Persistent corticotropin-releasing factor1 receptor desensitization and downregulation in the human neuroblastoma cell line IMR-32

Molecular Brain Research 92 (2001) 115–127 www.elsevier.com / locate / bres

Research report

Persistent corticotropin-releasing factor 1 receptor desensitization and downregulation in the human neuroblastoma cell line IMR-32 Patrick H. Roseboom b

a,b ,

*, Colleen M. Urben a , Ned H. Kalin a,c

a Department of Psychiatry, University of Wisconsin-Madison, 6001 Research Park Boulevard, Madison, WI 53719, USA Department of Pharmacology, University of Wisconsin-Madison, 6001 Research Park Boulevard, Madison, WI 53719, USA c Department of Psychology, University of Wisconsin-Madison, 6001 Research Park Boulevard, Madison, WI 53719, USA

Accepted 6 June 2001

Abstract Brain corticotropin-releasing factor (CRF) systems integrate various responses to stress. Pathological responses to stress may result from errors in CRF receptor regulation in response to changes in synaptic CRF levels. To establish an in vitro model to study brain CRF receptors, we characterized the CRF-induced modulation of CRF 1 receptors in the human neuroblastoma cell line, IMR-32. Treatment with CRF decreased CRF 1 receptor binding and desensitized CRF-induced increases in cAMP. The decrease in binding had an EC 50 of |10 nM, was maximal by 30 min, and was blocked by the CRF receptor antagonist [D-Phe 12 , Nle 21,38 , C a –MeLeu 37 ]CRF 12 – 41 . The desensitization was homologous as vasoactive intestinal polypeptide-induced increases in cAMP were unchanged, and elevation of cAMP did not alter CRF 1 receptor binding. Treatment with CRF for up to 24 h did not alter CRF 1 receptor mRNA levels, suggesting that a posttranscriptional mechanism maintains the decrease in receptor binding. Interestingly, recovery of CRF receptor binding and CRF-stimulated cAMP production was only partial following exposure to 100 nM CRF. In contrast, receptor binding recovered to control levels following exposure to 10 nM CRF. These data suggest that exposure to high doses of CRF result in permanent changes characterized by only partial recovery. Identifying the mechanisms underlying this partial recovery may provide insights into mechanisms underlying the acute and chronic effects of stress on CRF receptor regulation.  2001 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Receptor modulation, up- and down-regulation Keywords: Resensitization; Stress; Sauvagine; Adenylate cyclase; CRF41; Receptor regulation

1. Introduction Corticotropin-releasing factor (CRF) located in the hypothalamus is the principle peptide that stimulates synthesis and release of adrenocorticotropin from the pituitary [54]. CRF is also widely distributed throughout the brain where it is thought to integrate autonomic, endocrine, immune and behavioral responses to stress [9,25,26,29,37,53]. CRF produces its effects by interacting with at least two types of CRF receptors, termed CRF 1 and CRF 2 [13,55]. Several splice variants of the CRF 1 and CRF 2 receptors have been cloned, including CRF 2a , CRF 2b , and CRF 2g (for review see [28]). Both CRF 1 and *Corresponding author. Tel.: 11-608-263-0504; fax: 11-608-2659362. E-mail address: [email protected] (P.H. Roseboom).

CRF 2 receptors are coupled through G-proteins to the activation of adenylate cyclase. CRF also interacts with the CRF binding protein (CRF-BP) that binds CRF with an affinity similar to or greater than that of the CRF receptors [39]. The CRF-BP is thought to act as a negative regulator of the effects of CRF by preventing it from activating CRF receptors and may be involved in CRF clearance or degradation [2]. Data suggest that alterations in brain CRF systems are associated with certain psychopathologies such as depression and anxiety. In humans, CRF peptide levels are elevated in the brains of depressed patients [35] and CRF receptor levels are decreased in the brains of suicide victims [34]. Conversely, in Alzheimer’s disease CRF peptide levels are decreased and CRF receptors levels are elevated in the cerebral cortex [7]. Studies in rodents reveal that brain CRF systems are relatively resistant to

0169-328X / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00162-0

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change but can be modulated by chronic exposure to stress. For example, CRF receptor binding decreases in rat frontal cortex with chronic intermittent foot shock [1], and CRF 1 receptor mRNA levels decrease in rat frontal cortex and increase in the rat hippocampus following chronic unpredictable stress [24]. In tree shrews, chronic social stress increases or decreases CRF receptor binding depending on the brain region examined [12]. In rat pups, the stress of 24 h of maternal deprivation differentially regulates CRF 2 receptor mRNA levels in the hypothalamus and amygdala [10,11]. Nevertheless, other studies have failed to demonstrate changes in brain CRF receptors following chronic exposure to stress or corticosterone administration [17,18,58]. It is possible that the discrepancies between these studies result from differences in the type, severity and duration of the stressor to which the animals were exposed. In contrast to brain CRF receptors, pituitary CRF receptors appear to change more readily. Pituitary CRF receptors decrease as an adaptive response following chronic infusion of CRF or following hypersecretion of hypothalamic CRF as a result of adrenalectomy or chronic stress [16,18,56,57]. This reduction in CRF receptor number is associated with a decrease in CRF-stimulated cAMP accumulation and ACTH release in cultured pituitary cells. In addition, exposure to stressful events early in life appears to produce changes in the pituitary CRF system that are maintained into adulthood. For example, exposure of rat pups to maternal deprivation decreases anterior pituitary CRF receptor levels in adulthood [30]. Changes in CRF receptor levels likely represent adaptations to changes in the synaptic level of CRF. This is supported by the observation that repeated intracisternal administration of relatively high doses of CRF for 4 days decreased levels of rat amygdala CRF receptor binding [15]. In addition, in vitro studies show that incubation of primary cultures of extrahypothalamic brain cells with CRF for 1 to 3 days results in a loss of CRF receptor binding [27]. Depending on the length of agonist exposure, G-proteincoupled receptors can undergo desensitization or downregulation. Desensitization occurs following brief agonist exposure. Within minutes, the agonist-activated receptor becomes phosphorylated and the binding of an arrestin-like molecule uncouples the receptor from the G-protein, which prevents the receptor from activating second messenger systems. The receptor can then become internalized into vesicles where it can be recycled to the cell membrane following dephosphorylation [31]. Downregulation occurs following long-term agonist exposure. After hours or days, the internalized receptor is targeted for degradation and new protein synthesis is required to replace the receptors [51]. There are several well-documented examples in which derangements in receptor regulation can play a role in the etiology of certain cardiac and endocrine disorders [4,49].

Therefore, the possibility exists that derangements in CRF receptor regulation may result in maladaptive responses to stress that may ultimately lead to psychopathology. The understanding of CRF receptor regulation is aided by the use of in vitro cell lines. Recently, it was established that the human neuroblastoma cell line, IMR-32, expresses functional CRF 1 receptors [8,21]. This cell line was established in the late 1960s from an abdominal neuroblastoma tumor [52], and has an advantage over cells that are genetically engineered to express CRF 1 receptors because expression is controlled by the natural promoter instead of an artificial promoter used in stably transfected cells. The present study characterizes the regulation of CRF 1 receptors in IMR-32 cells in response to CRF exposure. We report that these receptors undergo a homologous desensitization that is associated with a decrease in CRF 1 receptor binding with no significant change in CRF 1 mRNA levels. Interestingly, receptor recovery is only partial following even brief exposure to CRF, and with prolonged exposure receptor binding remains significantly reduced for at least several days.

2. Materials and methods

2.1. Reagents [ 125 I]Tyr 0 sauvagine (2200 Ci / mmole) was from DuPont New England Nuclear (Boston, MA); Trizol and all cell culture reagents were from Life Technologies (Rockville, MD); aprotinin, bovine serum albumin, 59-bromo-29-deoxyuridine (BrDU), 3-isobutyl-1-methylxanthine (IBMX) and Triton X-100 were from Sigma–Aldrich (St Louis, MO); Hybond N membranes and the cAMP [ 125 I] scintillation proximity assay system were from Amersham Pharmacia Biotech (Piscataway, NJ); Bradford reagent was from Bio-Rad Laboratories (Hercules, CA); QuikHyb was from Stratagene (La Jolla, CA); and all peptides were from Peninsula Labs (Belmont, CA).

2.2. Cell culture IMR-32 cells were obtained from ATCC (Manassas, VA) and were cultured at 378C with 7.5% CO 2 in minimum essential medium containing Earle’s salts and non-essential amino acids and supplemented with 10% heat-inactivated fetal bovine serum, 4 mM L-glutamine, 100 units / ml penicillin and 100 mg / ml streptomycin. When cells reached |95% confluency, 2.5 mM BrDU was added and cells were differentiated for 10 days with media changed every third day. For receptor binding and Northern blot analysis cells were cultured in 75 cm 2 flasks containing 25 ml media. Drugs were added directly to the media in the flasks. Following treatment for recovery experiments, the cells were washed with 25 ml of media, then 25 ml of media with BrDU was added to the flasks and the cells

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were allowed to recover for various times. At the end of the incubation, media was removed, the cells were washed twice with ice cold phosphate-buffered saline (PBS), scraped from the flask into PBS and pelleted by centrifugation (which represents a third wash step) and stored at 2808C until assayed. For cAMP measurement, cells were cultured in 2 ml of media per well in 12-well plates. Following treatment, the cells were washed once with 2 ml of media, then 2 ml of media containing 1 mM IBMX to inhibit phosphodiesterase and various concentrations of CRF or VIP were added to each well and cells were treated for 1 h. Following treatment for the recovery experiment, cells were washed with 2 ml of media, and then incubated with 2 ml of fresh media with BrDU for 24 h prior to adding media with IBMX and various concentrations of CRF or VIP. Media was removed, wells were rinsed with PBS and intracellular cAMP was extracted overnight at 2208C with 1 ml acidified ethanol per well. The samples were dried by SpeedVac and stored at 2208C until assayed.

2.3. Receptor binding CRF 1 receptor levels were quantified using [ 125 I]tyr 0 sauvagine (2200 Ci / mmole). Frozen cell pellets were resuspended by trituration in 1 ml of buffer (50 mM Tris HCl, pH 7.2 at room temperature, 10 mM MgCl 2 , 2 mM EGTA), and homogenized by hand with 10 strokes of a glass / teflon homogenizer. An additional 5 ml of buffer was added, the samples were incubated on ice for 30 min, and the membranes were pelleted by centrifugation at 48,0003g for 20 min. The pellet was resuspended in 6 ml buffer by trituration and re-centrifuged at 48,0003g for 20 min. The final resuspension was by trituration in 0.7 ml of buffer followed by glass / Teflon homogenization. The protein concentration was determined by the method of Bradford and membrane suspensions were diluted with buffer to 1 mg / ml of protein. The binding reaction was performed on 100 mg of protein in siliconized 1.5 ml polypropylene tubes in 300 ml of buffer containing 0.2% bovine serum albumin and 100 kallikrein inhibitor units / ml aprotinin and [ 125 I]tyr 0 sauvagine. For saturation curve analysis, [ 125 I]tyr 0 sauvagine concentrations ranged from 0.05 nM to 2.0 nM, and for all other experiments [ 125 I]tyr 0 sauvagine was used at 0.2 nM. The samples were incubated for 2 h on a rotator at room temperature and bound ligand was separated from unbound by centrifugation at room temperature at 12,0003g for 10 min. Following removal of the supernatant, the pellet was washed once with 1 ml of ice-cold PBS containing 0.01% Triton X-100. Samples were spun again at room temperature at 12,0003 g for 5 min and the supernatant was removed. The tube bottoms were clipped off into 12375 polystyrene tubes and counted on a Cobra 5005 gamma counter (Packard Instrument Co., Meriden, CT). Non-specific binding was determined in the presence of 1 mM CRF.

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2.4. Northern blot Total RNA was extracted using Trizol, quantified by UV spectroscopy and equal amounts electrophoresed on a 1.5% agarose / 0.7 M formaldehyde gel as previously described [3]. Following capillary transfer to a Hybond N membrane, the blot was probed with a 32 P-labeled randomprimed fragment. The CRF 1 receptor probe corresponded to bases 1 to 363 of the human CRF 1 receptor cDNA (GenBank accession number L23333), the CRF probe corresponded to bases 660 to 1230 of the human CRF cDNA (GenBank accession number NM-000756), the CRF-BP probe corresponded to bases 561 to 1110 of the human CRF-BP cDNA (GenBank accession number XM003672), and the CRF 2 receptor probe corresponded to bases 1 to 355 of the human CRF 2 receptor cDNA (GenBank accession number U34587). The CRF 2 receptor probe can cross-hybridize with the three receptor subtypes (CRF 2a , CRF 2b , CRF 2g ). To confirm the integrity and equal loading of the RNA samples, the blot was then stripped and hybridized with a 32 P-labeled random-primed human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe prepared as previously described [46]. Blots were hybridized at 688C in QuikHyb and the final wash was at 608C in 0.13SSC containing 0.1% SDS for 15 min. Hybridized blots were imaged and analyzed using a Storm 860 with ImageQuant for Windows NT software (Molecular Dynamics, Sunnyvale, CA).

2.5. cAMP Intracellular levels of cAMP were measured by radioimmunoassay using the cAMP [ 125 I]scintillation proximity assay method following the manufacturer’s protocol for the non-acetylation assay. Samples were resuspended in 1 ml and 100 ml aliquots were assayed in triplicate. The assay had a sensitivity of 0.078 pmoles / tube, an EC 50 of 2.3 pmoles / tube, and intra- and inter-assay coefficients of variation of 3.3 and 9.4%, respectively.

2.6. Statistics With noted exceptions, the data were analyzed with oneor two-factor ANOVAs. Following significance in the ANOVAs, data were subject to analysis with Bonferonni post-tests, except for the concentration- and time-dependent effects of CRF on [ 125 I]tyr 0 sauvagine binding, which were analyzed using Dunnett’s multiple comparison test. For the equilibrium binding data, the Kd and Bmax values were compared using paired t-tests, and the initial effects of CRF treatment in the recovery experiments were analyzed by Student’s t-tests. Analysis was done using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA), except for the cAMP data, which were analyzed using SigmaStat 2.0 (SPSS Science, Chicago, IL).

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3. Results

3.1. BrDU differentiated IMR-32 cells express CRF1 receptors, but not CRF peptide, CRF2 receptors or CRF binding protein Treatment of IMR-32 cells with BrDU has been shown to induce neuronal differentiation in a cAMP-independent manner as evidenced by formation of long neurites and by the induction of tyrosine hydroxylase and catechol-Omethyltransferase [41]. In addition, the Bmax value reported for [ 125 I]tyr 0 ovine CRF binding to cells differentiated for 10 days with 2.5 mM BrDU was 142 fmoles / mg protein [21], which is approximately four-fold greater than the Bmax value of 32 fmoles / mg protein reported for undifferentiated cells [8]. For this reason all experiments were performed on cells that had been differentiated for 10 days with 2.5 mM BrDU. Previously published receptor binding data indicate that IMR-32 cells express CRF 1 but not CRF 2 receptors [8,21]. Consistent with this, we were unable to detect CRF 2 receptor mRNA in these cells by Northern blot analysis (Fig. 1). Additionally, we were able to displace .85% of [ 125 I]tyr 0 sauvagine specific binding to IMR-32 cells using the CRF 1 receptor-selective ligands SC241 [45] or

DMP696 [20]. Using radioimmunoassay, we were not able to detect CRF peptide in media or homogenates of differentiated IMR-32 cell cultures (data not shown) nor could we detect CRF mRNA expression in Northern blots of differentiated IMR-32 cell RNA (Fig. 1). In addition, we were not able to detect CRF-BP mRNA in IMR-32 cell RNA (Fig. 1). These findings indicate that IMR-32 cells express CRF 1 receptors but not CRF, CRF 2 or CRF-BP.

3.2. Treatment of IMR-32 cells with CRF produces concentration- and time-dependent decreases in CRF1 receptor binding but not CRF1 mRNA levels Incubation of IMR-32 cells with increasing concentrations of CRF for 4 h produced a concentration-dependent decrease in [ 125 I]tyr 0 sauvagine binding to cell membranes. Binding at each concentration of CRF was significantly lower than control (F3,15 574.94, P,0.0001; Fig. 2A). In contrast, CRF 1 mRNA levels were not altered. The EC 50 for this effect was approximately 1 nM, which is similar to the Ki (0.46 nM) for CRF competing for the high affinity binding site of CRF 1 receptors in these cells [21]. In addition, incubation of cells with 100 nM CRF for 4 h resulted in a time-dependent decrease in [ 125 I]tyr 0 sauvagine binding to the cell membranes (F6,29 5

Fig. 1. Detection of CRF system components by Northern blot analysis. IMR-32 cell RNA was isolated from differentiated cells, and each IMR-32 cell lane contains 1 mg poly A1 RNA, except the CRF 1 receptor lane which contains 25 mg total RNA. The adjacent lanes contain positive control RNA as follows: CRF, 1 mg monkey cortex poly A1 RNA; CRF 1 receptor, 25 mg human cerebellum total RNA; CRF 2 receptor, 30 mg rat septum total RNA; CRF-BP, 1 mg human amygdala poly A1 RNA. The Northern blots were stripped and probed for G3PDH to confirm RNA integrity (lower panel).

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Fig. 2. Concentration- and time-dependent effects of CRF treatment of IMR-32 cells on CRF 1 receptor binding and mRNA levels. IMR-32 cells were incubated for 4 h with various concentrations of CRF (A) or incubated with 100 nM CRF for various times (B). Cells were then harvested and receptor binding measured using 0.2 nM [ 125 I]tyr 0 sauvagine. Insets: Northern blot analysis of CRF 1 receptor mRNA levels in total RNA obtained from IMR-32 cells. Receptor binding values are the means of three to five independent determinations. Similar Northern blot data were seen in four different experiments. *P,0.05, ***P,0.001, different from control.

14.71, P,0.0001). Binding at each time-point was significantly lower than control, with the maximal effect occurring after 30 min (Fig. 2B). CRF 1 mRNA levels were not affected at any time-point examined. An intact cell preparation is required for the decrease in receptor binding because incubation of a broken cell preparation in the presence or absence of 100 nM CRF did not significantly affect the levels of 0.2 nM [ 125 I]tyr 0 sauvagine binding (Control, 21.862.7 fmoles / mg protein vs. CRF-treated 23.962.3 fmoles / mg protein). The lack of effect in a broken cell preparation indicates that the decrease in receptor binding observed with intact cells was not simply caused by competition from residual CRF carried over into the binding assay. In addition, the cells are washed three times during harvesting to remove any remaining CRF, and the membranes are washed twice, including a 30 min incubation step to allow CRF dissociation, making any CRF carryover into the receptor binding assay highly unlikely. To determine if the loss of CRF receptor binding resulted from a change in receptor affinity or number, saturation curve analysis was performed on IMR-32 cells

that were incubated for 4 h in the presence or absence of 100 nM CRF. The Bmax value was significantly lower in CRF-treated cells than in vehicle-treated cells (37.1610.8 fmoles / mg protein and 94.6622.9 fmoles / mg protein, respectively; four independent determinations; P,0.05). In contrast the Kd value was not significantly different between CRF-treated and vehicle-treated cells (0.6860.11 nM and 1.0560.13 nM, respectively).

3.3. The decrease in CRF1 receptor binding is blocked by a CRF receptor antagonist To determine if the CRF-induced decrease in CRF 1 receptor binding was mediated through activation of CRF receptors, the effects of incubating cells with 10 nM CRF were measured in cells that had been pretreated for 1 h in the presence or absence of the CRF receptor antagonist [D-Phe 12 , Nle 21,38 , C a –MeLeu 37 ]CRF 12 – 41 ( D-Phe). Pretreatment with the CRF antagonist (1 mM) partially blocked the decrease in receptor binding induced by incubating cells with 10 nM CRF (F1,32 511.9, P,0.002). CRF 1 receptor binding was significantly higher in the 10

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3.4. CRF treatment produces a homologous desensitization of CRF1 receptor-coupled adenylate cyclase

Fig. 3. Effects of the CRF receptor antagonist [D-Phe 12 , Nle 21,38 , C a – MeLeu 37 ]CRF 12-41 ( D-Phe) on the CRF-induced decrease in CRF 1 -receptor binding. Four different conditions were used: Control, cells were incubated in the absence of D-Phe or CRF; 1 mM D-Phe, cells were pre-incubated for 1 h with D-Phe followed by an additional 4 h; 10 nM CRF, cells were pre-incubated for 1 h in the absence of D-Phe followed by 4 h with 10 nM CRF; D-Phe / CRF, cells were pre-incubated for 1 h with D-Phe followed by 4 h with CRF and D-Phe. Cells were then immediately harvested and CRF receptor binding determined using 0.2 nM [ 125 I]tyr 0 sauvagine. Data represent the average of six independent determinations. **P,0.01, different from 10 nM CRF condition.

nM CRF / D-Phe-treated cells compared to cells that had been treated with 10 nM CRF alone (Fig. 3). Therefore, the antagonist was able to partially block the CRF-induced decrease in CRF 1 receptor binding, indicating that the effect is CRF 1 receptor-mediated.

We next determined if the decrease in receptor binding was associated with a decrease in CRF 1 receptor-mediated increases in cAMP. The ability of CRF treatment to increase intracellular cAMP levels in cells that were pretreated for 4 h with 100 nM CRF was significantly reduced compared to control cells (F2,61 510.04, P,0.001, Fig. 4). To determine if the effects of CRF were limited to the CRF 1 receptor we examined the ability of vasoactive intestinal polypeptide (VIP) to stimulate cAMP production in these cells. VIP-stimulated increases in cAMP were not significantly affected by prior CRF treatment (F2,146 52.56, n.s., Fig. 4). Therefore, incubating IMR-32 cells with CRF produces a desensitization of the CRF 1 receptor-coupled adenylate cyclase system. The finding that CRF pretreatment does not alter VIP-stimulated cAMP accumulation is consistent with a homologous or receptor-specific form of desensitization. If the desensitization is homologous it likely is mediated by a G-protein-coupled receptor kinase and not via cAMPdependent protein kinase (PKA) [32]. Therefore, elevations in cAMP, which activate PKA, should not be sufficient to reproduce the effects of CRF on the CRF 1 receptor. To test this, we measured CRF 1 receptor levels in cells treated with either VIP or forskolin, two agents that have been shown to increase cAMP in IMR-32 cells ([50]; Roseboom et al., unpublished observations). Incubation of cells for 4 h with five different concentrations of VIP ranging from 0.1 nM to 1 mM did not significantly alter CRF 1 receptor

Fig. 4. Effects of CRF-treatment on CRF- and VIP-stimulated increases in intracellular cAMP. The cAMP response to stimulation with CRF or VIP was measured in cells that had been treated for 4 h in the presence or absence of 100 nM CRF. The media was then replaced and cells were treated for 1 h with increasing concentrations of CRF or VIP. Intracellular levels of cAMP were measured by radioimmunoassay and the graph depicts CRF- or VIP-stimulated increases in cAMP over basal levels. For the CRF concentration effect curve basal levels were 9.561.5 pmoles / well for control and 39.465.1 pmoles / well for CRF treated; for the VIP concentration effect curve basal levels were 10.061.1 pmoles / well for control and 38.363.9 pmoles / well for CRF treated. CRF and VIP data represent the average of 9–12 and 21–27 independent determinations, respectively. ***P,0.001, different from corresponding control.

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binding (F5,29 50.307, n.s.). Likewise incubation of cells for 4 h with four different concentrations of forskolin ranging from 0.3 mM to 10 mM did not significantly alter CRF 1 receptor binding (F4,14 50.327, n.s.). The failure of VIP or forskolin to affect CRF 1 receptor binding indicates that an increase in cAMP, and the subsequent activation of PKA, is not sufficient to decrease CRF 1 receptor binding. This is consistent with a homologous form of desensitization.

3.5. Long-term treatment of the IMR-32 cells with CRF maintains receptor downregulation but does not alter CRF1 mRNA levels During prolonged exposure to an intense stressor, synaptic concentrations to CRF may be elevated for an extended

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period of time. Therefore we determined the effects of exposing cells to 100 nM CRF for up to 24 h. The decrease in CRF 1 receptor binding produced by 100 nM CRF was maintained up to 24 h with continuous treatment without a change in CRF 1 mRNA levels (Fig. 5). These data suggest that even with long-term CRF treatment, transcriptional control is not involved in maintaining the desensitized state.

3.6. Recovery of CRF receptor levels depends on the concentration of CRF treatment We next assessed the recovery of CRF receptor binding in IMR-32 cells following CRF treatment. Cells were exposed to 100 nM CRF for different periods of time, the cells were washed with CRF-free media and then CRF 1

Fig. 5. Effects of long-term exposure to CRF on CRF 1 receptor binding and mRNA levels. IMR-32 cells were incubated with or without 100 nM CRF for the indicated times. Media containing CRF and control media were replenished every 4 h to control for possible peptide degradation. One half of the cells from each flask were used for receptor binding analysis and the other half for Northern blot analysis. The graph depicts receptor binding determined with 0.2 nM [ 125 I]tyr 0 sauvagine. Inset: Northern blot analysis of CRF 1 mRNA levels in total RNA from IMR-32 cells. Receptor binding data represent the average of three determinations from a single experiment.

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receptor binding was measured at different recovery time points. Treatment of cells with 100 nM CRF for 10 min, 1 h or 6 h all produced a significant decrease in [ 125 I]tyr 0 sauvagine binding (Fig. 6). However, for the recovery time-points, [ 125 I]tyr 0 sauvagine binding to CRFtreated cells remained lower than binding to control cells (10 min: F1,38 55.48, P,0.05; 1 h: F1,8 58.96, P,0.05; 6 h: F1,19 535.5, P,0.0001). There was a trend for greater receptor recovery following the 10 min treatment compared to the 1 h or 6 h treatments. The average percent of control values for the recovery time-points was 77.261.8% for the 10 min treatment, 48.965.0% for the 1 h treatment and 54.463.8% for the 6 h treatment. Additionally,

receptor binding was still only 58.6610.5% of control, even up to 84 h following a 6 h exposure to CRF (Fig. 6). In contrast, receptor binding recovered to control levels following a 6 h exposure to a lower CRF concentration (10 nM). As seen in Fig. 6, there was a significant decrease in [ 125 I]tyr 0 sauvagine binding produced by 10 nM CRF treatment, but for the recovery time-points, [ 125 I]tyr 0 sauvagine binding did not differ between CRFpretreated cells and control cells (F1,19 51.13, n.s.). We next examined whether the partial recovery of CRF 1 receptor binding was associated with the maintenance of a desensitization of the CRF-stimulated cAMP response. In cells that had been incubated with 100 nM CRF for 4 h

Fig. 6. Dose-dependent differences in the recovery of IMR-32 cell CRF 1 receptor binding following exposure to 100 nM or 10 nM CRF. IMR-32 cells were exposed to 100 nM CRF for 10 min, 1 h or 6 h or 10 nM CRF for 6 h. The media was removed, the flasks were rinsed once with 25 ml of media and the media was replaced without CRF. Cells were then harvested after recovering for the indicated times. The level of CRF 1 receptor binding was measured with 0.2 nM [ 125 I]tyr 0 sauvagine. Values represent the average of three independent determinations for the 1 h, 6 h and 10 nM CRF data and five independent determinations for the 10 min data. *P,0.05, **P,0.01, different from corresponding control, [ main effect of CRF treatment (see text).

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Fig. 7. Partial recovery of CRF 1 receptor-stimulated cAMP production in IMR-32 cells 24 h after a 4 h exposure to 100 nM CRF. Cells were incubated in the presence or absence of CRF for 4 h, the cells were washed with 2 ml media, the media was replaced and cells were allowed to recover for 24 h. Cells were then stimulated for 1 h with increasing concentrations of CRF. Intracellular levels of cAMP were measured and the graph depicts the CRF-stimulated increase in cAMP over basal. Basal levels were 18.8462.35 pmoles / well for control and 28.1963.07 pmoles / well for CRF treated. These data are the average of eight to nine independent determinations. ***P,0.001, different from corresponding control.

and then allowed to recover for 24 h, there was a significantly lower cAMP accumulation induced by CRF treatment compared to control cells (F2,48 55.73, P,0.01). The responses seen with 1 nM and 10 nM CRF were significantly reduced in the CRF-pretreated cells compared to control cells (Fig. 7). This finding indicates that, similar to the incomplete recovery of CRF receptor binding, the cAMP response remains desensitized even after 24 h of recovery. It is likely that the impaired cAMP response results from a decreased number of CRF 1 receptors that are available to activate the signal transduction cascade.

4. Discussion The data presented here indicate that exposure of IMR32 cells to CRF results in a rapid concentration-dependent decrease in CRF 1 receptor binding that is associated with a desensitization of CRF 1 receptor-coupled adenylate cyclase. Both the time-course and concentration-dependence of the decrease in receptor binding and desensitization are similar to that reported for CRF 1 receptor regulation in the human retinoblastoma Y-79 cell line [14]. In addition, the decrease in CRF 1 receptor binding reported in the present study appears to be CRF 1 receptor-mediated

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because it is partially blocked by the CRF receptor antagonist D-Phe. Involvement of the CRF 2 receptor is unlikely because CRF 2 receptor binding was not detected, and CRF 2 receptor mRNA was not seen using a probe that can cross-hybridize with the three different splice variants of the CRF 2 receptor (CRF 2a , CRF 2b , CRF 2g ). It is unlikely that this partial blockade produced by D-Phe is due to insufficient antagonist concentration, because the 1 mM concentration used is 100-fold above the Ki for D-Phe (10 nM) at inhibiting [ 125 I]tyr 0 ovine CRF binding to these cells. Another possible explanation for the partial blockade is that the CRF is interacting with a low affinity binding site that has been reported on IMR-32 cells [21]. The Ki for CRF interacting with this site is 56 nM; thus, 10 nM CRF would be a sufficient concentration to partially bind to this site. The affinity of the D-Phe antagonist for this site is unknown. Therefore, it is possible that some of the effects of CRF on these cells that are not blocked by D-Phe may be mediated through this low affinity site. Whether this low affinity site represents a different affinity state of the CRF 1 receptor or an unidentified CRF receptor subtype remains to be determined. The desensitization appears to be homologous or receptor-specific because CRF treatment does not alter the ability of VIP to increase cAMP levels in these cells. In addition, increasing intracellular cAMP by another receptor (VIP) or by a receptor-independent mechanism (forskolin) did not alter CRF 1 receptor binding. Based on the well-characterized process for homologous desensitization of adrenergic receptors [32], the CRF 1 receptor desensitization is likely produced by a G-protein-coupled receptor kinase phosphorylation of agonist-occupied receptor. This would result in binding of an arrestin-like molecule, followed by uncoupling of the receptor from the G-protein, then receptor sequestration and finally internalization into vesicles. In addition, the internalized receptors are not detectable with the [ 125 I]tyr 0 sauvagine radioligand, which cannot enter the receptor-containing vesicles. Other receptor systems are not affected because only agonist occupied receptor is a substrate for the receptor kinase. This process would account for the decrease in [ 125 I]tyr 0 sauvagine binding reported here immediately following addition of CRF to the IMR-32 cell culture media, and the decrease in CRF-stimulated adenylate cyclase activity in the absence of any change in VIPstimulated adenylate cyclase activity. The suggestion that CRF 1 receptor desensitization is mediated by a G-protein-coupled receptor kinase (GRK) and not by cAMP-dependent protein kinase A (PKA) is consistent with the observation that CRF treatment of COS cells expressing recombinant CRF 1 receptors causes CRF 1 receptor phosphorylation, whereas forskolin treatment, which elevates cAMP, does not [19]. This also agrees with the finding in Y-79 cells that antisense-mediated depletion of GRK3 protein, which is one of the six known forms of GRK, significantly attenuates homologous desensitization

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of CRF 1 receptors [6]. It remains to be determined if this receptor phosphorylation results in the binding of an arrestin-like molecule to the phosphorylated form of the CRF 1 receptor. The processes that control the level of CRF 1 receptor binding most likely differ depending on the duration of agonist exposure. As stated above, the decrease in CRF 1 receptor binding seen within minutes of CRF exposure is due to receptor sequestration into synaptic vesicles. However, with longer exposure to CRF (6 h and 24 h), receptors may become targeted for degradation and the loss of receptor binding would represent loss of receptor number. This process, which is termed receptor downregulation, is not as well characterized as receptor desensitization, but most likely involves the proteolytic degradation of the receptor [51]. The pathway for downregulation of CRF receptors remains to be elucidated. It should be noted that no studies have examined the effects of brief in vivo exposure to high doses of CRF on changes in CRF-stimulated cAMP production. Brief exposure would be expected to produce receptor desensitization but not necessarily receptor downregulation. Therefore, the effects of brief CRF exposure may be seen as a decrease in the ability of CRF to stimulate cAMP production without an alteration in total CRF receptor number. It is interesting that a change in CRF 1 receptor steadystate mRNA levels is not detected even after 24 h of treatment. It should be noted that the changes in mRNA levels may be smaller that can be detected using the Northern blot technique. Nevertheless, these results are consistent with data reported for the regulation of CRF 1 receptors in the human retinoblastoma Y-79 cell line [14]. However, this is in contrast to the decreases in rat anterior pituitary CRF 1 receptor mRNA levels seen following several hours of in vitro or in vivo exposure to CRF [40,47] or following acute stress [33,42]. Additionally, a 50% decrease in CRF 1 mRNA levels has also been reported in the locus coeruleus-like cell line, CATH.a, after a 4 h treatment with CRF [23,24]. In contrast, exposure of rat pups to intracerebroventricular CRF, which decreases CRF 1 receptor binding in the frontal cortex, increases CRF 1 receptor mRNA levels in the frontal cortex and hippocampal CA3 [5]. This increase in mRNA may be necessary to replace receptors that have been degraded during the process of downregulation. Taken together, these data suggest that it is possible to alter CRF 1 receptor mRNA levels; however, our studies and those with the Y-79 cells, show that it is also possible to see a dramatic, sustained downregulation of CRF receptors in the absence of any change in mRNA levels. It should be mentioned at this point that one of the limitations of studying the CRF system using IMR-32 cells is that they do not represent neurons from a specific brain region. In vivo studies have shown that regulation of the CRF system can be brain-region specific. Some regionspecific regulation is dependent on whether a particular

neural circuit is activated by a given stimulus, but phenotypic differences between cells from different brain regions also contribute to differences in regulation. Because IMR32 cells express CRF 1 receptors that can effectively couple to the adenylate cyclase system as occurs in vivo, they represent one in vitro approach to understanding the regulation of the CRF 1 receptor-coupled adenylate cyclase system. However, results obtained with these cells must be confirmed with primary cultures of neurons and with in vivo studies. This absence of mRNA changes following prolonged agonist exposure is not unique to CRF 1 receptors. It has also been reported for other G-protein-coupled receptors, for example the thromboxane A 2 (TXA 2 ) receptor when expressed in a human astrocytoma cell line [48]. However, TXA 2 receptor mRNA levels do change following exposure to agonist in an endothelial cell line [43]. This suggests the possibility that G-protein-coupled receptor mRNA regulation may differ between neuronal and nonneuronal cells. We report here a concentration-dependent difference in the ability of CRF receptor levels to recover following CRF treatment. While receptor levels returned to control values following treatment with 10 nM CRF, they failed to recover following a 10 min, 1 h or 6 h treatment with 100 nM CRF. The recovery of CRF receptors following exposure to 10 nM CRF agrees with the rapid resensitization observed in primary cultures of rat pituitary cells where the cAMP response recovers 2 h after a 3 h exposure to 10 nM CRF [44]. A similar rapid recovery occurs in adrenergic peripheral cell lines following treatment with norepinephrine [32,38]. In comparison, recovery of CRF receptors in the human retinoblastoma Y-79 cell line is slow requiring approximately 24 h following a 4 h exposure to 10 nM CRF. The lack of complete recovery of CRF receptors in IMR-32 cells following treatment with 100 nM CRF suggests that at this dose CRF treatment produces a permanent decrease in the responsiveness of IMR-32 cells to subsequent CRF exposure. The physiological relevance of the 100 nM concentration needs to be addressed. Given that the Kd for CRF binding to CRF 1 receptors on IMR-32 cells is approximately 0.5 nM, it would be expected that a large fraction of the receptors would be occupied at 10 nM CRF (90– 95%). However, it would take 100 nM CRF to occupy virtually all of the receptors on the cell surface. Therefore, it is possible that there will be a somewhat greater second messenger response at the 100 nM versus 10 nM concentration. While the 100 nM CRF represents a very ‘high’ concentration, the data indicate that exposure to this high level for as short as 10 min is enough to produce a long-term downregulation. Given that the 100 nM CRF concentration is in the dynamic range for interacting with the receptors and that the synaptic concentration has to be at this level for only a relatively short period of time, it is not unreasonable to hypothesize that exposure to a very

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stressful event may result in a long-term alteration in CRF 1 receptor responsiveness in the brain. The question arises as to what is different about the 100 nM CRF versus the 10 nM CRF treatment that results in an inability of the CRF 1 receptors to fully resensitize. Resensitization of G-protein-coupled receptors is thought to be initiated by sequestration of phosphorylated receptors into endosomes where the receptors are then dephosphorylated and recycled back to the cell surface. The protein b-arrestin is thought to function, in part, as an adapter-like protein, by binding to the phosphorylated receptor and targeting it to the cellular endocytotic machinery [60]. The dissociation of the b-arrestin from the receptor is necessary for receptor dephosphorylation to take place. Recently it has been shown that for the vasopressin V2 receptor (V2R) that resensitizes very slowly, the b-arrestin remains associated with the V2R inside the endocytic vesicles thereby preventing receptor dephosphorylation. In contrast, for the b 2 adrenergic receptor that resensitizes rapidly, the b-arrestin dissociates from the receptor prior to internalization [36]. The high stability of the V2R-b–arrestin complex is thought to result from a high level of phosphorylation of serine residues in the C-terminal domain of the desensitized form of the V2R [22]. Although the distribution of serine residues in the C-terminal domain differs between CRF 1 and V2R receptors, it is possible that treatment with 100 nM CRF may result in a greater phosphorylation of the C-terminal domain of the CRF 1 receptor compared to treatment with 10 nM CRF. This would result in a more stable complex between the CRF 1 receptor and an arrestin-like molecule, preventing receptor dephosphorylation and resensitization. Future studies will determine if there are receptor phosphorylation differences following 10 nM and 100 nM CRF treatments. Another intriguing observation is that CRF 1 receptor numbers fail to recover to control levels even after 3.5 days despite unchanged levels of CRF 1 receptor mRNA. This suggests that the ability of the cell to make new CRF 1 receptors has been impaired. One possible explanation is a decreased translational efficiency of the CRF 1 receptor mRNA in the desensitized cells. The likelihood of such a mechanism is strengthened by the recent observation that a short upstream open reading frame in the 59-untranslated region of CRF 1 receptor mRNA can inhibit translation without altering CRF 1 receptor mRNA levels [59]. Another possible explanation is that the translated CRF 1 receptor protein cannot undergo the necessary posttranslational modifications to produce functional receptors on the cell surface. Future experiments will determine which of the above scenarios is occurring in the IMR-32 cell line and to what extent this mechanism of receptor regulation occurs in vivo. In conclusion, our studies suggest that treatment with 100 nM CRF produces a long-term decrease in the sensitivity of the CRF 1 receptor-coupled adenylate cyclase

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system. Receptor downregulation and desensitization are adaptive and well-known responses to receptor activation. However, it is hard to understand how longer-term decreases in receptors would play an adaptive role after stress. In vitro high dose CRF treatment may represent a model for the effects of overwhelming stress. If this is the case, our findings would suggest there is a potential for long-term CRF receptor downregulation to occur in vivo in response to the large increases in CRF release that occur with overwhelming stress. Such a postsynaptic effect could result in a net decrease in CRF signaling through this pathway. Alternatively, the presynaptic production and release of CRF could be enhanced secondarily to the persistent decrease in postsynaptic receptor levels in such a way that the CRF system would be functionally overactive. Experiments are currently in progress to determine the extent to which our in vitro observations occur in vivo and the extent to which they may participate in the etiology and maintenance of stress-related psychopathology.

Acknowledgements This work was supported by NIMH grant MH40855 (NHK), and funds from the University of Wisconsin HealthEmotions Research Institute and Meriter Hospital (Madison, WI). We thank Dr. Dimitri E. Grigoriadis (Neurocrine Bioscience, San Diego, CA) for providing us with human CRF 1 receptor, CRF 2 receptor and CRF-BP cDNA probes and for advice on experimental design. We also thank Drs. Robert C. Zaczek and Paul J. Gilligan (Dupont Pharmaceutical Company, Glenolden, PA) for the gifts of SC241 and DMP696, Mr. George A. Nash for technical assistance with the Northern blot analyses and Dr. Brian A. Baldo for advice regarding the statistical analyses.

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