selected for these studies because activation of the G{alpha}13 pathway in P19 cells and F9 embryonic cells, for example, stimulates differentiation (29, 41). Interruption of this pathway with dominant negative mutants of signaling elements blocked the ability of LPA to traffic beta2ARs. Bypassing LPA with constitutively active mutants of these same elements mimicked the effects of LPA-stimulated internalization of beta2ARs in the absence of the lipid mediator. These studies provide compelling and complementary evidence to support the schematic shown in Fig. 6. Input from activation of the G{alpha}13/p115RhoGEF/RhoA/JNK pathway to the trafficking of beta2ARs leads to a decrease in the cell surface complement of beta2AR that influences both the ability of beta-adrenergic agonists to activate cyclic AMP accumulation as well as to recover from agonist-induced desensitization. Stimulation of JNK by LPA not only activates the internalization of beta2ARs but also suppresses the recycling of internalized beta2ARs back to the cell membrane (Fig. 6). The ability of LPA to suppress the cyclic AMP accumulation mediated by beta2ARs as well by receptors for bradykinin suggests that the G{alpha}13/p115RhoGEF/RhoA pathway stimulated by LPA cross-talks to the signaling of the G{alpha}s/stimulatory adenylyl cyclase pathway by more than one GPCR. Whether or not the mechanism(s) for the LPA-induced suppression is common for both G{alpha}s protein-coupled receptors is not known. Likewise the details of how the LPA/LPAR/G{alpha}13/p115RhoGEF/RhoA pathway cross-talks to the cellular machinery that controls the trafficking of the beta2AR and bradykinin receptor remain to be established.

The observed ability of LPA, operating via G{alpha}13-based and MAP kinase-based cascades, to regulate the distribution and trafficking of the beta2AR may, in fact, be relevant to the inflammatory responses and the therapeutic potential of beta-adrenergic agonists. beta-Adrenergic agonists display the ability to suppress aspects of the inflammatory response both in vitro and in vivo (36, 37), but their ability to chronically suppress inflammation has been shown to be limited. We speculate one possibility, i.e. activation of the LPA signaling pathway, well known to be important in various inflammatory responses (44, 45), may counter the therapeutic effects of beta-adrenergic agonists at a proximal level, modulating the cell surface complement of beta2AR. LPA suppresses the steady-state level of cell surface receptors while increasing the amount of internalized beta2AR. This effect of LPA on beta2AR internalization mimics classical agonist-induced sequestration of the receptor and operates via a cross-talk from G{alpha}13/p115RhoGEF/RhoA pathway to the stimulatory adenylyl cyclase pathway but occurs in the absence of beta-adrenergic agonist.


   FOOTNOTES
 
* This work was supported by United States Public Health Service Grant DK42510 from NIDDK, National Institutes of Health (to C. C. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: Pharmacology-HSC, State University of New York, Stony Brook, NY 11794-8651. Tel.: 631-444-7873; Fax: 631-444-7696; E-mail craig{at}pharm.sunysb.edu.

3 The abbreviations used are: GPCR, G protein-coupled receptor; beta2AR, beta2-adrenergic receptor; JNK, c-Jun N-terminal kinase; GEF, guanine nucleotide exchange factor; MAPK, mitogen-activated protein kinase; MKK, MAPK kinase kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; Erk, extracellular signal-regulated kinase; LPA, lysophosphatidic acid; HEK, human embryonic kidney; HA, hemagglutinin; GFP, green fluorescent protein; siRNA, small interference RNA; Iso, isoproterenol; DN, dominant negative; KD, knockdown; CA, constitutively active; MAP, mitogen-activated protein; LPAR, LPA receptor. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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