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Prevents Desensitization of Gs-Coupled Receptors by Regulating GRK2 Association with the Plasma Membrane
Divisions of Clinical Pharmacology and Rheumatology, Department of Medicine, New York University School of Medicine, New York, New York
Received July 15, 2005; accepted December 29, 2005
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Abstract |
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increase expression and function of adenosine A2A receptors (A2ARs), although the increased function is disproportionate to the increment in expression. We therefore studied the effect of TNF- on A2A R function and desensitization in human monocytoid THP-1 cells. We observed that TNF- regulates activity of A2A Rs and other G protein-coupled receptors (GPCRs) by altering their ligand-mediated desensitization. Pretreatment of resting cells with the A2AR agonist 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) or the pan-adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine quickly desensitized cAMP responses to CGS 21680 restimulation, but TNF- treatment prevented A2AR desensitization. As expected, A2A R occupancy induced translocation of GPCR kinase-2 (GRK2) to the plasma membrane (PM). We were surprised to find that after TNF- treatment, A2AR occupancy not only failed to induce GRK2 translocation to PM but also decreased GRK2 association with PM. TNF- altered GRK2 translocation in response to the 
-adrenergic receptor agonist isoproterenol in a similar manner. Similar to GRK2, -arrestin associated with PM after A2A R stimulation in control cells but not in TNF--treated cells. C2 -ceramide, a downstream mediator in the sphingomyelinase (SMase)-dependent pathway, mimicked the effect of TNF- on GRK2 translocation. Moreover, inhibitors of the SMases and an inhibitor of c-Jun NH2-terminal kinase, also a downstream effector in the SMase pathway, reversed TNF--mediated effects on GRK2 translocation and A2A R desensitization. These results suggest a novel form of cross-talk between TNF- receptors and GPCRs; TNF- enhances GPCR function by preventing agonist-induced desensitization of GPCRs by diminishing agonist-dependent recruitment of GRK2 and -arrestin to PM by a SMase pathway-mediated mechanism.
; Ohta and Sitkovsky, 2001). We and others have demonstrated that the inflammatory cytokines IL-1 and TNF- increase the function and expression of adenosine receptors, especially A2ARs, in what seems to be feedback regulation of inflammation (Khoa et al., 2001, 2003; Bshesh et al., 2002; Trincavelli et al., 2002). However, in our prior studies on several human cell types, cytokine-induced changes in A2AR number are probably not sufficient to account for the increase in the receptor's activities (Khoa et al., 2001, 2003), leading us to speculate that TNF- and other inflammatory mediators may also increase receptor function by modulating the desensitization mechanisms of adenosine receptors.
After receptor activation, G protein-coupled receptors (GPCRs) rapidly lose the ability to stimulate intracellular signals in a process that is mediated by specific GPCR kinases (GRKs) that have the unique ability to recognize and phosphorylate their substrate only when they are in their active conformation state (Inglese et al., 1993). Of the seven GRKs cloned to date, GRK2 and the less abundant GRK3 (also known as -adrenergic receptor kinase-1 and -2, respectively) are widely distributed throughout the body (Benovic et al., 1991). GRK2 was first recognized as a kinase responsible for phosphorylation of adrenergic receptors but was later shown to be a ubiquitous member of the GRK family, which phosphorylates and desensitizes a variety of GPCRs, including adenosine receptors (Fredholm et al., 2001). Although the desensitization of GPCRs, in general, and adenosine receptors, in particular, seems to involve multiple processes (Chern et al., 1993; Palmer et al., 1994), GRK2 has been implicated as the mediator of desensitization, at least in the short term, of adenosine receptors, because targeted changes in cellular levels and/or membrane translocation of this kinase affect desensitization and activity of adenosine receptors (Mundell et al., 1997; Mundell and Kelly, 1998; Willets et al., 1999). Although GRK2 and other GRKs are regulated by both endogenous and exogenous factors (Penela et al., 2003), there has been some evidence that expression and activity of GRKs are regulated by inflammation and inflammatory cytokines in certain cells or tissues (Lombardi et al., 1999, 2001). More recent studies by Trincavelli et al. (2004) demonstrated that TNF- reduced agonist-dependent receptor phosphorylation and attenuated agonist-mediated desensitization of A2BRs in human astroglial cells. Precise mechanisms of TNF- regulation of adenosine receptor desensitization and the role of this cytokine in regulation of GPCR activity in general, however, remain unknown.
In this study on human monocytoid THP-1 cells, we determined whether TNF- treatment affects agonist-dependent desensitization of A2ARs and examined the effect of TNF- on expression and subcellular distribution of GRK2 and -arrestins in response to stimulation with agonists of A2ARs and -adrenergic receptors. In addition, we examined the signaling pathways mediating TNF- regulation of GRK2 and A2AR desensitization.
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was purchased from R&D Systems (Minneapolis, MN). CGS 21680 and 5'-N-ethylcarboxamidoadenosine (NECA) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-GRK2 and anti-A2AR antibodies and alkaline phosphatase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti--arrestin1 antibody was obtained from BD Biosciences (San Jose, CA). C2-ceramide (N-acetylsphingosine), C2-dihydro-ceramide, sphingosine, C6-ceramide (N-hexanoyl-D-sphingosine), desipramine, and GW4869 were obtained from Sigma-Aldrich. The JNK inhibitor SP 600125 was obtained from BIOMOL Research Laboratories (Plymouth Meeting, PA). The protein kinase A (PKA) inhibitor KT 5720, protein kinase C (PKC) inhibitor Ro 32-0432, MEK1/2 inhibitor PD 098059, and p38 MAPK inhibitor SB 202190 were purchased from Sigma Chemical.
Cell Culture. Human monocytoid THP-1 cells, obtained from American Type Culture Collection (Manassas, VA), were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics at 37°C in a humidified atmosphere consisting of 5% CO2. They were treated with 5 ng/ml TNF- overnight or medium alone before being subject to further treatments.
Protein Extraction and Crude Membrane Preparation. To prepare total cell lysates, cells were washed with ice-cold PBS and lysed in ice-cold lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% Nonidet P-40, and protease inhibitor cocktail (Sigma-Aldrich) for 15 to 20 min. The lysates were microcentrifuged at 14,000 rpm for 10 min at 4°C, and supernatants were collected. Crude membrane preparations were obtained after sonication of cells in a modification of a previously described technique (Arslan et al., 1997). In brief, cells were washed, resuspended in ice-cold PBS, and disrupted with a tissue homogenizer (three 10-s periods) in the presence of antiprotease cocktail (Sigma-Aldrich). Whole cells, nuclei, and other large debris were cleared by centrifugation at 300g for 10 min at 4°C, and the particulate fraction (crude membrane fraction) of the cleared supernatant was separated by microcentrifugation at maximum speed for 30 min. The protein content was determined by standard bovine serum albumin protein assay.
Western Immunoblot. Equal amounts of protein (10-40 µg/lane) were separated by 10% SDS-polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membranes. Nonspecific antibody binding was blocked with 3% skim milk in Tris-buffered saline (TBS). Subsequently, the blot membrane was incubated with a primary antibody for 1 to 4 h and then with an alkaline phosphatase-conjugated secondary antibody for another hour in TBS. After each antibody incubation, the blot was extensively washed (three to four times; 5 min each) with TBS containing 0.1% Tween 20. Finally, the blot was exposed to fluorescent ECF substrate (Amersham Pharmacia Biotech, Piscataway, NJ) and scanned using the Storm PhosphorImager system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The band intensity was then quantitated using ImageQuant software (GE Healthcare). To screen multiple proteins, after probing with an antibody, the blot was stripped off and reprobed for -actin or other proteins of interest.
Immunoprecipitation. Cultured cells (2-5 x 107 cells for each treatment) were collected and washed with ice-cold PBS. Cells were resuspended and lysed in 1 ml of lysis buffer. The whole-cell lysate (WCL) was precleared for 30 min at 4°C with 0.25 µg of appropriate control (normal) IgG, corresponding to the host species of the primary antibody, and 20 µl of protein A/G-agarose (Santa Cruz Biotechnology, Inc.). The precleared cell lysates were subsequently incubated with 2 µg of the primary antibody for 1 h before the addition of 20 µl of protein A/G-agarose beads, and the incubation was continued overnight at 4°C with constant rotation. Next, the agarose beads were pelleted by centrifugation at 2500 rpm (
1000g) for 5 min at 4°C. After extensive washes (four to five times) with ice-cold lysis buffer, the beads were resuspended in sample loading buffer, boiled for 4 to 5 min, and subject to SDS-polyacrylamide gel electrophoresis and Western immunoblot with antibodies of interest.
Study of Adenosine Receptor Desensitization. Cells were pretreated overnight with medium alone or TNF- and then stimulated with either 10 µM CGS 21680 for various times (0-60 min) or with 10 µM NECA (30 min). The medium was then removed, and cells were washed and resuspended with fresh medium and stimulated with 1 µM CGS 21680 for various times. Cells were then assayed for intracellular cAMP concentrations. cAMP Quantification. After appropriate treatment, culture supernates were discarded, and cells were washed with ice-cold PBS. Intracellular cAMP levels were determined using the Biotrak cAMP enzyme immunoassay system (Amersham Pharmacia Biotech). In brief, washed cells from each treatment were lysed in 200 µl of the lysis buffer provided with the kit. Cell debris was removed by brief centrifugation. The lysate was applied in duplicate to 96-well plates provided by the manufacturer, and all steps of the assay were carried out following the manufacturer's instructions. cAMP concentrations were normalized to the protein content measured by standard protein assay.
Data Analysis. All statistical analysis was performed using Prism software (GraphPad Software, Inc., San Diego, CA). Data are presented as mean ± S.E.M. when appropriate. One- or two-way analysis of variance (ANOVA) was used to determine overall statistical significance. Differences with a P value less than 0.05 were considered significant.
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Diminished Agonist-Induced A2AR Desensitization. Because A2ARs are generally coupled to Gs and activate adenylyl cyclase, increasing cAMP formation, we evaluated the agonist-induced desensitization of A2ARs in control and TNF--treated THP-1 cells by measuring cAMP accumulation in response to the selective A2AR agonist CGS 21680 (1 µM) after pretreatment of the cells with either 10 µM CGS 21680 or 10 µM NECA, a pan-adenosine receptor agonist. Analysis of the time course of agonist-mediated desensitization of adenosine A2A receptor-stimulated increases in intracellular cAMP demonstrated a significantly greater response of TNF- treated cells than the control cells to agonist stimulation whether or not they underwent desensitization with agonist (Fig. 1A). More importantly, these desensitization studies revealed that most of the A2ARs in control cells were quickly desensitized after 10 µM CGS 21680 pretreatment, whereas desensitization of A2ARs in TNF--treated cells was not only delayed but also greatly inhibited (P < 0.01; TNF--treated versus control cells; two-way ANOVA; Fig. 1, A and B). Likewise, after 30-min NECA pretreatment (10 µM), A2ARs in control cells seemed to be desensitized, and these cells were unresponsive to CGS 21680 stimulation, whereas TNF--treated cells were still responsive to CGS 21680 with an increase in cAMP level up to 127 ± 7% of basal level (Fig. 1C).
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on GRK2 Expression and Subcellular Localization. It has been previously demonstrated that Gs-coupled receptors, including A2ARs, are quickly phosphorylated by GRK2 and other GRKs and subjected to desensitization after agonist exposure. To determine the mechanism for TNF- inhibition of A2AR desensitization, we next studied the effect of TNF- on expression and subcellular distribution of GRK2 after agonist stimulation. Neither TNF- treatment nor CGS 21680 stimulation significantly changed the total cellular level of GRK2 (Fig. 2A; data not shown). Because there were no changes in total GRK2 levels and proximity to its receptor target is probably more important for the function of this kinase, we examined the effect of TNF- treatment on translocation of GRK2 from cytosol to the plasma membrane after agonist stimulation. As demonstrated by immunoblot, in control (untreated) THP-1 cells the level of GRK2 in the plasma membrane quickly increased after stimulation with CGS 21680 in a time-dependent manner and reached up to 124 ± 5% of baseline within 10 to 20 min (Fig. 2B). In contrast, in cells that were pretreated overnight with TNF-, agonist stimulation failed to induce GRK2 translocation to the plasma membrane and instead resulted in a time-dependent decrease in the membrane GRK2 level to 80 ± 3% of basal level within 10 to 20 min (Fig. 2, A and B). The difference in response to CGS 21680 between control and TNF--treated cells in terms of GRK2 translocation was highly significant (P < 0.001; TNF- versus control; two-way ANOVA). Incubation of cells with TNF- for shorter periods also significantly inhibited CGS 21680-induced translocation of GRK2 to the plasma membrane after short-term treatment (Fig. 2C; P < 0.01 for all time points versus control). The effect of TNF- on GRK2 translocation did not require protein synthesis because coincubation of cells with cycloheximide, a protein synthesis inhibitor, did not alter the inhibitory effect of TNF- on agonist-dependent GRK2 relocalization (Fig. 2D; P > 0.05; two-way ANOVA).
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To determine whether the regulatory effect of TNF- on agonist-induced GRK2 translocation is specific for the A2ARs or whether this regulatory mechanism is a more general phenomenon for other Gs-coupled receptors, we examined GRK2 relocalization in control and TNF--treated THP-1 cells in response to isoproterenol, a selective -adrenergic receptor agonist. As demonstrated in Fig. 3, isoproterenol, like CGS 21680, induced translocation of GRK2 to the plasma membrane in control cells, whereas TNF- treatment decreased the membrane-associated GRK2 level after isoproterenol stimulation (P < 0.01; TNF- versus control; two-way ANOVA).
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Inhibits Agonist-Induced Receptor-GRK2 Association. It was previously reported that activated Gs-coupled receptors bind to membrane-bound GRKs, resulting in kinase activation and phosphorylation of these receptors (Li et al., 2003; Pao and Benovic, 2005). To determine the effect of TNF- on interaction between A2ARs and GRK2, we immunoprecipitated A2ARs from the total cell lysates from untreated and treated cells and then probed for the presence of GRK2. As shown in Fig. 4A, without agonist stimulation little GRK2 was associated with A2ARs in either control or TNF--treated THP-1 cells. As expected, CGS 21680 stimulation clearly increased GRK2 association with A2ARs in control cells. However, in TNF--treated cells, the agonistinduced increase in A2AR-GRK2 association was almost abrogated, consistent with the TNF--induced decrease in the membrane-associated GRK2 level during agonist stimulation (Fig. 4).
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on -Arrestin Translocation. -Arrestin1/2 proteins bind phosphorylated Gs-coupled receptors and mediate their internalization. We next determined whether TNF--induced changes in GRK2 translocation after agonist stimulation lead to similar changes in membrane recruitment of -arrestin protein. As demonstrated by Western immunoblot analysis of crude membrane and cytosolic fractions of THP-1 cells, -arrestin1 was mostly detected in the cytosol (Fig. 5A). Nonetheless, in control cells CGS 21680 stimulation quickly and significantly increased -arrestin1 levels in the plasma membrane, up to 121 ± 5% of the basal level within 5 to 10 min (P < 0.01), whereas the membrane -arrestin1 level in TNF--treated cells was not significantly increased after CGS 21680 stimulation (Fig. 5). The difference in agonist-induced changes in membrane-bound -arrestin levels between control and TNF--treated cells was significant (P < 0.05; TNF- versus control; two-way ANOVA). Neither TNF- nor CGS 21680 induced any detectable changes in the cytosolic -arrestin1 levels (Fig. 5A; data not shown). These results indicate that TNF- treatment, consistent with its effect on GRK2 translocation, suppresses -arrestin recruitment to the plasma membrane during stimulation of Gs-coupled receptors.
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Involvement of Sphingomyelinase-Dependent Pathway in Mediating Effects of TNF- on GRK2 Translocation and A2AR Desensitization. Because the effect of TNF- on agonist-dependent GRK2 translocation does not depend on new protein synthesis (Fig. 2D), we examined the role of other signaling pathways for TNF- in the prevention of desensitization of A2ARs. TNF- has previously been shown to signal for cellular activation in monocytes and macrophage-like cells (Wiegmann et al., 1992; Dbaibo et al., 1993; MacKichan and DeFranco, 1999; Mallampalli et al., 1999) via activation of sphingomyelinases (SMases), which generate lipid-derived signaling elements such as C2-ceramide. Pretreatment with 20 µM C2-ceramide decreased GRK2 levels in the plasma membrane after CGS 21680 stimulation to 84 ± 5% of basal level (Fig. 6, A and B), mimicking the effect of TNF-. Treatment of the cells with C2-dihydroceramide, an inactive analog of C2-ceramide, did not change the effect of CGS 21680 on GRK2 translocation (Fig. 6A; data not shown). Of other ceramide isoforms tested, 50 µM sphingosine was toxic to the cells and 10 µM C6-ceramide did not clearly alter the agonist-induced membrane translocation of GRK2 (data not shown).
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To further understand the involvement of SMAse pathways in TNF- signaling, we examined the effects of selective inhibitors of acid and neutral SMases on the capacity of TNF- treatment to modulate TNF--mediated inhibition of GRK2 translocation to the plasma membrane. Cotreatment of THP-1 cells with TNF- and either desipramine, an inhibitor of acid SMase, or GW4869, an inhibitor of neutral SMase, partly but significantly reversed the TNF- effect on CGS 21680-dependent GRK2 translocation (P < 0.05 for both inhibitors; two-way ANOVA), whereas combination of both inhibitors almost completely reversed the TNF- effect (P < 0.01 versus no inhibitor; two-way ANOVA; Fig. 6C). These inhibitors did not interfere with agonist-mediated GRK2 translocation in control cells (Fig. 6C). To determine whether the reversal of TNF- inhibition of GRK2 translocation by SMAase inhibitors affects GRK2-mediated receptor desensitization, we studied the effect of desipramine and GW4869 on A2AR desensitization along with TNF- treatment. TNF- greatly inhibited A2AR desensitization after 30-min CGS 21680 pretreatment, as shown previously (Fig. 1), and this inhibitory effect of TNF- was almost completely abrogated in the presence of SMase inhibitors (P < 0.01 versus no inhibitor; two-way ANOVA; Fig. 6D).
Effects of Protein Kinase Inhibitors on GRK2 Translocation. It was previously reported that GRK2 activity and membrane targeting are also regulated by phosphorylation of GRK2 by other protein kinases, including PKA, PKC, and MAPKs (Penela et al., 2003). To determine whether any of these kinases are involved in mediating the TNF- effect on GRK2 translocation, we studied the effect of specific inhibitors of these kinases on the phenomenon. We found that inhibition of PKC by Ro 32-0432 moderately inhibited and PKA inhibitor KT 5720 slightly affected the capacity of CGS 21680 to stimulate GRK2 association with the plasma membrane in control cells (P < 0.05 for Ro 32-0432 and P > 0.05 for KT 5720 versus no inhibitor; two-way ANOVA; Fig. 7, A and B). None of the inhibitors, including those of p38 MAPK and MEK1/2, diminished the effect of TNF- on CGS 21680-induced GRK2 translocation (Fig. 7, A-D).
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and ceramides are known to activate JNK signaling via activating its upstream kinases (Sathyanarayana et al., 2002; Shen et al., 2003), and other downstream protein kinases mediate effects of adenosine and other GPCRs. Treatment of cells with the selective JNK inhibitor SP 600125 (25 µM) minimally enhanced CGS 21680-induced GRK2 translocation in control cells. More interestingly, in TNF--treated cells JNK inhibition significantly and greatly reversed the TNF- effect on CGS 21680-dependent GRK2 translocation (Fig. 7E; P < 0.05 for SP 600125 versus no inhibitor; two-way ANOVA). Moreover, the effect of SP 600125 on TNF- inhibition of GRK2 translocation correlated with the reversal of TNF- inhibition of A2AR desensitization of the cAMP response by this inhibitor (Fig. 7F).
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induces a 20 to 30% increase in A2AR expression. Although one would expect a shift in the dose-response curve for this receptor, we observed that there was, in fact, a threshold effect. Thus, concentrations of ligand that should stimulate maximal receptor function do not stimulate any detectable response unless the cells have been pretreated with TNF-. To better understand this apparent shift in the threshold for A2AR function, we carried out the current study. We found that TNF- inhibits agonist-dependent desensitization of A2ARs in human monocytoid THP-1 cells, an observation that further elucidates the TNF--stimulated change in receptor function. These findings provide a more solid explanation for the enhanced function and activity of A2ARs observed after TNF- and IL-1 treatment in our previous studies (Khoa et al., 2001, 2003). Together, these data suggest that TNF- and probably some other inflammatory cytokines promote A2AR signaling and function through multiple effects, not only enhancing the receptor number but also regulating mediators of receptor signaling and desensitization. Our demonstration that TNF- inhibits A2AR desensitization is also consistent with the previous report by Trincavelli et al. (2004) that TNF- inhibits desensitization of A2BRs and enhances the functions of these receptors in human astroglial cells. Moreover, the current study provides further mechanistic insight into TNF--mediated inhibition of receptor desensitization. Our results demonstrate that the cytokine promotes the agonist-dependent dissociation of GRK2, a kinase primarily responsible for phosphorylation and desensitization of GPCRs, from the plasma membrane and diminishes recruitment of -arrestin to the plasma membrane after stimulation with the agonists of A2ARs and -adrenergic receptors, leading to diminished phosphorylation and desensitization of these receptors. Findings from our studies and others that A2ARs, A2BRs, and -adrenergic receptors respond similarly to TNF- treatment suggest that this is a common regulatory mechanism for other Gs-coupled receptors.
Previous studies have demonstrated that the activity of GRKs decreases in lymphocytes of rheumatoid arthritis patients and that the proinflammatory cytokines interferon-
and IL-6 down-regulate cellular expression and activity of GRK2 and GRK6 and increase sensitivity to 2-adrenergic receptor activation (Lombardi et al., 1999). Lombardi et al. (2001) have also reported that inflammation induces a tissuespecific down-regulation of GRKs in the immune system of animals with adjuvant arthritis. Our results indicate that TNF- decreases the activity of GRK2 as the cytokine was shown to inhibit A2AR desensitization and to enhance receptor function, although this effect does not seem to result from decreased expression of GRK2 but rather from diminished translocation of GRK2 to the plasma membrane. In sum, our data and the previously published results indicate that decreased association of GRK2 and -arrestin with GPCRs in TNF--treated cells is responsible for decreased agonist-induced receptor desensitization and increased activity of corresponding GPCRs.
It is interesting that TNF- treatment not only prevents agonist-dependent membrane translocation of GRK2 but also seems to promote agonist-dependent dissociation of GRK2 from the membrane. Unlike most other members of the GRK family, which are generally associated with the plasma membrane (Stoffel et al., 1994; Pronin et al., 1998), GRK2 resides predominantly in the cytosol and translocates to the plasma membrane via a carboxy-terminal binding domain or by directly binding to phospholipids upon receptor ligation (Daaka et al., 1997; Carman et al., 2000). Multiple mechanisms have been shown to regulate the association of GRK2 with the plasma membrane and its activity, including regulation of GRK2 by other kinases, such as PKA, PKC, and MAPKs, during activation of GPCRs (Penela et al., 2003). Although inhibition of PKA and PKC might partly affect phosphorylation and membrane translocation of GRK2 in control cells, the inability of inhibitors of these kinases to alter the effect of TNF- on the ligand-induced association of GRK2 with the plasma membrane suggests that other mechanisms are responsible for ligand-induced desensitization of adenosine A2A receptors. Moreover, our results indicate involvement of the TNF--activated, sphingomyelinase-dependent pathway (signaling through the protein kinase JNK) (Dbaibo et al., 1993; Muller et al., 1998; Mallampalli et al., 1999) in the TNF--mediated blockade of A2A receptor desensitization. It is still unclear, however, how ceramide and/or JNK, activated by TNF-, modulate GRK2 subcellular localization during GPCR occupancy. An alternative explanation is suggested by the finding of Liu et al. (2002) that growth hormone-induced formation of ceramide is mediated by a G-dependent pathway and that ceramide formation is blocked by a mutated form of GRK2, which is a known G scavenger (Koch et al., 1994). Thus, it is possible that TNF- and its downstream effector ceramide might interfere with G-GRK2 binding and thus prevent GRK2 association with the plasma membrane during GPCR stimulation. Nonetheless, ceramide-mediated mechanisms might not be solely responsible for the TNF- effect on GRK2 translocation. Decreased activity and membrane availability of GRK2 in TNF- treated cells during GPCR activation may also result from increased binding of GRK2 to other cytosolic proteins, such as Raf kinase inhibitor protein, an inhibitor that can alternate binding to either Raf-1 or GRK2 (Lorenz et al., 2003). TNF- treatment may promote Raf kinase inhibitor protein-GRK2 binding and thereby retain GRK2 in the cytosol during GPCR ligation, and this hypothesis needs to be investigated.
TNF-, one of the most prominent inflammatory cytokines, plays a pivotal role in orchestrating host defense and inflammatory processes in vertebrates. Although critical for host defense, TNF-, in concert with other proinflammatory cytokines, such as IL-1 and IL-6, has been strongly implicated in the pathogenesis of inflammatory and autoimmune diseases, such as rheumatoid arthritis and inflammatory bowel disease, and it is responsible for progressive tissue injury and damage in those conditions (Feldmann et al., 1998). The development of TNF- and IL-1 inhibitors has therefore been one of the most active areas of drug development over the last decade for the treatment of inflammatory diseases (Vilcek and Feldmann, 2004). Although clinically effective, these inhibitors still pose serious side effects (Cush, 2004), some of which result from unexpected interactions between TNF- and other receptors. Our results suggest one such interaction between TNF- and Gs-linked receptors that could account for a distinct toxicity of the anti-TNF- therapies, exacerbation of congestive heart failure (CHF). In a rat model of CHF, Wang et al. (2005) demonstrated that density and protein content of 1-adrenergic receptors were increased, whereas GRK isoforms, including GRK2, and -arrestin1 were decreased in the membrane fraction of cells from failing hearts. Increased adrenergic tone is a compensatory mechanism in the failing heart, and interference with this compensation may exacerbate heart failure. A recent study has demonstrated increased production of TNF- and the accompanying up-regulation of A2AR in peripheral blood mononuclear cells from CHF patients (Capecchi et al., 2005). Thus, we speculate that drugs targeting inflammatory cytokines, such as TNF- or IL-1, which are beneficial in the treatment of inflammatory diseases, may undercut TNF--induced enhancement of -adrenergic tone, thereby exacerbating CHF.
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ABBREVIATIONS: IL, interleukin; TNF, tumor necrosis factor; A2AR, adenosine A2A receptor; GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; CGS 21680, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine; NECA, 5'-N-ethylcarboxamidoadenosine; JNK, c-Jun NH2-terminal kinase; PKA, protein kinase A; PKC, protein kinase C; MEK, mitogen-activated protein kinase kinase; MAPK, mitogen-activated protein kinase; SB 202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole; PBS, phosphate-buffered saline; WCL, whole cell lysate; TBS, Tris-buffered saline; ANOVA, analysis of variance; SMase, sphingomyelinase; CHF, congestive heart failure; WB, Western blot; GW4869, N, N'-bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3'-p-phenylene-bis-acrylamide dihydrochloride; SP600125, 1,9-pyrazoloanthrone anthra(1,9-cd)pyrazol-6(2H)-one; KT 5720, 2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-(9,10,12)-9,12-epoxy-1H-diindolo(1,2,3-fg: 3',2',1'-kl)pyrrolo (3,4-i)(1,6)benzodiazocine-10-carboxylic acid, hexyl ester; Ro 32-0432, (S)-3-(8-((dimethylamino)methyl)-6,7,8,9-tetrahydropyrido(1,2-a)indol-10-yl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrol-2,5-dione; PD 098059, 2'-amino-3'-methoxyflavone.
Address correspondence to: Dr. Bruce N. Cronstein, Department of Medicine, New York University School of Medicine, 550 First Ave., New York, NY 10016. E-mail: bruce.cronstein{at}nyumc.org
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