The FASEB Journal • Research Communication
PKC-dependent regulation of the receptor locus dominates functional consequences of cysteinyl leukotriene type 1 receptor activation Deepak A. Deshpande,* Rodolfo M. Pascual,* Si-Wei Wang,* Delrae M. Eckman,* Ellen C. Riemer,† Colin D. Funk,‡ and Raymond B. Penn*,1 *Department of Internal Medicine and Center for Human Genomics and †Department of Pathology, Wake Forest University Health Science, Winston-Salem, North Carolina; and ‡Departments of Physiology and Biochemistry, Queen’s University, Kingston, Ontario, Canada Leukotrienes are important lipid mediators of asthma that contribute to airway inflammation and bronchoconstriction. Critical mechanisms for physiological regulation of the main G protein-coupled receptor (GPCR) mediating the leukotriene responses in asthma, cysteinyl leukotriene type 1 receptor (CysLT1R), have not been delineated. Although desensitization of GPCRs is a well-established phenomenon, studies demonstrating its physiological relevance are lacking. Here, we demonstrate that relief of PKCmediated desensitization of endogenous CysLT1Rs augments multiple LTD4-stimulated cellular functions, with associated increases in intracellular signaling events. In analyses of airway smooth muscle contraction ex vivo, PKC inhibition augmented LTD4-stimulated contraction, and increased phosphoinositide hydrolysis and calcium flux in both murine and human airway smooth muscle cells. Similarly, for human monocytes, PKC inhibition augmented LTD4-stimulated calcium flux and cell migration assessed in transwell chamber experiments and also augmented LTD4-induced production of monocyte chemotactic protein assessed by ELISA. In contrast, PKC inhibition had no effect or slightly attenuated these cell functions and signaling events promoted by other receptor agonists, suggesting that despite antithetical effects on downstream events, desensitization of the CysLT1R is the principal mechanism by which PKC regulates the functional consequences of CysLT1R activation.—Deshpande, D. A., Pascual, R. M., Wang, S.-W., Eckman, D. M., Riemer, E. C., Funk, C. D., Penn, R. B. PKC-dependent regulation of the receptor locus dominates functional consequences of cysteinyl leukotriene type 1 receptor activation. FASEB J. 21, 2335–2342 (2007)
ABSTRACT
Key Words: G protein-coupled receptor 䡠 cysteinyl leukotriene 䡠 protein kinase C 䡠 desensitization 䡠 airway smooth muscle
functions important in inflammatory processes underlying numerous diseases, including asthma (1, 2) and cardiovascular disease (3). Despite its physiological importance and the fact that this receptor was cloned over 7 yr ago (4, 5), only one study to date has identified regulatory features of the CysLT1R. Naik et al. (6) established that unlike most G protein-coupled receptors (GPCRs), whose agonist-induced responsiveness and endocytosis are regulated by GPCR kinases (GRKs) and arrestins (7), the CysLT1R is unique in that both agonist-induced desensitization and internalization are dependent primarily on protein kinase C (PKC) activity. Because most studies of GPCR regulation involve analyses of overexpressed receptors in artificial expression systems such as COS or HEK293 cells, insight into the physiological significance of receptor regulation is often lacking. In only a handful of instances have paradigms of GPCR regulation been shown to be of physiological relevance through demonstration of their impact on cell function. The most compelling evidence comes from Koch, Rockman, and colleagues. Through a series of elegant studies using a combination of cell and in vivo systems, GRK-mediated desensitization of beta-adrenergic receptors in cardiomyocytes has been shown to not only mediate cardiac tachyphylaxis to infused or endogenous beta-agonist and diminish cardiac contractility but also constitutes a pathogenic mechanism of heart failure (8 –11). Because cellular functions are often the result of the integration of multiple signaling events, the influence of a specific mechanism regulating the GPCR locus on such functions can be constrained. Moreover, multiple, redundant mechanisms of receptor desensitization can exist, and signaling elements downstream of the receptor are also subject to regulation. Thus, the impact of
1
Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate cell 0892-6638/07/0021-2335 © FASEB
Correspondence: Center for Human Genomics, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA. E-mail:
[email protected] doi: 10.1096/fj.06-8060com 2335
receptor modifications and regulation of proximal signaling events on cellular functions can be offset or overshadowed by numerous other control systems. These factors, in addition to other homeostatic mechanisms that constrain responses of low abundance, endogenously expressed proteins in primary cell types, contribute to the difficulty in demonstrating the relevance of GPCR regulatory mechanisms that target the receptor locus. In the present study, we demonstrate that PKCmediated desensitization of the CysLT1R translates into the attenuation of leukotriene D4- (LTD4-) regulated cellular functions in multiple physiological systems. PKC inhibition is shown to enhance contraction of airway smooth muscle (ASM), as well as migration and chemokine production in monocytic cells in response to LTD4, whereas these same functions mediated by other receptors are either unaffected or slightly inhibited with PKC inhibition. Thus, PKC-dependent agonist-specific desensitization of the CysLT1R is a robust phenomenon of physiological relevance.
MATERIALS AND METHODS
Transgenic mice with smooth muscle-specific expression of the human CysLT1R (hCysLT1R) were generated as described previously (12). Transgene expression was assessed by PCR analysis of total genomic DNA isolated from tail clips, as described previously (12). Ex vivo measurement of tracheal contraction and isolation of ASM cells were performed under protocols approved by the Animal Care and Use Committee at Wake Forest University Health Sciences. Mice were sacrificed by CO2 inhalation and tracheae were excised and cleaned of surrounding connective tissue. For contractile studies, tracheae were sectioned (horizontally) in half, and each section mounted onto a multiwire myograph system consisting of myograph chambers, a block interface for connecting to force transducers, and Powerlab (ADInstruments, Colorado Springs, CO) for data recording. Data collection and analysis were carried out using Chart5 software for Windows (ADInstruments). Tracheal rings were bathed in Krebs-Henseleit solution (pH 7.40 –7.45) and maintained at 37°C with 5% CO2 and 95% O2 with frequent changing of the solution. The tracheal segments were allowed to stabilize and subsequently set at a preload tension of 0.5 g. The rings were stimulated with 10 M methacholine (MCh) for 5 min, then washed and reset to 0.5 g preload, which was maintained through a 30-min equilibration period. Rings were then pretreated with either 10 M bisindolylmaleimide (Bis) I, 10 M Bis V, 10 M RO-31– 8220, or vehicle (DMSO) for 10 min. The rings were then stimulated with various different concentrations of LTD4 or MCh, and the responses were recorded for 10 min. In a select set of experiments, the rings were washed thoroughly with K-H buffer and allowed to recover for 10 min. The rings were rechallenged with LTD4 or MCh, and tensions were recorded for 10 min. The net contractile responses were determined by subtracting the baseline tension from that of the peak tension on agonist stimulation, and the change in the tension was normalized to the wet weight of the tracheal ring segment. Vol. 21
August 2007
Human ASM cell cultures were generated from tissue obtained at autopsy, as described by Panettieri et al. (13) under a protocol approved by the Wake Forest University Health Sciences Institutional Review Board. For generation of murine ASM cultures, tracheae harvested from 4 or 5 mice were cleaned of surrounding tissues and pooled to isolate ASM cells using the above procedure for human ASM cultures with minor modifications as described previously (14). ASM cells were characterized by immunostaining using anti-␣-actin and anti- smooth muscle myosin heavy-chain antibodies. Purification of human monocytes from peripheral blood Blood was collected by venipuncture from healthy human volunteers and peripheral blood mononuclear cells (PBMCs) were isolated as described previously (15). PBMCs were plated onto a 10-cm dish, and monocytes were allowed to attach to the surface for 2 h. The adherent monocytes were washed twice with ice-cold PBS (pH 7.4) and detached by incubation with PBS/5 mM EDTA on ice for 15 min. The recovered cells were centrifuged at 2000 rpm and resuspended in plain RPMI medium. Cell count and viability were determined using an automated cell counter (Vi-CELL; Beckman Coulter, Fullerton, CA). Intracellular calcium ([Ca2ⴙ]i) measurements
Generation of transgenic mice, airway contractile measurements ex vivo
2336
Generation of murine and human ASM cultures
1st and 2nd passage human and murine ASM cells and freshly isolated human monocytes were used in experiments assessing regulation of CysLT1R-mediated Ca2⫹ flux. Cells grown on glass coverslips were washed and loaded with 5 M Fura-2 AM for 30 min at 37°C. The cells were washed and maintained in Hank’s Balanced Salt Solution containing 10 mM HEPES, 11 mM glucose, 2.5 mM CaCl2, and 1.2 mM MgCl2 (HBSS; pH 7.4). The coverslips were mounted onto an open slide chamber and [Ca2⫹]i flux was assessed using a dual excitation fluorescence photomultiplier system (IonOptix, Milton, MA) as described previously (6). The fluorescence intensities were converted into absolute calcium concentration using a calibration curve derived from maximum (ionophore) and minimum (EGTA) calcium flux in these cells, as per the software. The cells were treated with Veh or 10 M Bis I for 10 min, and basal [Ca2⫹]i was determined. The cells were subsequently stimulated with LTD4, histamine, or MCh. The net calcium response was calculated by subtracting the basal from peak [Ca2⫹]i on agonist stimulation. For analyses of human ASM cells and monocytes, experiments were repeated using cells obtained from different donors. Measurement of phosphoinositide (PI) generation First and second passage human and murine ASM cells were grown to confluence on 24-well plates and loaded with 2 Ci/ml myo-[3H]inositol for 18 h. After washing with phosphate-buffered saline the cells were incubated with DMEM containing 5 mM LiCl for 10 min. The cells were then treated with vehicle or 10 M Bis I for 10 min and stimulated with vehicle (basal), LTD4, histamine or MCh for 30 min. Reactions were quenched with 20 mM formic acid and inositol, and phosphoinositide (PI) fractions were separated by anion exchange chromatography as described previously (6). PI production was calculated by dividing PI fraction by total (PI ⫹ inositol) inositol fraction and the data were reported as fold basal (agonist-stimulated/vehicle).
The FASEB Journal
DESHPANDE ET AL.
Monocyte chemotaxis assay
RESULTS
Chemotaxis of monocytes was determined using Costar Transwell plates fitted with polycarbonate membrane of 5-M pore size, as described previously (16). Human monocytes isolated from peripheral blood were incubated with Veh or 10 M Bis I for 10 min. RPMI medium containing Veh, 100 nM LTD4, or 100 ng/ml monocyte chemotactic protein-1 (MCP-1) was added to the bottom chamber of the plate. 200,000 cells per well were added to the upper chamber also containing the pretreatment agent and allowed to incubate at 37°C for 1 h, after which cell number in the bottom chamber was determined.
PKC-dependent regulation of GPCR-mediated airway smooth muscle contraction, signaling
Regulation of MCP-1 production in THP-1 cells Because analysis of regulation of MCP-1 in monocytes production requires a large number of cells, experiments were performed using the human monocytic cell line THP-1. THP-1 cells grown in RPMI medium supplemented with 10% FBS were centrifuged and resuspended in serum-free RPMI media. The cells were treated with vehicle or 10 M Bis I for 10 min and subsequently stimulated with vehicle, 100 nM LTD4, or 100 ng/ml LPS. The supernatant was harvested before and 3 h after the addition of the stimulants and frozen at –20°C. MCP-1 levels were subsequently measured by ELISA (R&D Systems, MN) as per manufacturer’s protocol. Statistical analysis Data analysis was carried out using GraphPad Prism, and data were expressed as mean ⫾ se. Group comparisons were performed using one-way ANOVA or Student’s t test where appropriate, with a P value of ⬍ 0.05 sufficient to reject the null hypothesis.
To assess the functional consequences of CysLT1R regulation by PKC, we examined contractile properties of tracheal rings excised from transgenic mice expressing the human CysLT1R (12). Wild-type C57BL/6 mice express an extremely low level of endogenous CysLT1R, and tracheal rings exhibit a weak contractile response to LTD4. Transgenic expression of human CysLT1R at physiological levels (20 fmol/mg protein) increases LTD4-mediated ASM tension development to approximately one-half to two-thirds that stimulated by m3 mAChR activation (Fig. 1 and (12)). As shown in Fig. 1A, inhibition of conventional PKC isoforms by pretreatment with Bis I significantly increased LTD4stimulated tension development in tracheal rings. Pretreatment with the PKC inhibitor RO-31– 8220 had a similar effect (not shown), whereas pretreatment with Bis V (structurally similar to Bis I but lacking the ability to inhibit PKC) had no effect (Fig. 1B). Moreover, and in contrast to the effect on LTD4-stimulated contraction, pretreatment with Bis I caused a small but significant decrease in MCh-induced contraction (Fig. 1C). Interestingly, pretreatment with Bis I also significantly augmented LTD4-stimulated contraction of the endogenous murine CysLT1R (Fig. 1D), which possesses the same PKC-consensus sites (12) contained in the human CysLT1R (6). LTD4-mediated contraction of rings from transgenic mice was antagonized in a dose-dependent manner by the selective CysLT1R antagonist MK-571 (Fig. 1E).
Figure 1. Regulation of ASM contraction by PKC. Tracheal rings from transgenic mice expressing human CysLT1R were incubated with Veh (A–C) or 10 M Bis I (A, C) or Bis V (B) for 10 min, then stimulated with indicated concentrations of LTD4 or MCh. D) Tracheal rings from wild-type mice were incubated with Veh or 10 M Bis I for 10 min, then stimulated with 100 nM LTD4. E) Dose-dependent effect of MK571 on tension development stimulated by 100 nM LTD4. *P ⬍ 0.05, Bis I pretreatment group vs. Veh pretreatment group. In rings stimulated with 10 M MCh, a trend (P⫽0.10, n⫽11) was observed for Bis I-mediated inhibition of tension development. n ⫽ 11 (A), n ⫽ 8 (B and E), n ⫽ 11 (C), n ⫽ 4 (D). FUNCTIONAL CONSEQUENCES OF CYSLT1R REGULATION
2337
Further evidence of the capacity of PKC inhibition to attenuate the functional consequences of CysLT1R desensitization in ASM is provided when tracheal rings from hCysLT1R mice are repeatedly challenged with LTD4. In preparations pretreated with vehicle, a second challenge with LTD4 resulted in a loss of tension development (Fig. 2A, E). However, this loss was significantly attenuated in rings pretreated with Bis I (Fig. 2B, E). Conversely, MCh-stimulated tension development was maintained on rechallenge and was unaffected by PKC inhibition (Fig. 2C, D, E). Analysis of agonist-induced PI production and Ca2⫹ flux in early passage murine ASM cells from hCysLT1R mice demonstrates that the effects of PKC inhibition on contraction-promoting proximal signaling events are consistent with the functional effects observed in tracheae. Bis I pretreatment significantly increased both LTD4-stimulated PI hydrolysis and peak Ca2⫹ flux in cultures derived from murine trachea from hCysLT1R transgenic mice, whereas a trend toward inhibiting MCh-stimulated PI production (P⫽0.2) and Ca2⫹ flux (P⫽0.09, n⫽8) was observed (Fig. 3A, B). With human ASM cells CysLT1R expression wanes rapidly in culture (R. B. Penn, unpublished observation), but LTD4stimulated signaling can be assessed in early passage cells. As shown in Fig. 3C and D, PKC inhibition significantly increased LTD4-stimulated PI production and Ca2⫹ flux in human ASM cells whereas H1 histamine receptor signaling is slightly but not significantly inhibited. Collectively, these experiments demonstrate that despite what appears to be a capacity of PKC inhibition to inhibit downstream signaling that promotes PI generation and Ca2⫹ flux in ASM, the relief of
PKC-mediated desensitization of the CysLT1R is sufficient to promote an increase in LTD4-stimulated Ca2⫹ flux that translates into an increase in ASM tension development. Functional consequences of CysLT1R regulation in human monocytes Previous studies have shown that human monocytes express CysLT1Rs (16, 17) and that LTD4 stimulates monocyte chemotaxis that is inhibited by the CysLT1R antagonist monteleukast (18). To characterize regulation of LTD4-mediated chemotaxis, human monocytes were isolated from peripheral blood and PKC-dependent regulation of migration assessed in classical Transwell chamber experiments. As shown in Fig. 4A, Bis I pretreatment significantly increased LTD4-mediated migration, while slightly inhibiting migration promoted by MCP-1 (P⫽0.209, n⫽11). LTD4-stimulated Ca2⫹ flux in monocytes was also significantly increased in human monocytes by pretreatment with Bis I (Fig. 4B). LTD4 can also stimulate induction of MCP-1 production in monocytic cells, an effect reversed by the CysLT1R selective antagonist pranlukast (19). We therefore characterized PKC-dependent regulation of LTD4-stimulated MCP-1 production in the monocytic cell line THP-1. Pretreatment with Bis I significantly increased LTD4-stimulated MCP production by ⬎2 fold (Fig. 4C). Conversely, MCP-1 production stimulated by LPS was unaffected by Bis I pretreatment. LTD4-stimulated MCP-1 production in THP-1 cells was antagonized in a dose-dependent manner by the selective CysLT1R antagonist MK-571 (Fig. 4D).
Figure 2. Effects of PKC inhibition on contraction elicited by repeated challenge with LTD4, methacholine. Rings were pretreated with vehicle (A, C) or Bis I (B, D), challenged with 100 nM LTD4 (A, B) or 10 M MCh (C, D), washed, then rechallenged with 100 nM LTD4 (A, B) or 10 M MCh (C, D). Graphs depict raw tracings of absolute tension not normalized to tissue weight. E) Responses were calculated as the ratio of tension development stimulated by the second challenge (S2) to that stimulated by the first challenge (S1). *P ⬍ 0.05, Bis I pretreatment group vs. Veh pretreatment group, n ⫽ 9. 2338
Vol. 21
August 2007
The FASEB Journal
DESHPANDE ET AL.
Figure 3. PKC-dependent regulation of agonist-stimulated Ca2⫹ flux and PI generation in murine and human ASM cells. Early passage murine (A, B) and human (C, D) ASM cells were pretreated with Veh or 10 M Bis I and stimulated with 100 nM LTD4, 1 M MCh, or 1 M histamine, and net Ca2⫹ flux and PI generation was measured as described in Methods. *P ⬍ 0.05, Bis I pretreatment group vs. Veh pretreatment group. In murine ASM stimulated with MCh, a trend was observed for Bis I-mediated inhibition of net Ca2⫹ flux (P⫽0.09, n⫽8) and PI generation (P⫽0.20, n⫽4). n ⫽ 8 (A–C), n ⫽ 4 (D)
DISCUSSION In the present study, we establish the physiological relevance of PKC-dependent regulation of the CysLT1R. Three different cellular or tissue functions mediated by the CysLT1R—ASM contraction, monocyte migration, and chemokine production—are all increased by inhibition of intracellular PKC. Importantly, when these same functions are stimulated by activation of other receptors, inhibition of PKC has either no effect or slightly decreases the receptor-dependent actions, demonstrating the relative importance of the CysLT1 receptor locus in PKC-mediated regulation of these functions. We previously demonstrated the role of PKC in regulating both the responsiveness and internalization of the LTD4-stimulated CysLT1R (6). Employing both COS and HEK293 cells as expression systems for recombinant CysLT1R constructs, we found that inhibition of conventional PKC isoforms (but not arrestin inhibition, or inhibition of other protein kinases) significantly enhanced multiple measures of CysLT1R signaling while dramatically inhibiting receptor internalization. Moreover, mutation of putative PKC phosphorylation sites in the C terminus of the CysLT1R had the same effect and were redundant with pharmacological PKC inhibition. The dependence on COS and HEK293 cells for expression of the (difficult to express) CysLT1R limited our studies to analyses of receptor trafficking and signaling events. Although agonist-specific modification and desensitization is a well-documented phenomenon for the majority of GPCRs, establishing its relevance in physiological systems has been problematic. Several possible reasons exist for the failure to demonstrate a clear impact of GPCR regulatory processes on cell/tissue/ organism function. One is that those regulatory features characterized in more artificial systems may be obscured or absent in primary cell types because of FUNCTIONAL CONSEQUENCES OF CYSLT1R REGULATION
differences in regulatory molecule:receptor stoichiometries. Another involves the influence of compartmentalization effects that can be absent or overwhelmed in overexpression systems. In both artificial and physiological systems, the redundancy of GPCR desensitization mechanisms (GRK- and second messenger kinase-dependent mechanisms are often concomitantly evoked) and the molecules they employ (many cells express multiple GRK and arrestin isoforms) is also well established, making it difficult to implicate a specific mechanism/molecule. Perhaps most important is the presence of other, more dominant control systems that serve critical cell or organism needs. Indeed, this latter phenomenon tends to constrain the effects of experimental perturbations in many primary cell types, reflecting a hierarchical system of controls absent in unor de- differentiated cells that constitute convenient expression systems. Technical issues also contribute, in that endogenous levels of many receptors and regulatory proteins are low, are not easily characterized with existing reagents (antibodies), and provide poor signalto-noise ratios for receptor-mediated signaling events and cellular functions. The application of transfection or infection strategies in primary cell types and tissues is often problematic, thus limiting the scope of approaches for understanding regulatory mechanisms. The impact of antithetical control systems invoked by a common upstream effector is evident in our current studies. In both the regulation of calcium flux and calcium-dependent functions, PKC activity has a positive regulatory role through actions on signaling pathway elements downstream of the receptor. This is most clearly evidenced by the ability of PKC inhibition to reduce calcium flux and contraction in airway smooth muscle cells stimulated with either histamine or MCh. Previous studies have demonstrated that the activities of phospholamban, myosin light chain, heat shock protein 27, myosin light chain kinase, caldesmon, CPI-17 of 2339
Figure 4. PKC-dependent regulation of chemotaxis, Ca2⫹ flux in human monocytes and MCP-1 production in THP-1 cells. A) Human monocytes isolated from peripheral blood were treated with Veh or 10 M Bis I for 10 min and loaded into the top well of the transwell plates containing Veh and 10 M Bis I, respectively. Migration of cells into the bottom chamber containing plain DMEM medium with vehicle (basal), 100 nM LTD4 or 100 ng/ml MCP was determined by cell counting after 1 h. P ⬍ 0.05, Bis I pretreatment group vs. Veh pretreatment group, LTD4 stimulation, n ⫽ 11. P ⫽ 0.209, Bis I pretreatment group vs. Veh pretreatment group, MCP stimulation, n ⫽ 11. B) Human monocytes isolated as in (A) were loaded with Fura-2-AM, stimulated with 100 nM or 1 M LTD4 for assessment of net intracellular Ca2⫹ flux, as described in Methods. C) THP-1 cells were treated with Veh or 10 M Bis I for 10 min and then stimulated with 100 nM LTD4 or 100 ng/ml LPS for 3 h. MCP-1 levels in culture media were measured by ELISA. D) THP-1 cells were pretreated 10 min with Veh or the indicated concentration of MK-571, stimulated 3 h with Veh or 100 nM LTD4, and MCP-1 levels in culture media were measured as in (C ). *P ⬍ 0.05, Bis I pretreatment group vs. Veh pretreatment group.
myosin phosphatase, TRPC channels, and nonselective cation channels are modulated by PKC-dependent phosphorylation (20 –23). However, relief of PKC-mediated CysLT1R desensitization is sufficient to override these downstream mechanisms and effect an increase in LTD4-stimulated PI generation, Ca2⫹ flux, and consequently, contraction (Fig. 5). The inability of PKC inhibition to affect the H1 HR and m3 mAChR loci has been previously established in studies demonstrating that agonist-specific desensitization of these receptors is dependent on GRKs and arrestins (24 –26). Despite the paucity of studies directly linking mech-
anisms of GPCR desensitization to functional consequences in cells or more integrative systems, progress in two specific areas is noteworthy. For both cardiac and neuronal function, considerable evidence suggests that homologous desensitization of GPCRs mediated by GRKs and arrestins plays an important role. Koch, Rockman, and colleagues have demonstrated through use of transgenic mice a clear role for GRK-mediated AR desensitization in the diminished contractile response of failing heart to endogenous and exogenous beta-agonist. In murine models of heart failure, elevated levels of circulating catecholamines induce
Figure 5. Proposed model of differential regulation of GPCR signaling and function by PKC. 2340
Vol. 21
August 2007
The FASEB Journal
DESHPANDE ET AL.
increased expression of GRK2 and promote AR desensitization; inhibition of GRK-mediated AR desensitization by transgenic expression of the C-terminus of GRK2 (GRK2-CT) reverses AR desensitization, improves beta-agonist-stimulated cardiac function, and reverses disease phenotype (8 –10). With respect to neuronal function, several labs have now demonstrated that knockout of various GRK or arrestin isoforms attenuates desensitization or internalization of various GPCRs expressed in neuronal cells and alters behavioral phenotypes. For example, ablation of the arrestin3 (also termed beta-arrestin2) gene impairs agonist-induced mu opioid receptor desensitization, increases the antinociceptive effect of morphine, and deters the onset of opioid tolerance (27, 28). Similarly, opioid-induced analgesic tolerance is significantly less in GRK3-knockout mice (29, 30). Ablation of the GRK5 gene attenuates muscarinic (primarily m2 mAChR) receptor desensitization in mouse brain and is associated with enhanced cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation (31), whereas GRK6 knockout enhances striatal D2 dopamine receptor-G protein coupling and is associated with supersensitivity to the locomotor-stimulating effect of cocaine and amphetamine (32). Although these studies suggest an important role for GRKs and arrestins in mediating neuronal function, it should be noted that the phenotypes observed cannot be unequivocally linked to altered receptor regulation per se, in light of the (increasingly appreciated) pleiotropic nature of GRKs and arrestins (33–35). The obvious benefit of understanding the relative importance of the multiple regulatory mechanisms affecting GPCR-modulated functions is in establishing the feasibility of selectively targeting mechanisms in order to deter disease progression or improve treatment efficacy. Findings from the current study suggest that PKC helps quench CysLT1R activation but otherwise promotes downstream calcium-dependent signaling in ASM, suggesting that any antiasthma therapy targeting PKC in ASM should strive to block PKC-aided calcium-release and signaling while preserving PKCmediated desensitization of the CysLT1R. Toward this end, advances in gene/drug delivery systems enabling precise regulation of protein-protein interactions or chaperone functions (36, 37) could ultimately provide the means for selectively targeting these antithetical actions of PKC. This study was funded by the National Institutes of Health AI059755 (to R.B.P.). C.D.F holds a Tier I Canada Research Chair in Molecular, Cellular and Physiological Medicine and is a Career Investigator of the Heart and Stroke Foundation of Canada and is supported by a Canadian Institutes of Health Research grant (MOP-68930).
2. 3. 4. 5.
6.
7. 8.
9.
10.
11. 12.
13.
14.
15. 16.
17.
18.
19.
REFERENCES 20. 1.
Beller, T. C., Friend, D. S., Maekawa, A., Lam, B. K., Austen, K. F., and Kanaoka, Y. (2004) Cysteinyl leukotriene 1 receptor
FUNCTIONAL CONSEQUENCES OF CYSLT1R REGULATION
controls the severity of chronic pulmonary inflammation and fibrosis. Proc. Natl. Acad. Sci. U. S. A. 101, 3047–3052 Austen, K. F. (2005) The mast cell and the cysteinyl leukotrienes. Novartis. Found. Symp. 271, 166 –175 Funk, C. D. (2005) Leukotriene modifiers as potential therapeutics for cardiovascular disease. Nat. Rev. Drug. Discov. 4, 664 – 672 Metters, K. M., Gareau, Y., Lord, A., Rochette, C., and Sawyer, N. (1994) Characterization of specific binding sites for cysteinyl leukotrienes in sheep lung. J. Pharmacol. Exp. Ther. 270, 399 – 406 Lynch, K. R., O’Neill, G. P., Liu, Q., Im, D. S., Sawyer, N., Metters, K. M., Coulombe, N., Abramovitz, M., Figueroa, D. J., Zeng, Z., et al. (1999) Characterization of the human cysteinyl leukotriene CysLT1 receptor. Nature 399, 789 –793 Naik, S., Billington, C. K., Pascual, R. M., Deshpande, D. A., Stefano, F. P., Kohout, T. A., Eckman, D. M., Benovic, J. L., and Penn, R. B. (2005) Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling. J. Biol. Chem. 280, 8722– 8732 Penn, R. B., and Benovic, J. L. (1998) Regulation of G proteincoupled eceptors. In Handbook of Physiology (Conn, P. M., Ed.). Oxford Univ. Press, New York, pp 125–164 Koch, W. J., Rockman, H. A., Samama, P., Hamilton, R. A., Bond, R. A., Milano, C. A., and Lefkowitz, R. J. (1995) Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a beta ARK inhibitor. Science 268, 1350 –1353 Rockman, H. A., Choi, D. J., Akhter, S. A., Jaber, M., Giros, B., Lefkowitz, R. J., Caron, M. G., and Koch, W. J. (1998) Control of myocardial contractile function by the level of beta- adrenergic receptor kinase 1 in gene-targeted mice. J. Biol. Chem. 273, 18180 –18184 Rockman, H. A., Chien, K. R., Choi, D. J., Iaccarino, G., Hunter, J. J., Ross, J., Jr., Lefkowitz, R. J., and Koch, W. J. (1998) Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc. Natl. Acad. Sci. U. S. A. 95, 7000 –7005 Penn, R. B., Pronin, A. P., and Benovic, J. L. (2000) Regulation of G protein-coupled receptor kinases. Trends. Cardiovasc. Med. 10, 81– 89 Yang, G., Haczku, A., Chen, H., Martin, V., Galczenski, H., Tomer, Y., Van Besien, C. R., Evans, J. F., Panettieri, R. A., and Funk, C. D. (2004) Transgenic smooth muscle expression of the human CysLT1 receptor induces enhanced responsiveness of murine airways to leukotriene D4. Am. J. Physiol. Lung Cell. Mol. Physiol. 286, L992–L1001 Panettieri, R. A., Murray, R. K., DePalo, L. R., Yadvish, P. A., and Kotlikoff, M. I. (1989) A human smooth muscle cell line that retains physiological responsiveness. Am. J. Physiol. Cell Physiol. 256, C329 –C335 Deshpande, D. A., White, T. A., Guedes, A. G., Milla, C., Walseth, T. F., Lund, F. E., and Kannan, M. S. (2005) Altered airway responsiveness in CD38-deficient mice. Am. J. Respir. Cell Mol. Biol. 32, 149 –156 Loza, M. J., Foster, S., Peters, S. P., and Penn, R. B. (2006) Beta-agonists modulate T cell functions via direct actions on type 1 and type 2 cells. Blood 107, 2052–2060 Thivierge, M., Stankova, J., and Rola-Pleszczynski, M. (2001) IL-13 and IL-4 up-regulate cysteinyl leukotriene 1 receptor expression in human monocytes and macrophages. J. Immunol. 167, 2855–2860 Figueroa, D. J., Breyer, R. M., Defoe, S. K., Kargman, S., Daugherty, B. L., Waldburger, K., Liu, Q., Clements, M., Zeng, Z., O’Neill, G. P., et al. (2001) Expression of the cysteinyl leukotriene 1 receptor in normal human lung and peripheral blood leukocytes. Am. J. Respir. Crit. Care. Med. 163, 226 –233 Thivierge, M., Stankova, J., and Rola-Pleszczynski, M. (2006) Toll-like receptor agonists differentially regulate cysteinyl-leukotriene receptor 1 expression and function in human dendritic cells. J. Allergy. Clin. Immunol. 117, 1155–1162 Ichiyama, T., Hasegawa, M., Ueno, Y., Makata, H., Matsubara, T., and Furukawa, S. (2005) Cysteinyl leukotrienes induce monocyte chemoattractant protein 1 in human monocytes/ macrophages. Clin. Exp. Allergy. 35, 1214 –1219 Tani, E., and Matsumoto, T. (2004) Continuous elevation of intracellular Ca2⫹ is essential for the development of cerebral vasospasm. Curr. Vasc. Pharmacol. 2, 13–21
2341
21. 22.
23.
24. 25.
26.
27. 28.
29.
2342
Murphy, S., and Frishman, W. H. (2005) Protein kinase C in cardiac disease and as a potential therapeutic target. Cardiol. Rev. 13, 3–12 Harnett, K. M., Cao, W., and Biancani, P. (2005) Signaltransduction pathways that regulate smooth muscle function I. Signal transduction in phasic (esophageal) and tonic (gastroesophageal sphincter) smooth muscles. Am. J. Physiol. Gastrointest. Liver. Physiol. 288, G407–G416 Braz, J. C., Gregory, K., Pathak, A., Zhao, W., Sahin, B., Klevitsky, R., Kimball, T. F., Lorenz, J. N., Nairn, A. C., Liggett, S. B., et al. (2004) PKC-alpha regulates cardiac contractility and propensity toward heart failure. Nat. Med. 10, 248 –254 Iwata, K., Luo, J., Penn, R. B., and Benovic, J. L. (2005) Bimodal regulation of the human H1 histamine receptor by G proteincoupled receptor kinase 2. J. Biol. Chem. 280, 2197–2204 Hosey, M. M., Benovic, J. L., DebBurman, S. K., and Richardson, R. M. (1995) Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors. Life. Sci. 56, 951–955 Willets, J. M., Challiss, R. A., Kelly, E., and Nahorski, S. R. (2001) G protein-coupled receptor kinases 3 and 6 use different pathways to desensitize the endogenous M3 muscarinic acetylcholine receptor in human SH-SY5Y cells. Mol. Pharmacol. 60, 321–330 Bohn, L. M., Lefkowitz, R. J., Gainetdinov, R. R., Peppel, K., Caron, M. G., and Lin, F. T. (1999) Enhanced morphine analgesia in mice lacking beta-arrestin 2. Science 286, 2495–2498 Bohn, L. M., Gainetdinov, R. R., Lin, F. T., Lefkowitz, R. J., and Caron, M. G. (2000) Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence. Nature 408, 720 –723 McLaughlin, J. P., Myers, L. C., Zarek, P. E., Caron, M. G., Lefkowitz, R. J., Czyzyk, T. A., Pintar, J. E., and Chavkin, C. (2004) Prolonged kappa opioid receptor phosphorylation me-
Vol. 21
August 2007
30.
31.
32.
33. 34. 35. 36. 37.
diated by G-protein receptor kinase underlies sustained analgesic tolerance. J. Biol. Chem. 279, 1810 –1818 Terman, G. W., Jin, W., Cheong, Y. P., Lowe, J., Caron, M. G., Lefkowitz, R. J., and Chavkin, C. (2004) G-protein receptor kinase 3 (GRK3) influences opioid analgesic tolerance but not opioid withdrawal. Br. J. Pharmacol. 141, 55– 64 Gainetdinov, R. R., Bohn, L. M., Walker, J. K., Laporte, S. A., Macrae, A. D., Caron, M. G., Lefkowitz, R. J., and Premont, R. T. (1999) Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice. Neuron. 24, 1029 –1036 Gainetdinov, R. R., Bohn, L. M., Sotnikova, T. D., Cyr, M., Laakso, A., Macrae, A. D., Torres, G. E., Kim, K. M., Lefkowitz, R. J., Caron, M. G., and Premont, R. T. (2003) Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice. Neuron. 38, 291–303 Von Zastrow, M. (2004) A cell biologist’s perspective on physiological adaptation to opiate drugs. Neuropharmacology. 47, Suppl. 1, 286 –292 Shenoy, S. K., and Lefkowitz, R. J. (2005) Seven-transmembrane receptor signaling through beta-arrestin (Online). Sci. STKE. 2005, cm10 Premont, R. T. (2005) Once and future signaling: G proteincoupled receptor kinase control of neuronal sensitivity. Neuromolecular. Med. 7, 129 –147 Milligan, G., and White, J. H. (2001) Protein-protein interactions at G-protein-coupled receptors. Trends. Pharmacol. Sci. 22, 513–518 Dorn, G. W., 2nd, and Mochly-Rosen, D. (2002) Intracellular transport mechanisms of signal transducers. Annu. Rev. Physiol. 64, 407– 429
The FASEB Journal
Received for publication December 29, 2006. Accepted for publication March 1, 2007.
DESHPANDE ET AL.
