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Receptor-Extracellularly Regulated Kinase Signal Transduction Pathway: The Critical Role of Ser301 for Signaling Switch and Selectivity
Sir Quinton Hazell Molecular Medicine Research Centre (N.P., J.C., H.S.R., D.K.G.), Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom; Division of Pediatrics (M.A.L.), The Children’s Hospital of The Cleveland Clinic Foundation, Cleveland, Ohio 44195; and The Medical School (E.W.H.), University of Leeds, Leeds LS2 9NL, United Kingdom
Address all correspondence and requests for reprints to: Dr. D. Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Department of Biological Sciences, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: d.grammatopoulos{at}warwick ac.uk.
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ABSTRACT |
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receptors activates both the Gs and Gq, leading to activation of the adenylyl cyclase/protein kinase A (PKA) and the phospholipase C/protein kinase C and ERK1/2 signaling pathways, respectively. The overall result of these signals is often unpredictable, as these two signaling pathways can interact in many cellular systems, with either potentiation or inhibition of ERK1/2 activity. In the present studies we investigated potential signaling interactions after stimulation of CRH-R1 receptors in human cultured pregnant myometrial cells or HEK293 cells overexpressing recombinant CRH-R1 receptors. We found that the adenylyl cyclase/PKA pathway has the capacity to markedly decrease UCN-induced ERK1/2 activation, and that these effects were due in part to the ability of PKA to phosphorylate the CRH-R1 at position Ser301 in the third intracellular loop. Mutant CRH-R1 receptors with substitutions at position Ser301, which is the only potential PKA phosphorylation site, were resistant to PKA-dependent phosphorylation and showed altered signaling characteristics, which were dependent upon the amino acid substitution at this position.
We conclude that Ser301, which is located in the third intracellular loop of CRH-R1, is critical for efficient coupling of the receptor to G proteins and to second messenger generation. Phosphorylation by PKA prevents maximal coupling of the CRH-R1 to Gq-protein, and thereby reduces activation of ERK 1/2.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The CRH-Rs belong to the class B receptor superfamily, which includes receptors for CRH/PTH/calcitonin/pituitary adenylate cyclase-activating polypeptide/GHRH/glucagon/glucagon-like peptide/secretin. In most target tissues CRH and related peptides exert their effects by activation of adenylyl cyclase (AC), with increased cAMP levels resulting in stimulation of protein kinase A (PKA) activity. Depending upon the tissue, CRH-Rs can activate multiple G proteins (5, 6, 7), which can result in generation of a variety of second messengers, consistent with the suggestion that CRH and CRH-related peptides can regulate diverse signaling pathways (4).
In human myometrium, CRH (and CRH-related peptides) may play important roles in the regulation of myometrial contractility during pregnancy and labor (8). Activation of CRH-R, in response to CRH or UCN binding, can trigger activation of AC and increased cAMP levels (9), a pathway that has been linked to inhibition of myosin light chain phosphorylation and increased myometrial relaxation (10). Interestingly, in cultured human pregnant myometrial cells and human embryonic kidney (HEK)293 cells individually expressing CRH-R subtypes, we have previously shown that UCN or sauvagine, but not CRH, could phosphorylate and activate ERK1/2, a pathway that has been proposed to be involved in the development of myometrial contractility by uterotonins (11, 12). This effect was mediated by the R1 and R2ß receptor subtypes and was exerted primarily, but not exclusively, via activation of the Gq-inositol 1,4,5-triphosphate (InsP3)-protein kinase C (PKC) pathway (13).
There is little evidence about the mechanism controlling the ability of the CRH-R1 to switch between signaling cascades. Evidence from other G protein-coupled receptors (GPCRs) suggests that receptor phosphorylation by intracellular protein kinases might be important in this process. Phosphorylation by PKA of the ß2-adrenergic receptor switches its coupling from Gs to Gi, leading to stimulation of the ERK1/2 signaling pathway (14), whereas PKA potentiates InsP3-Ca2+ response of the mGluR1 by inducing a sustained coupling of mGluR1 to the InsP3 pathway even in the absence of agonist (15). Furthermore, PKA can modulate the functional activity of multiple intracellular proteins involved in ERK1/2 signaling; for example, in B-Raf-negative cells, PKA-induced Rap1 activation leads to inhibition of Ras-Raf-1 interaction and ERK1/2 signaling. In contrast, in cells expressing B-Raf, PKA stimulates ERK1/2 phosphorylation via sequential activation of Rap-1 and B-Raf (16). In addition, in some tissues, such as the myometrium, PKA can inhibit receptor/Gq protein-coupled stimulation of phospholipase C (PLC)ß3 by phosphorylation of Ser1105 of PLCß3 (17).
The present study was designed to investigate potential interactions between CRH-R-activated Gs/AC/PKA and Gq/PLC/PKC/ERK1/2 signaling cascades in cells derived from human pregnant myometrium and HEK293 cells overexpressing CRH-R1. Using the established model of UCN-induced ERK1/2 activation, we focused on the CRH-R1 as a potential target of PKA actions in an effort to identify and elucidate changes in CRH-R1 signaling properties.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), without altering the total amount of immunoreactive ERK. Pregnant myometrial cells express both R1 and R2 CRH-R subtypes; therefore, we used selective CRH-R subtype antagonists (antalarmin and antisauvagine-30) (18, 19) to identify which of these subtypes mediate the UCN effects. The CRH-R1 receptor antagonist antalarmin (1 µM) was able to reduce the UCN effect by 74 ± 7% (Fig. 1A), whereas the CRH-R2 receptor antagonist antisauvagine-30 (1 µM) exerted only a weak (23 ± 5%) inhibitory effect on UCN-stimulated ERK1/2 phosphorylation, indicating that UCN action was primarily mediated via activation of CRH-R1 receptors. In human myometrium UCN primarily activates the Gs/AC/PKA cascade; therefore, potential cross-talk between the PKA and ERK1/2 signaling cascades was further investigated. For this purpose, forskolin, which directly activates AC, and PKA inhibitors (H-89 and myr-PKAi) were used. Results showed that pretreatment of myometrial cells with forskolin (500 µM) significantly reduced (38 ± 6%) whereas inhibition of PKA by H-89 (1 µM) or myr-PKAi (10 µM) markedly increased (44 ± 5%) UCN-induced, but not basal, ERK1/2 activity (Fig. 1B).
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subtype was transiently expressed in HEK293 cells (293-tR1), and the interactions between AC/PKA and ERK1/2 pathways were analyzed.
UCN-Induced ERK1/2 Activation in 293-tR1 Cells: Effects of PKA Modulation
Our previous studies in HEK293 cells stably expressing CRH-R1 receptor, showed that UCN was able to increase phospho-ERK immunoreactivity via a mechanism involving Gq, PLC, and PKC (13). Experiments using 293-tR1 cells showed that UCN (100 nM) was able to induce a 5- to 7-fold activation of ERK1/2 (Fig. 2A), and that Ro31–8220, a nonspecific PKC inhibitor (used at 1 µM for 10 min), reduced the UCN effect by 75 ± 8%, confirming that UCN activates ERK1/2 predominantly via a PKC-mediated phenomenon. The effects of modulation of the AC/PKA cascade by pretreatment with forskolin or H-89 on UCN-induced ERK1/2 activation were investigated in 293-tR1 cells. The effect of these modulators on PKA functional activity was evaluated initially by a PKA activity assay. These assays showed significant increases in PKA catalytic activity after treatment with 100 nM UCN (2-fold) or 500 µM forskolin (3.5-fold) (Fig. 2B), effects that could be almost completely abrogated by pretreatment of 293-tR1 cells with H-89 (1 µM for 30 min). In agreement with our studies in myometrial cells, it was found that activation of AC by forskolin significantly reduced (by 40 ± 5%) UCN-induced, but not basal, ERK1/2 activation, whereas inhibition of PKA by H-89 augmented (by 53 ± 7%) UCN-induced, but not basal, ERK1/2 activity (data not shown). In addition, inhibition of AC by SQ22536 pretreatment, increased UCN-induced ERK1/2 activity by 33 ± 2% (Fig. 2C). These data confirmed the suitability of the 293-tR1 cellular system to study cross-talk between the PKA and ERK1/2 signaling cascades.
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cells with H-89 and Ro31–8220 to simultaneously block PKA and PKC, respectively, before UCN-induced stimulation of ERK1/2 activity. As shown in Fig. 3, H-89 and Ro31–8220 exerted inhibitory effects with similar potency (by 73 ± 6%) on UCN-induced ERK1/2 phosphorylation, confirming that activation of PKC was primarily required for UCN action in PKA-deficient cells.
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Functional Activity subtype contains a single potential site for PKA phosphorylation located at position Ser301 in the third intracellular (IC) loop (19), which might represent a potential target of PKA action. To address this, mutant CRH-R1 receptors were constructed by site-directed mutagenesis, and Ser301 was replaced by either an uncharged residue [valine-(S301V)CRH-R1], an acidic residue [glutamate-(S301E)CRH-R1], or a basic residue [arginine-(S301R)CRH-R1] (Fig. 4) to render the mutant receptors resistant to PKA phosphorylation. Wild-type (w.t) and mutant CRH-R1 receptors were transiently expressed in HEK293 cells. Immunofluorescence studies with a specific CRH-R1/2 antibody (Ab) (Fig. 5A) and determination of maximum binding site concentrations (Bmax) demonstrated similar levels of expression in the plasma membrane for w.t and mutant receptors (Table 1). Similarly, radioreceptor binding assays showed that all mutant CRH-R1 receptors were able to bind [125I]tyr°-UCN with binding affinity similar to that of the w.t CRH-R1, as assessed by Scatchard analysis (data not shown). In vitro phosphorylation assays showed that the w.t CRH-R1 was indeed a target of PKA-induced phosphorylation, as in 32P-labeled 293-tR1 cells, forskolin treatment induced incorporation of 32P into a protein with an apparent molecular mass of approximately 45 kDa, which was immunoprecipitated by specific CRH-R1/2 antibodies (Abs). The molecular mass of this protein was consistent with previously published data (20) (Fig. 5B). In contrast, no phosphorylated proteins were immunoprecipitated by anti-CRH-R1/2 Abs after similar treatment of HEK293 cells expressing (S301V)CRH-R1 (Fig. 6B) or any other mutant CRH-R1 (data not shown), confirming that Ser301 was indeed a PKA phosphorylation site. To confirm the specificity of Abs used, cell membranes were solubilized and immunoprecipitated with receptor antiserum; Western blot analysis using the CRH-R1/2 Ab showed a single immunoreactive protein with an apparent molecular mass of approximately 45 kDa, in both 293-tR1 and 293-t(S301V)R1 cell membranes. No immunoreactive proteins were detected when a synthetic CRH-R blocking peptide (1 µM) was added, thus confirming the specificity of these data (Fig. 5B). No phosphorylated proteins were immunoprecipitated by anti-CRH-R1/2 Abs after similar treatment of untransfected HEK293 cells (data not shown). In addition, UCN treatment of 32P-labeled 293-tR1 cells induced incorporation of 32P into a 45-kDa immunoreactive protein (Fig. 5C), an effect that could be only partially blocked by pretreatment with the PKA inhibitor H-89, suggesting the potential involvement of multiple protein kinases (such as PKA, PKC, or GPCR kinases) in agonist-induced CRH-R1 phosphorylation (20, 21). Similar results were obtained with the AC inhibitor SQ22536 (data not shown).
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receptors to activate the ERK1/2 cascade was not affected by modulation of the AC/PKA pathway. Neither direct activation of AC by forskolin (Fig. 6A
) nor inhibition of PKA by H-89 altered UCN-induced, ERK1/2 activity (Fig. 6B) in 293-t(S301V)CRH-R1 cells. On the other hand, inhibition of PKC activity with Ro31–8220 significantly reduced UCN-induced ERK1/2 phosphorylation, confirming that UCN action was primarily mediated by PKC. As expected, simultaneous inhibition of PKA and PKC activity (by H-89 and Ro31–8220, respectively) before UCN-induced stimulation of ERK1/2 activity did not show any differences on UCN-induced ERK1/2 phosphorylation compared with the effect of Ro31–8220 alone (Fig. 6C).
One remarkable feature of the mutant CRH-R1 receptors was that they exhibited differential activation of ERK1/2 in response to UCN despite similar levels of expression and UCN binding in HEK293 cells expressing recombinant receptors. As seen in Fig. 7, UCN-induced stimulation of ERK1/2 in 293-t(S301V)CRH-R1 cells was significantly less than in 293-tCRH-R1 cells. However, UCN-induced phosphorylation of ERK1/2 was similar in 293-t(S301R)CRH-R1, 293-t(S301E)CRH-R1, and 293-tCRH-R1 cells. This led us to hypothesize that replacement of Ser301 might induce significant changes in the signaling properties of the CRH-R1 receptor.
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Receptors proteins with different replacements at Ser301, we studied the effect of UCN on G protein activation and second messenger stimulation in HEK293 cells transiently expressing recombinant w.t or mutant CRH-R1 receptors. G protein activation was investigated by using the nonhydrolyzable GTP analog [32P]GTP-AA to label receptor-activated G proteins (22). A single concentration of UCN (100 nM) was used, as this concentration had been previously shown to induce maximal incorporation of 32P-GTP-AA into CRH-R1-activated G proteins (5, 23). As shown in Fig. 8, UCN treatment of 293 cells expressing w.t receptors led to activation of the four types of G proteins tested (Gs, Gq/11, Go, Gi/1/2), with an order of potency Gs (8-fold above basal) > Gq/11 = Go (4.7- to 5.5-fold above basal) > Gi/1/2 (2.2-fold above basal), in agreement with previous results (23). Similar results were obtained with receptors containing Glu301, but receptor activation of Gs and Gq/11 proteins was reduced by approximately 50% when Ser301 was replaced by valine. Remarkably, receptors containing Arg301 showed an approximately 50% reduction in coupling to Gs but normal activation of Gq. Interestingly, these mutations exerted different effects on CRH-R1 coupling to Gi1/2 and Go proteins; Gi1/2 activation was abolished when Ser301 was replaced by glutamate but was not affected when serine was replaced by valine or arginine. In contrast, replacement of serine by valine, arginine, or glutamate completely abolished CRH-R1-Go-protein coupling. We also assessed the effect of forskolin-induced activation of PKA on the CRH-R1-G-protein coupling pattern. Our results showed that in 293-tR1 cells, PKA activation significantly reduced (58 ± 6%) UCN-induced Gq protein activation (Fig. 9) but did not affect receptor coupling to any other G protein, suggesting a PKA action targeting selectively the CRH-R1-Gq-protein coupling. Most importantly, in HEK293 cells expressing (S301E)CRH-R1 (Fig. 9) or other mutant receptors (data not shown), the potency of UCN to activate Gq was not altered by PKA activation, again confirming the important role of Ser301 in mediating PKA actions.
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receptors in HEK293 cells. No differences were found in basal AC activation between w.t and mutant CRH-R1 (Table 1); however, the replacement of Ser301 by either valine or arginine reduced the CRH-R1 ability to activate AC by 40–60%, whereas Glu301 receptors showed w.t potency, in complete agreement with the results of Gs-protein radiolabeling experiments (Table 1 and Fig. 10A). We also evaluated the possibility that the decreased ability of mutant (S301V) and (S301R)CRH-R1 receptors to activate AC, was partly due to increased Gi activity. For that purpose, we used pertussis toxin (PTX) (2 h, final concentration, 100 ng/ml) pretreatment to inactivate Gi proteins via ADP ribosylation, before UCN-induced stimulation of AC activity. Pertussis treatment of 293-tR1 cells resulted in a small increase (10–15%) in UCN-stimulated cAMP production (Fig. 10B). Similar results were obtained with mutant (S301V) and (S301R)CRH-R1 receptors; however, the AC activation potency of (S301E)CRH-R1 (a mutant receptor unable to couple to Gi proteins) was not affected by PTX treatment, confirming our G protein labeling data.
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significantly increased InsP3 production (maximum 170 ± 21% of basal), with a maximum response at 100 nM UCN. Consistent with our G protein labeling data, UCN-induced production of InsP3 was reduced by approximately 60% when Ser301 was replaced by valine but was not affected by the S
E or SR substitutions (Fig. 11).
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DISCUSSION |
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Current evidence suggests that the mechanism(s) of CRH-R1-induced activation of ERK appears to be tissue and ligand specific; for example, in neuronal cells such as AtT-20 or Y79 neuroblastoma cells, ERK activation is dependent upon the AC/PKA system, allowing cells to respond to both CRH and UCN. In contrast, in myometrial and HEK293 cells overexpressing CRH-R1 receptors, ERK phosphorylation is PKC dependent and can be activated by UCN but not CRH (13). Moreover, our current study demonstrates that UCN activates multiple (both conventional and novel) isoforms of PKC similar to that which occurs in other signaling systems (28). Importantly, our present results provide novel evidence of an inhibitory pathway that modulates UNC activation of ERK and that involves PKA-induced phosphorylation of the CRH-R1 receptor. Inhibition of the AC/PKA pathway, which is the principal signaling cascade activated by CRH-R1 agonists in both types of cells tested, leads to augmented ERK response. A single serine residue (Ser301) present in the third IC loop appears to be the main target of PKA phosphorylation, and replacement of this residue allows the receptor to stimulate the ERK1/2 cascade independently of PKA activity (Fig. 12).
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. PKA-induced phosphorylation plays a role in modifying activity of many other GPCRs (e.g. ß1-adrenergic, ß2-adrenergic, MC2R, and MC4R) and is involved in receptor desensitization and internalization (29, 30). However, previous studies have shown that PKA is not involved in homologous desensitization of the CRH-R1 (Ref. 31 and our unpublished results). Our present data indicate that the CRH-R1 is a phosphorylation target of PKA, and that this modification regulates CRH-R1 signaling selectivity, particularly in cells where ERK activation does not require the AC/PKA cascade. The involvement of PKA is receptor specific, as cells expressing ß-adrenergic receptors require receptor phosphorylation by PKA to effect a G protein coupling switch and ERK activation (14). These events are likely to have significant physiological significance, as ERK activation plays a major role in cell proliferation, and the cross-talk between the AC/PKA and the ERK cascades involving PKA-mediated regulation of various downstream intracellular molecules is well established (16, 17). For example, CRH activation of the CRH-R1/cAMP/PKA pathway in the Ishikawa human endometrial adenocarcinoma cell line has been shown recently to inhibit cell growth and proliferation (32). Although not examined in this study, it is conceivable that the inhibitory effects of CRH and PKA on proliferation of Ishikawa cells might be due to inhibition of ERK activity.
The human pregnant myometrium provides an even more relevant example of the potential importance of CRH-R1 signal switching. UCN affects ERK activation in this tissue primarily via the CRH-R1 receptors (see Results), and ERK has been proposed to be involved in the regulation of myometrial contractility by uterotonins (12, 33). Previous studies have shown that UCN enhances prostaglandin F2-induced myometrial contractility (34), an effect that could be mediated by ERK. On the other hand, pregnancy is associated with increased myometrial PKA activity (35), which might provide a mechanism for preventing inappropriate ERK activation by CRH-like agonists to maintain uterine quiescence before the timely onset of labor. Indeed, our preliminary studies of CRH-R-G protein coupling during different stages of pregnancy have shown that activation of Gq proteins by CRH and UCN was evident in term (39 wk of gestation) but not preterm (33 wk of gestation), myometrial tissue, which coincided with a reduction in the ability of CRH and UCN to activate Gs protein (36). These results should be interpreted with caution, however, as progression of pregnancy toward term is also associated with an altered expression profile of CRH-R isoforms (21, 37).
The precise mechanism by which PKA-dependent phosphorylation of the CRH-R1 reduces full stimulation of the ERK pathway is unknown. Because replacement of the target residue for PKA phosphorylation in recombinant CRH-R1 led to corresponding losses of PKA phosphorylation of the receptor and inhibition of ERK1/2 activation, it is unlikely that other phosphorylated proteins are involved. Our G protein coupling and signaling data suggest that PKA activation results in selective impairment of CRH-R1-Gq protein coupling; therefore, it is possible that phosphorylation of Ser301 may prevent optimal interaction of the CRH-R1 with specific G proteins, such as Gq, that are involved in ERK activation; alternatively, PKA-mediated receptor phosphorylation may promote association with negative regulatory proteins that can attenuate G protein action, such as regulators of G protein signaling. It should be emphasized that these two hypotheses are not mutually exclusive and, in fact, both mechanisms may act synergistically. The former hypothesis is supported by our studies of mutant CRH-R1 receptors, which demonstrated the crucial role of this serine residue for efficient receptor-G protein interaction as well as regulation of signaling selectivity. This is not surprising given the critical role of the third IC loop in the coupling of many other GPCRs to G proteins. Our results suggest that Ser301 is important for interaction of the CRH-R1 with all four G proteins that we tested, and the nature of residue present in position 301 plays a significant role in determining the G protein activation efficiency of the CRH-R1. The three mutant CRH-R1 receptors exhibited completely different G protein activation profiles, a finding that elucidates the different requirements of each G protein for full activation. The Go protein activation was found to be the most sensitive to amino acid substitutions at this position as all mutant receptors failed to induce Go protein activation. Replacement of serine by the uncharged amino valine led to significant decreases in coupling of the mutant CRH-R1 to Gs and Gq proteins with corresponding decreases in cAMP and InsP3 generation. On the other hand, CRH-R1 proteins that contained the charged basic amino acid arginine in position showed normal Gq protein coupling but were unable to couple efficiently to Gs. Finally, receptors that contained the acidic amino acid glutamate at position 301 showed normal coupling to both Gq and Gs proteins, thus reflecting the ability of the acidic residue to substitute for the serine at this position in the agonist-activated CRH-R1 and providing some clues about the particular requirements of each G protein for optimal activation by the CRH-R1. Interestingly, the weakly activated Gi was sensitive only to the presence of a basic residue at position 301 of the third IC loop. Because little is known about the three-dimensional characteristics of the CRH-R1 and the critical domains for G protein activation, it is possible that introduction of these residues might disrupt the potential amphipathic -helix of the third IC loop or block specific amino acid-to-amino acid interactions between CRH-R1 and G proteins.
In conclusion, CRH-R1 signaling is modified by a self-regulatory mechanism that involves PKA phosphorylation of Ser301, which results in attenuation of ERK activity. This molecular mechanism enables the CRH-R1 to couple differentially to various G proteins and to thereby modify signaling toward distinct intracellular pathways. In the human myometrium this mechanism could account for the ability of CRH-R1 agonists to exhibit the contrasting roles of maintaining myometrial relaxation during preterm pregnancy and stimulating contractility (8) during labor.
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MATERIALS AND METHODS |
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-32P]GTP and reagents for enhanced chemiluminescence (ECL) were obtained from Amersham International (Little Chalfont, Buckinghamshire, UK). 4-Azidoanilide-HCl and 1-(3-dimethylamino propyl)-3-ethylenecarbodiimide hydrochloride were purchased from Aldrich Chemical Co. (Dorset, UK). The DNA 3'-end labeling kit was purchased from Boehringer Mannheim (Bell Lane, UK). Synthetic oligonucleotide probes, PCR and cloning reagents, culture media, and enzymes were purchased from GIBCO (Invitrogen Ltd, Paisley, UK). All other chemicals were purchased from Merck Eurolab Ltd (Poole, Dorset, UK).
Subjects and Culture of Myocytes
Pregnant myometrial biopsies (n = 10) were obtained from women undergoing elective cesarean section at term before the onset of labor for nonmaternal problems. The biopsy site was standardized to the upper margin of the lower segment of the uterus in the midline. This provides the closest approximation to the upper segment of the uterus. These studies were approved by the local ethical committee, and informed consent was obtained from all patients.
The tissue was immediately placed in 20 ml of ice-cold DMEM culture medium containing antibiotics (200 IU penicillin/ml and 200 mg streptomycin/ml). Myocytes were prepared by enzymatic dispersion as previously described (13). The cells were cultured at 37 C in a humidified atmosphere of 95% air and 5% CO2 until confluent.
Site-Directed Mutagenesis and Transfection of w.t/mutant CRH-R1 to HEK293 Cells
Human CRH-R1 cDNA was subcloned into the mammalian cell expression vector pcDNA3.1(-) (Invitrogen) and was used as a template for mutagenesis using the PCR overlap extension method (38) with Pfu polymerase (Stratagene, La Jolla, CA). The entire regions amplified by PCR were sequenced to ensure the fidelity of the mutant cDNAs and confirm presence of mutations. DNA sequence analysis was performed by the Core facility of the Department of Biological Sciences, University of Warwick.
Human w.t or mutant CRH-R1 cDNAs were transfected in HEK293 cells using Lipofectamine reagent (GIBCO, Invitrogen Ltd) as previously described (13). Cells were grown in medium tissue culture flasks in a culture medium consisting of high-glucose DMEM (GIBCO, Invitrogen Ltd) containing Glutamax, 25 mM HEPES, 10% fetal calf serum (FCS), 1% penicillin/streptomycin.
CRH-R Radioreceptor Assay
HEK293 cells transiently expressing w.t or mutant CRH-R1 were washed with PBS and lysed with 0.2% NaCl. The cells were homogenized in extraction buffer A [10 mM Tris-HCl, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF), 10 mM MgCl2, 0.1% BSA, and 0.1% bacitracin; pH 7.2]. Cell membranes were prepared as previously described (13). For receptor binding assays, membranes (40–50 µg of protein) were incubated with 125I-labeled UCN (0.1–1 nM) and unlabeled human UCN (1000 molar excess) as previously described (13). The binding data were analyzed using the EBDA program (39) and LIGAND (40).
ERK1/2 Activation and Immunoblot Analysis
Cells (myometrial or HEK293 expressing w.t or mutant CRH-R1) were cultured in six-well dishes for 2 d in DMEM containing 0.5% FCS. The confluent cells were washed with DMEM containing 0.5% FCS and incubated in fresh medium for an additional 30 min before addition of intracellular modulators and/or agonists for the specified period. At the end of the incubation the medium was aspirated, and the cells were washed twice with PBS containing 1 mM NaF. Cells were lysed and cell extracts were solubilized and electrophoresed as previously described (13). The resolved proteins were transferred to polyvinylidene difluoride membrane, and phospho- and total ERK levels were detected by immunoblot analysis and ECL as previously described (13).
Second Messenger Studies
HEK293 cells transiently expressing w.t or mutant CRH-R1 were plated in 12-well dishes and incubated with various concentrations of human UCN (1–100 nM) for 2 or 15 min (for IP3 or cAMP measurement, respectively). Assays for levels of cAMP and InsP3 were carried out as previously described (13). In some experiments cells were pretreated with PTX (2 h; final concentration, 100 ng/ml) as previously described (13).
Receptor Phosphorylation Assay
HEK293 cells transiently expressing w.t or mutant CRH-R1 (
5 x 109 per cell) were incubated in phosphate-free DMEM containing 300 µCi/ml [32P] orthophosphate for 3 h at 37 C, before the addition of vehicle or 500 µM forskolin or UCN (100 nM) for 15 min at 37 C in the presence or absence of (1 µM H-89, 30 min) or SQ22536 (10 µM for 30 min). At the end of the incubation period, cells were scraped into ice-cold buffer containing 10 mM Tris, pH 7.4, 5 mM EGTA, 5 mM EDTA, 1 mM PMSF, 10 mg/ml benzamidine, 5 mg /ml leupeptin, 10 mM sodium pyrophosphate, 10 mM NaF, 0.1 mM sodium orthovanadate, 100 nM okadaic acid, followed by centrifugation at 40,000 x g for 45 min. The resulting pellet was resuspended in 1 ml PBS containing 1% Triton X-100, 0.05% sodium dodecyl sulfate, 1 mM EGTA, 1 mM EDTA, 1 mM PMSF, 10 mg/ml benzamidine, 5 mg/ml leupeptin, 10 mM sodium pyrophosphate, 10 mM NaF, 0.1 mM sodium orthovanadate, and 100 nM okadaic acid, and samples were solubilized for 2 h on ice. Solubilized material was preincubated with preimmune serum (1:200) for 1 h and CRH-R1s were immunoprecipitated with 25 µl CRH-R Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 50 µl protein A Sepharose beads (4 C overnight). Samples were resuspended in sodium dodecyl sulfate loading buffer, and were subjected to 10% SDS-PAGE and autoradiography (-70 C, 10–14 d) using intensifying screens. Untransfected HEK-293 cells were used as negative controls. In some experiments resolved proteins were electrophoretically transferred to nitrocellulose filters, as previously described (41). The filters were incubated with primary Ab for the CRH-R1/2 (Santa Cruz) before incubation with the secondary antirabbit horseradish peroxidase-conjugated Ig for 1 h at room temperature and further washing for 30 min with PBS-0.1% Tween. As a negative control, the primary Ab was preabsorbed with a synthetic receptor peptide (1 µM, Santa Cruz). Ab complexes were visualized by ECL as previously described (23).
GTP-AA Photolabeling and Immunoprecipitation of G Subunits
[-P32]-GTP-AA was synthesized following a method described previously (13), with an overall yield of 40–55%. HEK293 cell membranes (100 µg) were incubated with or without human UCN (100 nM) for 5 min at 30 C before the addition of 5 µCi [-P32]GTP-AA, and after incubation at 30 C membranes were collected by centrifugation, resuspended, placed on ice, and exposed to UV light (254 nm). GTP-AA labeled cell membranes were precipitated by centrifugation, solubilized, immunoprecipitated, and electrophoresed following a method described previously (13). In some experiments cells were pretreated with forskolin, 500 µM) for 20 min and were collected in ice-cold buffer containing 10 mM Tris (pH 7.4), 5 mM EGTA, 5 mM EDTA, 1 mM PMSF, 10 mg/ml benzamidine, 5 mg /ml leupeptin, 10 mM sodium pyrophosphate, 10 mM NaF, 0.1 mM sodium orthovanadate, and 100 nM okadaic acid. The gels were then stained with Coomassie blue, dried using a slab gel dryer, and exposed to Fuji x-ray film at -70 C for 5–7 d.
Receptor Immunofluorescence
HEK293 cells were grown on four-chamber glass slides (Nunc, Inc., Naperville, IL), fixed in methanol-acetone (50:50) for 90 sec, air dried, and postfixed in acetone for 5 min. In some cases, before the coverslips were mounted, the nuclei were stained with diamidinophenylindole (Sigma). Fixed cells were washed in PBS and incubated for 60 min in 10% normal donkey serum (Santa Cruz) in PBS, to block nonspecific binding sites. Then, specimens were incubated with the primary goat polyclonal anti-CRH-R1/2 cross-reactive Ab (Santa Cruz) at a 1:100 dilution for 16–18 h at 4 C in a humidified chamber, and washed three times for 5 min in PBS containing 3% BSA. Next, specimens were incubated with the secondary antigoat IgG-fluorescein isothiocyanate-conjugated Ab (Santa Cruz), at a 1:25 dilution. After thorough washes in PBS (four 5-min washes), coverslips were mounted using 90% glycerol/PBS or Vectashield antifade mounting media (Vector Laboratories Inc., Burlingame, CA). The results were viewed under a fluorescent microscope using appropriate filters.
PKA Activity Assay System
A nonradioactive PKA assay kit (Calbiochem) was used to measure the PKA activity in 293-tR1 cells. Cells were seeded in six-well dishes and cultured until 90–95% confluency (107 cells per well). Cells pretreated with or without H89 (10 µM for 30 min), were incubated with UCN (100 nM) or forskolin (500 µM) in DMEM, for 10 min at 37 C and were then collected in ice-cold PBS. After centifugation the pellet was suspended in sample preparation buffer containing 50 mM Tris-HCl, 10 mM benzamidine, 5 mM EDTA, 10 mM EGTA, 50 mM ß-mercaptoethanol, 1 mM PMSF (pH 7.5), followed by sonication on ice four to five times for 10 sec. Samples were centrifuged at 100,000 x g for 60 min at +4 C, and the PKA activity in each sample was measured according to the manufacturer’s instruction.
Statistical Analysis
Data are shown as the mean ± SEM of each measurement. Comparison between group means was performed by ANOVA, and P < 0.05 was considered significant. The relative density of the bands was measured by OD scanning using the software Scion Image-Beta 3b for Windows (Scion Corp., Frederick, MD).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: Abs, Antibodies; AC, adenylyl cyclase; CRH-R, CRH receptor; ECL, enhanced chemiluminescence; FCS, fetal calf serum; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; IC loop, intracellular loop; InsP3, inositol 1,4,5-triphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipases C; PMSF, phenylmethylsulfonyl fluoride; PTX, pertussis toxin; UCN, urocortin; w.t, wild-type.
Received for publication September 18, 2003. Accepted for publication November 20, 2003.
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