QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.050088v1 306/2/794    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hauger, R. L. Articles by Dautzenberg, F. M. Search for Related Content PubMed PubMed Citation Articles by Hauger, R. L. Articles by Dautzenberg, F. M.

NEUROPHARMACOLOGY

Mediation of Corticotropin Releasing Factor Type 1 Receptor Phosphorylation and Desensitization by Protein Kinase C: A Possible Role in Stress Adaptation

Department of Veterans Affairs Healthcare System and Department of Psychiatry, University of California, San Diego, La Jolla, California

Received February 5, 2003; accepted May 5, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Protein kinase C (PKC)-mediated desensitization of the corticotropin releasing factor type 1 (CRF₁) receptor was investigated in human retinoblastoma Y79 and transfected COS-7 cells. Because stimulation of Y79 cells with CRF resulted in large (30-fold) increases in intracellular cAMP accumulation without changing inositol phosphate levels, the CRF₁ receptor expressed in retinoblastoma cells couples to G_s, but not to G_q, and predominantly signals via the protein kinase A cascade. Direct activation of PKC by treatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) or 1,2-dioctanoyl-sn-glycerol (DOG) desensitized CRF₁ receptors in Y79 cells, reducing the maximum for CRF- (but not forskolin)-stimulated cAMP accumulation by 56.3 ± 1.2% and 40.4 ± 2.1%, respectively (p < 0.001). Pretreating Y79 cells with the PKC inhibitor bisindolylmaleimide I (BIM) markedly inhibited PMA's desensitizing action on CRF-stimulated cAMP accumulation, but did not affect homologous CRF₁ receptor desensitization. Retinoblastoma cells were found to express PKC, I, II, , , and RACK1. When and isoforms of PKC were down-regulated 80 to 90% by a 48-h PMA exposure, PMA-induced CRF₁ receptor desensitization was abolished. In transfected COS-7 cells the magnitude of CRF₁ receptor phosphorylation after a 5-min exposure to PMA was 2.32 ± 0.21-fold greater compared with the basal level. Pretreating COS-7 cells with BIM abolished PMA-induced CRF₁ receptor phosphorylation. These studies demonstrate that protein kinase C (possibly and isoforms) has an important role in the phosphorylation and heterologous desensitization of the CRF₁ receptor.

The neuropeptide CRF regulates behavioral, neuroendocrine, and autonomic responses to stress by acting at two high-affinity CRF receptors (CRF₁ and CRF₂) in the amygdala and its extended neurocircuits, as well as in adrenocorticotropin-secreting pituitary corticotropes (Grigoriadis et al., 2001; Dautzenberg and Hauger, 2002; Perrin and Vale, 2002). CRF₁ receptors are rapidly desensitized in retinoblastoma Y79, neuroblastoma IMR-32, and transfected fibroblast Ltk cells exposed to high agonist concentrations of CRF or urocortin1 (Dieterich et al., 1996; Hauger et al., 1997; Dautzenberg et al., 2001a, 2002; Roseboom and Kalin, 2001). Our data indicate that GRK3 plays an important role in the homologous desensitization of CRF₁ receptors. First, in Y79 cells, uptake of a GRK3 antisense oligonucleotide or transfection of a GRK3 antisense cDNA construct decreased GRK3 expression by 55% and inhibited homologous CRF₁ receptor desensitization by 65% (Dautzenberg et al., 2001a). In addition, a large increase in GRK3 expression occurs in Y79 cells during the emergence of CRF-induced CRF₁ receptor desensitization (Dautzenberg et al., 2002). We have also observed that epitope-tagged CRF₁ receptors expressed in COS-7 cells are rapidly phosphorylated following exposure to a saturating concentration of CRF (Hauger et al., 2000). Since COS-7 cells express GRK2, but not GRK3 (Menard et al., 1997), it appears that GRK2 also has a role in desensitizing CRF₁ receptors. These findings suggest that GRK3, and possibly GRK2, regulate CRF₁ receptor signaling when the release of endogenous agonists in the central nervous system is increased by stress.

In contrast to homologous desensitization, heterologous mechanisms can attenuate receptor responsiveness independent of agonist binding. Typically, heterologous desensitization occurs when an unactivated receptor is phosphorylated via the catalyzing action of a specific second messenger kinase that has been stimulated by another GPCR (Penn and Benovic, 1998; Pierce et al., 2002). Several studies have shown that protein kinase A (PKA) desensitizes many G_s-coupled GPCRs by phosphorylating intracellular consensus sites (Krupnick and Benovic, 1998; Penn and Benovic, 1998; Pierce et al., 2002; Kohout and Lefkowitz, 2003). Because the agonist-induced "active" conformation of the CRF₁ receptor couples to G_s, which in turn activates the cAMP-PKA pathway, it was initially predicted that PKA would have a predominant role in CRF₁ receptor desensitization (Chen et al., 1993). However, neither maximal PKA stimulation nor PKA inhibition influenced CRF₁ receptor phosphorylation and desensitization (Hauger et al., 2000; Dautzenberg et al., 2001a; Roseboom and Kalin, 2001). Thus, PKA does not appear to mediate homologous or heterologous desensitization of the CRF₁ receptor.

Protein kinase C (PKC) can also mediate homologous or heterologous GPCR desensitization. PKC-mediated phosphorylation of GPCR and adenylyl cyclase proteins contributes to desensitization, internalization, and down-regulation of many receptors (Penn and Benovic, 1998; Oppermann et al., 1999; Olivares-Reyes et al., 2001; Bhattacharyya et al., 2002; Mandyam et al., 2002; Pierce et al., 2002). PKC-mediated mechanisms have important roles in regulating ion channel sensitivity and other aspects of postsynaptic neurotransmission, including long-term potentiation and depression (Tanaka and Nishizuka, 1994). PKC-related signaling abnormalities have also been implicated in the pathophysiology of a wide variety of human illnesses (Tanaka and Nishizuka, 1994; Dempsey et al., 2002).

Two potential PKC phosphorylation sites (Ser³⁸⁶ and Ser⁴⁰⁸) are present in the cytoplasmic C-terminal tail of the CRF₁ receptor (Chen et al., 1993; Dautzenberg et al., 2001b). A recent study has shown that oxytocin receptor activation initiates heterologous desensitization of CRF-stimulated cAMP accumulation in human myometrial cells via a PKC-dependent mechanism (Grammatopoulos and Hillhouse, 1999). Because CRF₁ receptors have been shown to couple to G_q proteins and signal via the PKC cascade (Ulisse et al., 1990; Kiang et al., 1994; Dieterich et al., 1996; Karteris et al., 2000), PKC may also contribute to homologous CRF₁ receptor phosphorylation and desensitization. The primary goal of this study was to test the hypothesis that protein kinase C mediates phosphorylation and desensitization of the CRF₁ receptor.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Reagent purchases were as follows. 1) Bovine serum albumin (BSA, fraction V), isobutylmethylxanthine, forskolin, and other highly pure chemicals: Sigma-Aldrich (St. Louis, MO); 2) aprotinin (Trasylol), phorbol 12-myristate 13-acetate (PMA), 4-phorbol 12-myristate 13-acetate (an inactive PMA isomer), 1,2-dioctanoyl-sn-glycerol (DOG), bisindolylmaleimide I (BIM), staurosporine: Calbiochem (San Diego, CA); 3) defined fetal bovine serum (SH30070.03): Hyclone Laboratories (Logan, UT). The University of California, San Diego Cell Culture Core Facility supplied all other cell culture reagents (Mediatech, Herndon, VA). Ovine CRF (CRF: Bachem California, Torrance, CA; purity >98%) was used to stimulate cAMP accumulation in all experiments and to desensitize CRF₁ receptors. All SDS-PAGE reagents were purchased from Invitrogen-Novex (Carlsbad, CA). For CRF₁ receptor phosphorylation experiments, the following reagents were used: 1) protein A-Sepharose (PrA-Seph) (Oncogene Research Products, San Diego, CA); 2) HA.11 mouse monoclonal anti-HA antibody (Babco, Richmond, CA).

Cell Culture and Transfection. Suspension human retinoblastoma Y79 cultures were grown at a density of 5 to 8 x 10⁷ cells/flask in RPMI 1640 and used between passages 4 and 25 as previously described (Hauger et al., 1997; Dautzenberg et al., 2001a). COS-7 cells were seeded at 6 x 10⁵ cells/10-cm dish in DMEM containing 10% (v/v) fetal bovine serum, 100 µg/ml streptomycin, and 100 IU/ml penicillin (COS-7 cell medium). COS-7 cells were cultured for 3 days before transfection using 5 ml of OptiMEM containing 10 µg/ml LipofectAMINE (Invitrogen, Carlsbad, CA) and 5 µg of HA-tagged CRF₁ receptor cDNA for 6 h at 37°C as described (Hauger et al., 2000). After changing to fresh COS-7 cell medium, the cells were cultured for a further 2 days before use.

Second Messenger Assays. Following extensive cell washing, intracellular cAMP levels were measured in ether-extracted and acetylated cell lysates using a double-antibody radioimmunoassay kit (cAMP [¹²⁵I] assay system, RPA 509; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK), as previously described (Hauger et al., 1997; Dautzenberg et al., 2001a). For inositol phosphate (IP) experiments, Y79 cells were metabolically labeled with myo-[2-³H]inositol (5 µCi/ml) overnight (18 h). After labeled cells were washed twice with a large volume (40 ml) of myoinositol-free RPMI 1640, they were preincubated in myoinositol-free Medium 199(E) with 10 mM lithium chloride for 30 min at 37°C. After cells were again washed, centrifuged, and resuspended in myoinositol-free Medium 199(E) with 10 mM lithium chloride, they were maximally stimulated with 1 µM CRF for 20 min. After the reaction was stopped by adding 10 mM formic acid, formation of IP₂ and IP₃ was measured by anion exchange chromatography using Bio-Rad AG 1-X8 columns, as previously described (Olivares-Reyes et al., 2001).

Western Blot Quantitation of PKC Protein Expression. Lysates of Y79 cells (20–30 µg per lane) were loaded onto 4 to 12% Tris-glycine gradient gels (Invitrogen-Novex) and proteins were resolved in a NOVEX Xcell II Mini-Cell System using SDS-PAGE under reducing conditions (Invitrogen-Novex Tris-glycine SDS sample buffer containing 5.0% -mercaptoethanol) at a fixed 125 V (current 35–40 amps) for 90 min according to the method of Laemmli (Dautzenberg et al., 2001a; Dautzenberg and Hauger, 2001). After Western transfer of resolved retinoblastoma proteins onto polyvinylidine difluoride membranes (Invitrogen-Novex) was completed (Dautzenberg et al., 2001a; Dautzenberg and Hauger, 2001), blots were blocked for 1 h in a solution of Tris-buffered saline (20 mM Tris pH 7.5, 500 mM NaCl) with 0.2% Tween 20 and 4% BSA (TTBS-BSA) with constant shaking at room temperature. Blots were then washed with TTBS-BSA and immunoprobed overnight (18 h) at 4°C with constant shaking with one of the following antibodies: 1) a mouse monoclonal antibody targeting PKC (610108; 1:1,000), PKC (611158; 1:1,000), PKC (610397; 1:500), PKC (610085; 1:1,000), PKC (610814; 1:250), PKC (610089; 1:250), PKC (610175; 1:250), PKC (610207; 1:250), RACK1 (610178; 1:2,500), or DGK (610930; 1:250) (BD Biosciences PharMingen Transduction Laboratory; San Diego, CA); 2) a mouse monoclonal targeting PKCI (E-3/sc-8049; 1:100) or a rabbit polyclonal targeting PKCII (C-18/sc-210; 1:200) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); or 3) a rabbit polyclonal antibody (P500; 1:3,000, kindly provided by Dr. A. Newton, Department of Pharmacology, University of California, San Diego) targeting the phosphorylated activation loop of protein kinase CII (Dutil and Newton, 2000). After the membranes were washed in TTBS-BSA (six 10-min washes), blots were incubated for 1 h at room temperature with constant shaking with one of the following antibodies (in TTBS-BSA): 1) sheep anti-mouse IgG-HRP (NA931, 1:5,000; Amersham Biosciences Inc., Piscataway, NJ) or 2) donkey anti-rabbit IgG-HRP (NA934, 1:5,000; Amersham Biosciences Inc.). After membranes were washed extensively in TTBS (six 20-min washes), chemiluminescent detection of Western blots was performed using ECL⁺ Plus (Amersham Biosciences Inc.).

CRF₁ Receptor Phosphorylation Assay. Phosphorylation of the CRF₁ receptor was determined as previously described (Hauger et al., 2000). Briefly, transfected COS-7 cells in 10-cm dishes were metabolically labeled for 4 h at 37°C in 5 ml of P_i-free DMEM containing 0.1% (w/v) BSA and 100 µCi/ml ³²P_i. Cells were then pretreated with vehicle or BIM for 30 min followed by a 5-min exposure to PMA for 5 min. Treated cells were lysed in buffer (LB: 50 mM Tris, pH 8.0, 100 mM NaCl, 20 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 10 mg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml pepstatin, 10 µg/ml benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 µM okadaic acid) and probe-sonicated twice for 20 s. After removal of nuclei at 750g, membranes were pre-extracted by the addition of an equal volume of LB containing 2 M NaCl and 8 M urea followed by overnight tumbling at 4°C. The membranes were then collected at 200,000g and solubilized in LB+ [LB supplemented with 1% (v/v) NP 40, 1% (w/v) sodium deoxycholate, and 0.1% (w/v) SDS] with Dounce homogenization. After clarification at 14,000g, solubilized membranes were precleared by being incubated with 2% (v/v) protein A-Sepharose for 1 h at 4°C. Immunoprecipitation of CRF₁ receptors was performed by adding 1 µl of HA.11 antibody and 2% (v/v) protein A-Sepharose and incubating overnight at 4°C. After washing of the Sepharose-bound immune complexes in LB+ lacking protease inhibitors, ³²P-labeled phospho-HA-CRF₁ receptors were eluted in Laemmli sample buffer for 1 h at 48°C and resolved by SDS-PAGE (8–16% gradient resolving gel) before visualization in a PhosphorImager (Amersham Biosciences Inc.).

Data Reduction and Statistical Analyses. CRF₁ receptor desensitization data were calculated as percentage of control values as previously described (Hauger et al., 1997). Data reduction for the cyclic AMP radioimmunoassay was performed using a log-logit program. Analyses of variance (ANOVAs) across experimental groups were performed on a MacIntosh PC using PRISM Version 2.0 (GraphPad Software Inc., San Diego, CA). If the one-way ANOVA was statistically significant, planned post hoc analyses were performed using Bonferroni's multiple comparison tests to determine individual group differences. Immunoreactive PKC protein bands on Western blots were quantitated and analyzed on the STORM imager using ImageQuant software (Amersham Biosciences Inc.) (Dautzenberg et al., 2001a).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Time Course for PMA-Induced CRF₁ Receptor Desensitization in Y79 Cells. We began our study by investigating potential PKC regulation of CRF₁ receptor signaling in human retinoblastoma Y79 cells. Although PMA-induced PKC activation augments the ability of CRF to stimulate cAMP accumulation and adrenocorticotropin release in anterior pituitary cells (Abou-Samra et al., 1987), the magnitude of cAMP accumulation stimulated by 100 nM CRF was decreased by 50% when Y79 cells were coincubated with 100 nM PMA during the 15-min stimulation period (Fig. 1A). When Y79 cells were stimulated with 1 µM CRF for 20 min, inositol phosphate levels were not increased above basal (i.e., no CRF stimulation) values (Fig. 1B) in contrast to a 29.5 ± 0.9-fold increase in cAMP formation observed in CRF-stimulated cells (p < 0.0001; data not shown). Previous studies have shown that PKC participates in both homologous and heterologous GPCR desensitization by promoting the phosphorylation of serines and threonines within consensus sites located in a receptor's C terminus and/or other intracellular loops (Penn and Benovic, 1998; Pierce et al., 2002). When Y79 cells were pretreated for 1 h with 1 µM PMA to maximally activate PKC, the magnitude of cAMP accumulation following subsequent restimulation with 100 nM CRF was decreased by 50% (p < 0.0001) (Fig. 1C), thereby providing evidence for PKC mediation of CRF₁ receptor desensitization. When the reversibility of PMA-induced desensitization of CRF₁ receptors was investigated, we found that CRF-stimulated cAMP stimulation failed to recover from the desensitized state in Y79 cells that were exposed to 1 µM PMA for 1 h, washed extensively, and then cultured for an additional 4 h (data not shown). The specificity of PMA-induced CRF₁ receptor desensitization was confirmed by demonstrating that CRF-stimulated cAMP accumulation was not significantly reduced following a 1-h pretreatment with 1 µM 4-phorbol, a PMA analog that is incapable of activating PKC (Fig. 1C). Although Y79 cells were washed thoroughly at the end of the pretreatment period, basal cAMP levels were significantly increased (p < 0.001) following a 1-h exposure to PMA but not 4-phorbol (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Effect of PMA exposure on CRF-stimulated cAMP accumulation in Y79 cells. A, effect of coincubation with 100 nM CRF and/or 100 nM PMA during the 15-min stimulation period. Each bar represents mean ± S.E.M. values of cAMP levels (n = 12 per group). By ANOVA, there were significant differences across the groups for changes in cAMP levels (F = 747.4, p < 0.0001). The following post hoc differences were found to be statistically significant between cell groups: a, p < 0.001 versus basal; b, p < 0.001 versus 100 nM PMA; c, p < 0.05 versus basal; d, p < 0.001 versus 100 nM CRF. B, effect of incubation with 1 µM CRF on inositol phosphate generation during a 20-min stimulation period. No significant differences (F = 2.52; p = 0.10) in IP2/IP3 levels were observed in CRF-stimulated or basal cells. C, effect of pretreatment with 1 µM PMA or 1 µM 4-phorbol for 1 h before a 15-min stimulation with 100 nM CRF. Each bar represents mean ± S.E.M. values of cAMP levels (n = 5 per group). Retinoblastoma CRF1 receptors were desensitized by 46% during the 1-h exposure to PMA, but not 4-phorbol. By ANOVA, there were significant differences across the groups for changes in cAMP levels (F = 117.7, p < 0.0001). The following post hoc differences were found to be statistically significant between cell groups: a, p < 0.001 versus basal; b, p < 0.001 versus control-CRF; c, p < 0.001 versus 4-phorbol-CRF; d, p < 0.001 versus control-basal or 4-phorbol-basal.

When the time course of the desensitizing effect of PMA pretreatment was analyzed, CRF-stimulated cAMP accumulation was found to decrease progressively in Y79 cells exposed to 1 µM PMA for 5 min (22.7 ± 1.6%), 10 min (48.9 ± 2.2%), 15 min (48.8 ± 2.6%), and 30 min (58.2 ± 2.6%) (Fig. 2A). The magnitude of PMA-induced CRF₁ receptor desensitization was similar in Y79 cells exposed to PMA for 30 min or 1 h (56.3 ± 1.2%) (Fig. 2A). However, the reduction in CRF-stimulated cAMP accumulation was significantly less following exposure to PMA for 3 h (42.2 ± 1.2%; p < 0.01) compared with 1 h (Fig. 2A). Although PMA pretreatment markedly reduced cAMP accumulation in Y79 cells rechallenged with CRF, it did not significantly decrease forskolin-stimulated cAMP accumulation during the same time period (Fig. 2A). When full CRF concentration-response curves were generated, the maximum CRF-stimulated cAMP accumulation was decreased 50% in Y79 cells exposed to 1 µM PMA (14.0 pmol/106 cells) compared with control cells (28.8 pmol/106 cells) (Fig. 2B).

View larger version (27K): [in this window] [in a new window]   Fig. 2. Time course for CRF1 receptor desensitization in Y79 cells exposed to 1 µM PMA. After pretreatment with PMA for the 5 min to 3 h was completed, retinoblastoma cells were extensively washed and stimulated with 100 nM CRF for 15 min. A, PMA-induced desensitization: data are mean ± S.E.M. of values expressed as percentage of control collected in 19 separate experiments (n = 3–12 replicates per treatment group). In control cells (i.e., no PMA pretreatment), basal and CRF-stimulated cAMP levels were 0.68 ± 0.13 and 22.57 ± 0.79 pmol/106 cells, respectively. By ANOVA, there were significant differences across the groups (F = 182.4, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: a, p < 0.001 versus control; b, p < 0.001 versus 5-min PMA; c, p < 0.05 versus 5-min PMA; d, p < 0.01 versus 1-h PMA. B, effect of pretreating Y79 cells with 1 µM PMA for 1 h on the sensitivity (EC50) and maximum for the dose-response stimulation of intracellular cAMP accumulation by CRF. In this representative experiment, cAMP levels (picomoles per 106 cells) were measured in duplicate in PMA-pretreated and control cells stimulated with 0 to 100 nM CRF.

The time course for homologous CRF₁ receptor desensitization in Y79 cells exposed to 1 µM CRF for 5 min to 3 h revealed progressive reductions in CRF-stimulated cAMP accumulation (p < 0.0001) (Fig. 3). However, in contrast to the time course of PMA-induced CRF₁ receptor desensitization (Fig. 2A), the reduction in CRF-stimulated cAMP accumulation was significantly greater in Y79 cells exposed to 1 µM CRF for 3 h (65.4 ± 1.8%; p < 0.05) compared with 1 h (49.3 ± 2.8%) (Fig. 3). The stimulation of cAMP accumulation by 50 µM forskolin in Y79 cells desensitized by CRF did not differ from cAMP responses to forskolin in control cells (data not shown), as previously observed (Hauger et al., 1997).

View larger version (65K): [in this window] [in a new window]   Fig. 3. Homologous CRF1 receptor desensitization induced by 1 µM CRF: data are mean ± S.E.M. of values expressed as percentage of control collected in seven separate experiments (n = 4–13 replicates per treatment group). In control cells (i.e., no CRF pretreatment), basal and CRF-stimulated cAMP levels were 0.63 ± 0.06 and 22.41 ± 0.75 pmol/106 cells, respectively. By ANOVA, there were significant differences across the groups (F = 102.3, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: a, p < 0.05 versus control; b, p < 0.001 versus control; c, p < 0.001 versus 5-min CRF; d, p < 0.01 versus 5-min CRF; e, p < 0.05 versus 1-h CRF.

Concentration-Dependent Characteristics of PMA-Induced CRF₁ Receptor Desensitization in Y79 Cells. We next established the concentration-dependence of the desensitizing effect of a 15-min PMA exposure on the ability of CRF to stimulate cAMP accumulation. CRF-stimulated cAMP accumulation was first observed to decrease at 10 nM PMA (80.8 ± 2.0% control; p < 0.001) and reached a nadir at 100 nM PMA (48.1 ± 3.6% control; p < 0.001) (Fig. 4). The EC₅₀ values for the PMA-induced CRF₁ receptor desensitization data were 15 nM. When Y79 cells were coincubated for 15 min with 10 nM CRF (which alone did not significantly desensitize CRF₁ receptors) and 10 nM PMA, the magnitude of CRF₁ receptor desensitization (48.3 ± 2.6%; p < 0.05) was significantly greater than that caused by 15-min pretreatment with 10 nM PMA (32.8 ± 3.7%) (Fig. 5). Concentration-dependent CRF₁ receptor desensitization was also observed in Y79 cells exposed to PMA (0–1 µM) for 1 h (p < 0.0001; data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Concentration-dependence of PMA-induced CRF1 receptor desensitization in Y79 cells for a 15-min pretreatment period. Data are mean ± S.E.M. of values expressed as percentage of control replicated in two independent experiments (n = 4–18 replicates per treatment group). In control cells (i.e., no PMA pretreatment), basal and CRF-stimulated cAMP levels were 0.77 ± 0.07 and 20.85 ± 0.36 pmol/106 cells, respectively. By ANOVA, there were significant differences across the groups (F = 52.6, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: a, p < 0.001 versus control; b, p < 0.001 versus 10 nM PMA; c, p < 0.05 versus 20 nM PMA.

View larger version (17K): [in this window] [in a new window]   Fig. 5. CRF1 receptor desensitization magnitudes resulting from a 15-min exposure to 10 nM PMA and/or 10 nM CRF. Data are mean ± S.E.M. of values expressed as percentage of control (n = 10 replicates per treatment group). These data were replicated in two independent experiments. By ANOVA, there were significant differences across the groups (F = 51.99, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: a, p < 0.001 versus control; b, p < 0.001 versus 10 nM CRF 15 min; c, p < 0.05 versus 10 nM PMA 15 min.

Effect of 1,2-Dioctanoyl-sn-glycerol (DOG) on CRF-Stimulated cAMP Accumulation. DOG structurally resembles the diacylglycerols formed when membrane phosphoinositides are hydrolyzed during G_q-coupled GPCR activation, and represents a more "physiological" signal stimulating PKC translocation to the membrane. Y79 cells pretreated with 100 µM DOG for 30 min (12.7 ± 0.4 pmol/10⁶ cells; p < 0.001) and 1 h (10.0 ± 0.3 pmol/10⁶ cells; p < 0.001) exhibited time-dependent reductions in the magnitude of CRF-stimulated cAMP accumulation compared with control cells (17.0 ± 0.4 pmol/10⁶ cells) (Fig. 6). The magnitude of CRF₁ receptor desensitization resulting from a 1-h DOG exposure (40.4 ± 2.1%; p < 0.05) was significantly greater than that observed after a 3-h DOG exposure (29.0 ± 2.7% decrease) (Fig. 6).

View larger version (54K): [in this window] [in a new window]   Fig. 6. Time courses for CRF1 receptor desensitization in Y79 cells exposed to 100 µM 1,2-dioctanoyl-sn-glycerol (DOG). Data are mean ± S.E.M. of values expressed pmol/106 cells for the stimulation of cAMP accumulation by 100 nM CRF (n = 10 per group) replicated in two independent experiments. By ANOVA, there were significant differences across the groups (F = 206.8, p < 0.0001). Significant cAMP responses to 100 nM CRF (p < 0.001) were observed in control cells and cells pretreated with DOG for 5 min, 30 min, 1 h, and 3 h. The following post hoc differences were also found to be statistically significant between groups: a, p < 0.001 versus control; b, p < 0.01 versus DOG 30 min; c, p < 0.05 versus DOG 3 h.

Expression of Protein Kinase C Isoforms in Y79 Cells. Retinoblastoma cell lysates were immunoprobed with antibodies that selectively recognize specific PKC isoforms to determine the pattern of protein kinase C expression in Y79 cells. Well defined retinoblastoma lysate bands migrated to positions consistent with the known molecular weights of PKC (Fig. 7A), PKCI (Fig. 7, B and C), and PKC (Fig. 7D). Expression of PKCII, PKC, PKC ((the mouse homolog of PKC), and RACK1 (a receptor protein that anchors protein kinase C to the membrane) was also detected in Y79 cells (data not shown). However, no immunoreactive bands were detected with specific antibodies for PKC, PKC, PKC, PKC, and DGK (data not shown). When additional immunoblotting experiments were performed using a polyclonal phospho-specific antibody (P500) targeting the phosphorylated activation loop of all PKC isoforms (Dutil and Newton, 2000), several bands migrating to 75–80 kDa were detected in Y79 cells (data not shown). Consequently, phosphorylated "mature" PKC species are present in the retinoblastoma cytosol. Only PKC isoforms with a phosphorylated activation loop are capable of catalyzing PKC-mediated phosphorylation of cellular proteins (Dutil and Newton, 2000).

View larger version (56K): [in this window] [in a new window]   Fig. 7. Down-regulation of - and I-isoforms of protein kinase C in Y79 cells during exposure to 1 µM PMA. Representative immunoblots (20–30 µg cell protein per lane) depicting changes in the expression of PKC (A), PKCI (B and C), or PKC (D) in Y79 cells exposed to 1 µM PMA, 1 µM CRF, or vehicle. Retinoblastoma PKC, PKCI, or PKC antibody-immunodetected bands comigrated in parallel with rat cerebrum standards at 80 to 82 kDa, consistent with the known molecular weights of these two PKC isoforms. Lysates of rat cerebrum (-, I-, and -isoforms) were used as internal controls for each immunoblot. These data were replicated in three independent experiments.

The levels of PKC and I proteins were depleted in Y79 cells exposed to PMA for 24 h or 48 h compared with control cells (Fig. 7, A–C). In contrast, incubating Y79 cells with 1 µM CRF for 24 h or 48 h did not decrease the expression of PKC (data not shown) or PKCI (Fig. 7C). This duration of CRF treatment produces >90% desensitization of retinoblastoma CRF₁ receptors and a 2- to 3-fold up-regulation of GRK3 expression (Hauger et al., 1997; Dautzenberg et al., 2002).

Effect of Down-Regulating PKC Expression on PMA-Induced CRF₁ Receptor Desensitization. Down-regulation of PKC by chronic phorbol ester treatment is another method for confirming that protein kinase C plays a role in receptor desensitization. Prolonged exposure of cells to PMA induces proteolysis, thereby depleting classical and novel, but not atypical, isoforms of PKC (Tanaka and Nishizuka, 1994). When retinoblastoma cells were pretreated with 1 µM PMA for 1 h, the magnitude of CRF-stimulated cAMP accumulation (11.4 ± 0.3 pmol/10⁶ cells; p < 0.001) again decreased by 60% compared with the CRF-stimulated cAMP response in control cells (Fig. 8). However, a large decrement in the ability of PMA to desensitize CRF₁ receptors was observed in Y79 cells chronically exposed to 1 µM PMA for 24 h (61.8 ± 4.9% of control) before 1-h PMA pretreatment and subsequent 15-min CRF stimulation (Fig. 8A). The similar magnitudes of CRF-stimulated cAMP accumulation in control Y79 cells (20.1 ± 0.6 pmol/10⁶ cells) and cells subjected to the 48-h PMA exposure (22.0 ± 0.5 pmol/10⁶ cells) indicated that chronic PMA treatment abolished PMA-induced CRF₁ receptor desensitization (Fig. 8A). Exposure of Y79 cells to PMA for 1 h, 24 h, or 48 h did not alter forskolin-stimulated cAMP accumulation (Fig. 8, A and B). As described above, the levels of PKC, I, and II proteins were depleted in Y79 cells exposed to PMA for 24 h or 48 h compared with control cells, while PKC expression did not change (Fig. 7, A–C). Since PKC down-regulation by 48-h PMA treatment abolished PMA-induced CRF₁ receptor desensitization, typical but not atypical PKC isoforms desensitize retinoblastoma CRF₁ receptors without altering adenylyl cyclase activity. Finally, in preliminary experiments, the magnitudes of homologous CRF₁ receptor desensitization did not differ in control Y79 cells and cells in which PKC was down-regulated by 48-h PMA exposure (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 8. Effect of down-regulating protein kinase C expression on CRF1 receptor desensitization in Y79 cells induced by a 1-h exposure to 1 µM PMA. A, data (mean ± S.E.M.) expressed as percentage of control for the stimulation of cAMP accumulation by 100 nM CRF (n = 6–16 per group) were collected in three independent experiments. By ANOVA, there were significant differences across the groups (F = 52.5, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: a, p < 0.001 versus control; b, p < 0.01 versus PMA 24 h; c, p < 0.001 versus PMA 48 h; d, p < 0.001 versus PMA 24 h-forskolin. B, representative experiment for 48-h PMA exposure. Data are mean ± S.E.M. of basal and CRF- and forskolin-stimulated cAMP levels (pmol/106 cells) (n = 6 per group). By ANOVA, there were significant differences across the groups (F = 288.4, p < 0.0001). Significant cAMP responses to 100 nM CRF (p < 0.001) and 50 µM forskolin (p < 0.001) were observed in control cells and cells pretreated with PMA for 1, 24, and 48 h. In addition, forskolin-stimulated cAMP accumulation was significantly greater than CRF-stimulated cAMP accumulation in control and PMA-treated cells (p < 0.001). However, the 48-h PMA exposure abolished PMA-induced CRF1 receptor desensitization. The following post hoc differences were also found to be statistically significant between groups: a, p < 0.001 versus control; b, p < 0.001 versus PMA 48 h.

Effect of Protein Kinase C Inhibition on Homologous and Heterologous CRF₁ Receptor Desensitization. The PKC inhibitors BIM (Fig. 9A) and staurosporine (500 nM; data not shown) had no effect on the homologous desensitization of retinoblastoma CRF₁ receptors. A 15-min exposure to 100 nM CRF resulted in similar reductions in the magnitude of cAMP accumulation following subsequent restimulation with 100 nM CRF in Y79 cells pretreated for 30 min with vehicle (30.2 ± 2.3%) or 2 µM BIM (36.9 ± 1.8%) (Fig. 9A). In contrast, pretreating Y79 cells with BIM significantly inhibited PMA-induced CRF₁ receptor desensitization (Fig. 9B). CRF-stimulated cAMP accumulation decreased in Y79 cells subjected to a 15-min exposure to 100 nM (53.9 ± 1.5% of control; p < 0.001) or 1 µM PMA (44.1 ± 2.6% of control; p < 0.001) (Fig. 9B). A 30-min pretreatment with 2 µM BIM inhibited CRF₁ receptor desensitization 56.6 ± 2.2% (p < 0.001) and 53.5 ± 2.5% (p < 0.001) during a 15-min exposure to 100 nM or 1 µM PMA, respectively (Fig. 9B).

View larger version (27K): [in this window] [in a new window]   Fig. 9. A, effect of pretreating Y79 cells with the PKC inhibitor bisindolylmaleimide I (2 µM) for 30 min on homologous CRF1 receptor desensitization induced by 15-min exposure to 100 nM CRF. Data are mean ± S.E.M. of values expressed as percentage of control (n = 19–20 replicates per treatment group) obtained in two separate experiments. By ANOVA, there were significant differences across the groups (F = 66.7, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: a, p < 0.001 versus control. B, effect of pretreating Y79 cells with the PKC inhibitor bisindolylmaleimide I (2 µM) for 30 min on PMA-induced CRF1 receptors in Y79 cells. Data are mean ± S.E.M. of values expressed as percentage of control (n = 7–17 replicates per treatment group) obtained in two separate experiments. After the 30-min pretreatment with 2 µM BIM was completed, Y79 cells were exposed to 100 nM PMA or 1 µM PMA for 15 min. By ANOVA, there were significant differences across the groups (F = 80.2, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: a, p < 0.001 versus control; b, p < 0.05 versus 100 nM PMA-no PKC inhibitor; c, p < 0.001 versus 100 nM PMA-no PKC inhibitor; d, p < 0.001 versus 1 µM PMA-no PKC inhibitor.

Effect of Protein Kinase C Inhibition on PMA-Induced CRF₁ Receptor Phosphorylation. We have previously reported that a high degree of CRF₁ receptor phosphorylation develops when COS-7 cells transiently transfected with an HA-epitope-tagged CRF₁ receptor cDNA are treated with CRF (Hauger et al., 2000). A 1.82 ± 0.09-fold increase (p < 0.001) in the density of the CRF₁ receptor phosphoprotein band (M_r 70,000) was again observed in transfected COS-7 cells stimulated for 5 min with 1 µM CRF compared with the basal phosphorylation level (data not shown). PMA-induced PKC activation in transfected COS-7 cells strongly phosphorylated the CRF₁ receptor (Fig. 10A). In a total of three experiments, the magnitude of CRF₁ receptor phosphorylation after a 5-min exposure to 200 nM PMA was 2.32 ± 0.21-fold greater compared with basal phosphorylation in control cells not exposed to PMA (p < 0.001) (Fig. 10B). A 30-min pretreatment with 2 µM BIM completely abolished PMA-induced CRF₁ receptor phosphorylation (Fig. 10). In addition, the basal level of CRF₁ receptor phosphorylation in untreated control cells was significantly decreased by BIM pretreatment (p < 0.05; Fig. 10B).

View larger version (25K): [in this window] [in a new window]   Fig. 10. Effect of the protein kinase C inhibitor bisindolylmaleimide I on PMA-stimulated HA-CRF1-receptor phosphorylation. A, after a 30-min pretreatment period with vehicle or 2 µM BIM was completed in a representative experiment, 32P-labeled HA-CRF1R-expressing COS-7 cells were exposed to media (control) or 1 µM PMA for 5 min. Phospho-HA-CRF1 receptors were immunoprecipitated and resolved by SDS-PAGE. The 60,000 to 70,000 Mr band representing the phosphorylated CRF1 receptor was 2.5-fold greater in PMA-treated cells (lane 3) compared with the band obtained from cells exposed to vehicle (lane 1). BIM pretreatment abolished PMA-induced HA-CRF1 receptor phosphorylation (lane 4). B, data (mean ± S.E.M.) from three separate experiments are presented as percentage of increase above basal HA-CRF1 receptor phosphorylation in control cells (transfected with HA-CRF1 receptor cDNA but not exposed to PMA). Pretreatment of transfected cells with 2 µM BIM for 30 min lowered basal receptor phosphorylation and blocked the ability of PMA to stimulate receptor phosphorylation. By ANOVA, there were significant differences across the groups (F = 35.5, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: a, p < 0.001 versus PMA-no PKC inhibitor; b, p < 0.05 versus control-no PKC inhibitor; c, p < 0.001 versus PMA-BIM.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study establishes that CRF₁ receptors endogenously expressed in human retinoblastoma Y79 cells undergo rapid PKC-mediated desensitization. In a series of experiments, PMA (0–1 µM) desensitized retinoblastoma CRF₁ receptors in a time- and concentration-dependent manner during a 5-min to 1-h pretreatment period. In addition, coincubation of Y79 cells for 15 min with 10 nM PMA (i.e., a concentration producing 20% desensitization) and 10 nM CRF (which did not result in any significant desensitization) before the 15-min CRF stimulation period desensitized CRF₁ receptors in an additive manner. Since forskolin-stimulated cAMP accumulation did not decrease when Y79 cells were exposed to PMA, PKC-induced CRF₁ receptor desensitization does not appear to involve a direct action of protein kinase C on adenylyl cyclase activity. When full CRF stimulation dose-response curves were generated, the maximum for CRF-stimulated cAMP accumulation decreased by more than 50% in Y79 cells exposed to 1 µM PMA for 1 h. The inability of 4-phorbol, which cannot activate PKC, to desensitize retinoblastoma CRF₁ receptors during a 1-h pretreatment period confirmed the specificity of PMA-induced CRF₁ receptor desensitization. Pretreatment of Y79 cells for 30 min or 1 h with DOG, a more "physiological" PKC activator, also caused a large degree of CRF₁ receptor desensitization.

Protein kinase C isoforms comprise a family of serine/threonine kinases that phosphorylate many cellular proteins, including GPCRs (Tanaka and Nishizuka, 1994; Penn and Benovic, 1998; Dempsey et al., 2002; Pierce et al., 2002). Retinoblastoma cells were found to express three conventional PKCs (, I, II), RACK1, which selectively binds PKCII (Dempsey et al., 2002), and one atypical PKC (). Diacylglycerols and phorbol esters bind to the N-terminal C1 regulatory domain of PKC, I, and II (but not PKC) and down-regulate these conventional PKC proteins when cells are chronically exposed to PKC activators (Tanaka and Nishizuka, 1994). When the duration of PMA or DOG exposure was extended to3hinour study, the magnitude of CRF₁ receptor desensitization was significantly less than that occurring with a 1-h pretreatment, presumably due to increased proteolytic degradation of PKC proteins. Subsequent experiments confirmed this possibility by demonstrating that PMA-induced CRF₁ receptor desensitization was abolished in Y79 cells where PKC and PKCI protein levels were depleted by a 48-h PMA exposure. PKC protein levels were not reduced in Y79 cells chronically exposed to PMA. Chronic PMA treatment can also down-regulate expression of the novel isoform PKC (Tanaka and Nishizuka, 1994), which is expressed in Y79 cells. Therefore, our data indicate that -, -, and possibly -isoforms of PKC desensitize retinoblastoma CRF₁ receptors.

BIM acts as a competitive inhibitor at the ATP binding site within the catalytic domains of many PKC isoforms including PKC, PKC, PKC, PKC, and PKC (Tanaka and Nishizuka, 1994). Although 2 µM BIM pretreatment did not alter homologous CRF₁ receptor desensitization, it markedly inhibited PMA-induced CRF₁ receptor desensitization. This suggests that a PKC-dependent mechanism contributes to heterologous, but not homologous, desensitization of CRF₁ receptors.

CRF₁ receptors in brain and transfected mouse fibroblast cells can couple to both G_s and G_q proteins, thereby activating both cAMP and phospholipase C pathways (Dieterich et al., 1996; Hillhouse et al., 2002). In Leydig cells and placenta, CRF₁ receptors may signal via G_q-mediated stimulation of phospholipase C and formation of IPs without generating an intracellular cAMP-dependent signal (Ulisse et al., 1990; Karteris et al., 2000). Taken together, these findings suggest that the CRF₁ receptor can couple to G_q and signal via the protein kinase C cascade in certain cell types (Kiang et al., 1994; Dautzenberg and Hauger, 2002). However, inositol phosphate generation was not increased in Y79 cells stimulated with CRF and inhibition of PKC in Y79 cells by BIM pretreatment failed to block homologous CRF₁ receptor desensitization. Thus, it seems likely that the CRF₁ receptor expressed in retinoblastoma cells couples to G_s but not to G_q. Similar findings have been observed for the pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC₁). The PAC₁ receptor exclusively couples to G_s in Y79 cells (Olianas et al., 1996; Dautzenberg et al., 1999), while the PAC₁ receptor couples to G_s and G_q in other cells (Harmar et al., 1998).

We determined whether an HA-epitope-tagged CRF₁ receptor recombinantly expressed in COS-7 cells could be phosphorylated by protein kinase C to establish a possible mechanism for PMA-induced CRF₁ receptor desensitization. A high degree of CRF₁ receptor phosphorylation was detected in transfected COS-7 cells during a 5-min exposure to PMA—an effect that was completely abolished by 30-min pretreatment with BIM. We previously observed that CRF exposure, but not forskolin-induced PKA activation or ionomycin-induced stimulation of Ca²⁺/calmodulin-dependent kinases, causes phosphorylation of the CRF₁ receptor in COS-7 cells, presumably due to a G protein receptor kinase mechanism (Hauger et al., 2000). Similarly, stimulation of PKA activity by forskolin or dibutyryl cAMP failed to desensitize CRF₁ receptors in Y79 and IMR-32 cells (Dautzenberg et al., 2001a; Roseboom and Kalin, 2001).

Recently, mice with a targeted deletion of the PKC gene were found to exhibit a deficit in fear conditioning (Weeber et al., 2000). In BALB/c mice, context-dependent fear conditioning was augmented by injecting CRF or activating PKC in the hippocampus, while pretreatment with a PKC inhibitor impaired acute stress-induced enhancement of context-dependent fear conditioning (Blank et al., 2002, 2003). PKC inhibitors also block CRF-induced increases in firing rates of hippocampal neurons in BALB/c mice (Blank et al., 2003). Consequently, PKC-mediated CRF₁ receptor signaling may contribute to the expression of contextual fear. We have previously hypothesized that deficient GRK-mediated CRF₁ receptor desensitization may trigger long-term psychopathology by increasing the sensitivity of and/or prolonging agonist-stimulated CRF₁ receptor signaling in brain pathways mediating fear and anxiety (Dautzenberg and Hauger, 2002). The present data suggest that abnormalities in PKC-mediated phosphorylation and desensitization of CRF₁ receptors may also result in CRF₁ receptor supersensitivity and sensitization of brain CRF neurotransmission, thereby reducing the activation threshold and increasing the magnitude and duration of fear and anxiety responses.

In conclusion, we have demonstrated that rapid desensitization of retinoblastoma CRF₁ receptors occurred when protein kinase C is activated. The presence of two potential PKC phosphorylation sites (Ser³⁸⁶ and Ser⁴⁰⁸) in the C-terminal cytoplasmic tail of the CRF₁ receptor suggests that the phosphorylation of the CRF₁ receptor by protein kinase C observed in this study may mediate this desensitization process (Chen et al., 1993; Dautzenberg et al., 2001b). However, depending on the cellular background, PKC-mediated CRF₁ receptor desensitization may occur via heterologous cross-talk with one or more G_q-coupled GPCRs or via a homologous mechanism whereby the agonist-activated CRF₁ receptor couples to the G_q protein and subsequently activates its phosphorylation by PKC. In a recent study it was found that activation of oxytocin receptors causes heterologous desensitization of CRF-stimulated cAMP accumulation in human myometrial cells via a PKC-dependent mechanism (Grammatopoulos and Hillhouse, 1999). Based on previous reports of cross-talk between receptor signaling pathways (Selbie and Hill, 1998; Budd et al., 1999; Thakker and Standifer, 2002), activation of heterologous GPCRs may result in cross-phosphorylation, desensitization, and possibly internalization of CRF₁ receptors, thereby shifting the stress response to other receptor mechanisms. Future studies will be directed at the identification of GPCRs that desensitize retinoblastoma CRF₁ receptors via PKC-mediated cross-talk.

	Acknowledgements

 
We thank Drs. A. Newton and P. Insel for helpful advice. We also gratefully acknowledge J. Hernandez-Aranda for expert assistance with experiemnts, A. Turken for performing cAMP assays, and Dr. Roger Smith for superb help with the initial CRF₁ receptor phosphorylation experiments. S. Shew graciously helped with editing of the manuscript.

	Footnotes

 
Dr. Hauger was supported by a Veterans Affairs (VA) Merit Review grant, the VA Mental Illness Research, Education and Clinical Center (MIRECC) of VISN22, and National Institutes of Health/National Institute on Drug Abuse Grant R01-DA13769-01A1. Dr. Catt was funded by the intramural program of the National Institutes of Health, where he is Chief of the Endocrinology and Reproductive Research Branch (ERRB) of the National Institute of Child Health and Human Development (NICHD). Dr. Olivares-Reyes was supported by a Postdoctoral Visiting Fellow award from the National Institutes of Health.

DOI: 10.1124/jpet.103.050088.

ABBREVIATIONS: GPCR, G protein-coupled receptor; GRK, GPCR kinase; CRF, corticotropin releasing factor; CRF₁, CRF receptor type 1; PKA, protein kinase A; G_s, stimulatory GTP binding protein; PKC, protein kinase C; BSA, bovine serum albumin; PMA, phorbol 12-myristate 13-acetate; DOG, 1,2-dioctanoyl-sn-glycerol; BIM, bisindolylmaleimide I; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate; ANOVA, analysis of variance; RACK, receptors for activated C-kinase; HA, hemagglutinin; PAC₁, pituitary adenylate cyclase-activating polypeptide type 1 receptor.

¹ Visiting Postdoctoral Fellow, ERRB, NICHD, National Institutes of Health.

² Current address: Departamento de Bioquimica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional, A. P. 14-740, Mexico 07000 D. F., Mexico.

³ Chief, ERRB, NICHD, National Institutes of Health.

⁴ Current address: CNS Discovery, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium.

Address correspondence to: Dr. Richard L. Hauger, VA Healthcare System and Department of Psychiatry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0603. E-mail: rhauger{at}ucsd.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Abou-Samra A-B, Harwood JP, Manganiello VC, Catt KJ, and Aguilera G (1987) Phorbol 12-myristate 13-acetate and vasopressin potentiate the effect of corticotropin-releasing factor on cyclic AMP production in rat anterior pituitary cells. J Biol Chem 262: 1129–1136.[Abstract/Free Full Text]

Bhattacharyya S, Puri S, Miledi R, and Panicker MM (2002) Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms. Proc Natl Acad Sci USA 99: 14470–14475.[Abstract/Free Full Text]

Blank T, Nijholt I, Eckart K, and Spiess J (2002) Priming of long-term potentiation in mouse hippocampus by corticotropin-releasing factor and acute stress: implications for hippocampus-dependent learning. J Neurosci 22: 3788–3794.[Abstract/Free Full Text]

Blank T. Nijholt I, Grammatopoulos DK, Randeva HS, Hillhouse HS, and Spiess J (2003) Modulation of neuronal excitability and associative learning by different corticotropin releasing factor-signaling cascades in mouse hippocampus. J Neurosci 23: 700–707.[Abstract/Free Full Text]

Budd DC, Challiss RAJ, Young KW, and Tobin AB (1999) Cross talk between m3-muscarinic and 2-adrenergic receptors at the level of receptor phosphorylation and desensitization. Mol Pharmacol 56: 813–823.[Abstract/Free Full Text]

Chen R, Lewis KA, Perrin MH, and Vale WW (1993) Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA 90: 8967–8971.[Abstract/Free Full Text]

Cliang A, Laporte SA, Caron MG, and Lefkowitz RJ (2002) Endocytosis of G protein-coupled receptors: roles of G protein-coupled receptor kinases and -arrestin proteins. Prog Neurobiol 66: 61–79.[CrossRef][Medline]

Dautzenberg FM, Braun S, and Hauger RL (2001a) GRK3 mediates homologous desensitization of CRF1 receptors: a potential mechanism regulating stress adaptation. Am J Physiol Regul Integr Comp Physiol 280: R935–R946.[Abstract/Free Full Text]

Dautzenberg FM and Hauger RL (2001) G protein receptor kinase 3 and protein kinase C mediate the homologous and heterologous desensitization, respectively, of the PACAP receptor, type 1 in human Y-79 retinoblastoma cells: absence of protein kinase A action in PAC₁ receptor desensitization. Neuropharmacology 40: 394–407.[CrossRef][Medline]

Dautzenberg FM and Hauger RL (2002) The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol Sci 23: 71–77.[CrossRef][Medline]

Dautzenberg FM, Kilpatrick GJ, Hauger RL, and Moreau J-L (2001b) Molecular biology of the CRH receptors—in the mood. Peptides 22: 753–760.[CrossRef][Medline]

Dautzenberg FM, Mevenkamp G, Wille S, and Hauger RL (1999) N-terminal splice variants of the type I PACAP receptor: isolation, characterization and ligand binding/selectivity determinants. J Neuroendocrinol 11: 941–949.[CrossRef][Medline]

Dautzenberg FM, Wille S, Braun S, and Hauger RL (2002) GRK3 regulation during CRF- and urocortin-induced CRF₁ receptor desensitization. Biochem Biophys Res Commun 298: 303–308.[CrossRef][Medline]

Dempsey EC, Newton AC, Mochly-Rosen D, Fields AP, Reyland ME, Insel PA, and Messing RO (2002) Protein kinase C isozymes and the regulation of diverse cell responses. Am J Physiol Lung Cell Mol Physiol 279: L429–L438.

Dieterich KD, Grigoriadis DE, and DeSouza EB (1996) Homologous desensitization of human corticotropin-releasing factor1 receptor in stable transfected mouse fibroblast cells. Brain Res 710: 287–292.[CrossRef][Medline]

Dutil EM and Newton AC (2000) Dual role of pseudosubstrate in the coordinated regulation of protein kinase C by phosphorylation and diacylglycerol. J Biol Chem 275: 10697–10701.[Abstract/Free Full Text]

Grammatopoulos D and Hillhouse EW (1999) Activation of protein kinase C by oxytocin inhibits the biological activity of the human myometrial corticotropin-releasing hormone receptor at term. Endocrinology 140: 585–594.[Abstract/Free Full Text]

Grigoriadis D, Haddach M, Ling N, and Saunders J (2001) The CRF receptor: structure, function and potential for therapeutic intervention. Curr Med Chem–CNS Agents 1: 63–97.

Harmar AJ, Arimura A, Gozes I, Journot L, Laburthe M, Pisegna JR, Rawlings SR, Robberecht P, Said SI, Sreedharan SP, et al. (1998) International union of pharmacology. XVIII. Nomenclature of receptors for vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptides. Pharmacol Rev 50: 265–270.[Abstract/Free Full Text]

Hauger RL, Dautzenberg FM, Flaccus A, Liepold T, and Spiess J (1997) Regulation of corticotropin-releasing factor receptor function in human Y-79 retinoblastoma cells: rapid and reversible homologous desensitization but prolonged recovery. J Neurochem 68: 2308–2316.[Medline]

Hauger RL, Smith RD, Braun S, Dautzenberg FM, and Catt KJ (2000) Rapid agonist-induced phosphorylation of the human CRF receptor, type 1: a potential mechanism for homologous desensitization. Biochem Biophys Res Commun 268: 572–576.[CrossRef][Medline]

Karteris E, Grammatopoulos D, Randeva H, and Hillhouse EW (2000) Signal transduction characteristics of the corticotropic-releasing hormone receptors in the feto-placental unit. J Clin Endocrinol Metab 85: 1989–1996.[Abstract/Free Full Text]

Kiang JG, Wang X, and McClain (1994) Corticotropin-releasing factor increases protein kinase C activity by elevating membrane-bound and isoforms. Chin J Physiol 37: 105–110.[Medline]

Kohout TA and Lefkowitz RJ (2003) Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol 63: 9–18.[Free Full Text]

Krupnick JG and Benovic JL (1998) The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu Rev Pharmacol Toxicol 38: 289–319.[CrossRef][Medline]

Mandyam CD, Thakker DR, Christensen JL, and Standifer KM (2002) Orphanin FQ/nociceptin-mediated desensitization of opioid receptor-like 1 receptor and m opioid receptors involves protein kinase C: a molecular mechanism for heterologous cross-talk. J Pharmacol Exp Ther 302: 502–509.[Abstract/Free Full Text]

Menard L, Ferguson SSG, Lefkowitz RJ, Caron MG, and Barak LS (1997) Synergistic regulation of ₂-AR receptor sequestration: intracellular complement of ARK and -arrestin determine kinetics of internalization. Mol Pharmacol 51: 800–808.[Abstract/Free Full Text]

Olianas MC, Ennas MG, Lampis G, and Onali P (1996) Presence of pituitary adenylate cyclase-activating polypeptide receptors in Y-79 retinoblastoma cells. J Neurochem 67: 1293–1300.[Medline]

Olivares-Reyes JA, Smith RD, Hunyady L, Shah BH, and Catt KJ (2001) Agonist-induced signaling, desensitization and internalization of a phosphorylation-deficient AT_1A angiotensin receptor. J Biol Chem 276: 37761–37768.[Abstract/Free Full Text]

Oppermann M, Mack M, Proudfoot AEI, and Olbrich H (1999) Differential effects of CC chemokines on CC chemokine receptor 5 (CCR5) phosphorylation and identification of phosphorylation sites on the CCR5 carboxyl terminus. J Biol Chem 274: 8875–8885.[Abstract/Free Full Text]

Penn RB and Benovic JL (1998) Mechanisms of receptor regulation, in Handbook of Physiology, Section 7: Endocrinology (Conn PM ed) vol I, pp 125–164, Oxford University Press, New York.

Perrin MH and Vale W (2002) Corticotropin-releasing factor receptors, in Understanding G Protein-Coupled Receptors and Their Role in the CNS (Pangalos MN and Davies CH eds) pp 505–526, Oxford University Press, New York.

Pierce KL, Premont RT, and Lefkowitz RJ (2002) Seven-transmembrane receptors. Nature (Lond) Rev 3: 639–650.

Roseboom PH and Kalin N (2001) Persistent corticotropin-releasing factor(1) receptor desensitization and downregulation in the human neuroblastoma cell line IMR-32. Brain Res Mol Brain Res 92: 115–127.[Medline]

Selbie LA and Hill SJ (1998) G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signaling pathways. Trends Pharmacol Sci 19: 87–93.[CrossRef][Medline]

Tanaka C and Nishizuka Y (1994) The protein kinase C family for neuronal signaling. Annu Rev Neurosci 17: 551–567.[CrossRef][Medline]

Thakker DR and Standifer KM (2002) Induction of G protein-coupled receptor kinases 2 and 3 contributes to the cross-talk between m and ORL1 receptors following prolonged agonist exposure. Neuropharmacology 43: 979–990.[CrossRef][Medline]

Ulisse S, Fabbri A, Tinajero JC, and Dufau ML (1990) A novel mechanism of action of corticotropin releasing factor in rat Leydig cells. J Biol Chem 265: 1964–1971.[Abstract/Free Full Text]

Weeber EJ, Atkins CM, Selcher JC, Varga AW, Mirnikjoo B, Paylor R, Leitges M, and Sweatt JD (2000) A role for the isoform of protein kinase C in fear conditioning. J Neurosci 20: 5906–5914.[Abstract/Free Full Text]

This article has been cited by other articles:

				R. H. Oakley, J. A. Olivares-Reyes, C. C. Hudson, F. Flores-Vega, F. M. Dautzenberg, and R. L. Hauger Carboxyl-terminal and intracellular loop sites for CRF1 receptor phosphorylation and beta-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R209 - R222. [Abstract] [Full Text] [PDF]

				D. Markovic, N. Papadopoulou, T. Teli, H. Randeva, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos Differential Responses of Corticotropin-Releasing Hormone Receptor Type 1 Variants to Protein Kinase C Phosphorylation J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1032 - 1042. [Abstract] [Full Text] [PDF]

				E. W. Hillhouse and D. K. Grammatopoulos The Molecular Mechanisms Underlying the Regulation of the Biological Activity of Corticotropin-Releasing Hormone Receptors: Implications for Physiology and Pathophysiology Endocr. Rev., May 1, 2006; 27(3): 260 - 286. [Abstract] [Full Text] [PDF]

				K. Kageyama, K. Hanada, T. Moriyama, T. Nigawara, S. Sakihara, and T. Suda G Protein-Coupled Receptor Kinase 2 Involvement in Desensitization of Corticotropin-Releasing Factor (CRF) Receptor Type 1 by CRF in Murine Corticotrophs Endocrinology, January 1, 2006; 147(1): 441 - 450. [Abstract] [Full Text] [PDF]

				T. Teli, D. Markovic, M. A. Levine, E. W. Hillhouse, and D. K. Grammatopoulos Regulation of Corticotropin-Releasing Hormone Receptor Type 1{alpha} Signaling: Structural Determinants for G Protein-Coupled Receptor Kinase-Mediated Phosphorylation and Agonist-Mediated Desensitization Mol. Endocrinol., February 1, 2005; 19(2): 474 - 490. [Abstract] [Full Text] [PDF]

				H. P. Jedema and A. A. Grace Corticotropin-Releasing Hormone Directly Activates Noradrenergic Neurons of the Locus Ceruleus Recorded In Vitro J. Neurosci., October 27, 2004; 24(43): 9703 - 9713. [Abstract] [Full Text] [PDF]

				D. Wietfeld, N. Heinrich, J. Furkert, K. Fechner, M. Beyermann, M. Bienert, and H. Berger Regulation of the Coupling to Different G Proteins of Rat Corticotropin-releasing Factor Receptor Type 1 in Human Embryonic Kidney 293 Cells J. Biol. Chem., September 10, 2004; 279(37): 38386 - 38394. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.050088v1 306/2/794    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hauger, R. L. Articles by Dautzenberg, F. M. Search for Related Content PubMed PubMed Citation Articles by Hauger, R. L. Articles by Dautzenberg, F. M.