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Endocrinology, doi:10.1210/en.2003-1023
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Endocrinology Vol. 145, No. 4 1718-1729
Copyright © 2004 by The Endocrine Society

Specificity and Regulation of Extracellularly Regulated Kinase1/2 Phosphorylation through Corticotropin-Releasing Factor (CRF) Receptors 1 and 2ß by the CRF/Urocortin Family of Peptides

Bhawanjit K. Brar, Alon Chen, Marilyn H. Perrin and Wylie Vale

The Clayton Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, California 92037

Address all correspondence and requests for reprints to: Wylie Vale, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037. E-mail: address: vale{at}salk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Corticotropin-releasing factor (CRF) receptor (CRFR)-mediated activation of the ERKs 1/2-p42 and -44) has been reported for CRF, urocortin (Ucn)-I, and sauvagine. Recently two new members of the CRF/Ucn family of peptides have been identified, Ucn-II/stresscopin-related peptide and Ucn-III/stresscopin. Using Chinese hamster ovary cells stably expressing CRFR1 and CRFR2ß, we show that Ucn-I, Ucn-II and Ucn-III activate ERK1/2-p42, 44 via CRFR2ß. CRF and Ucn-I but not Ucn-II or Ucn-III activates ERK1/2-p42, 44 in Chinese hamster ovary cells stably expressing CRFR1. The selectivity of the ligands for CRFR1 and CRFR2ß is shown in a time- and dose-dependent manner. The regulatory mechanisms for ERK1/2-p42, 44 activation by both receptor types are dependent on phosphatidylinositol-3 OH kinase, MAPK kinase 1, and phospholipase C. Raf-1 kinase, tyrosine kinases, and possibly intracellular Ca2+ provide regulatory roles for Ucn-I activation of ERK1/2-p42, 44 by CRFR1 and CRFR2ß. Studies of the regulation of ERK1/2-p42, 44 by Ucn-I were extended to cell lines that endogenously express CRFR1 (AtT-20 and CATHa cells) and CRFR2 (A7r5 and CATHa cells). Use of the Gi and Go protein inhibitor pertussis toxin showed that ERK1/2-p42, 44 activation by Ucn-I via CRFR1 and CRFR2ß are both Gi and/or Go protein dependent. Based on the data in this study, we present putative signaling pathways by which the CRF/Ucn family of peptides activate ERK1/2-p42, 44 by CRFRs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CORTICOTROPIN-RELEASING FACTOR (CRF) was isolated and characterized in 1981 as the major factor that orchestrates the endocrine stress response in mammals (1). Urocortin (Ucn)-I (2), a mammalian CRF-related peptide, and two other members of the CRF peptide family, Ucn II/stresscopin-related peptide (3, 4) and Ucn III/stresscopin (4, 5) have been detected in both humans and mice. The actions of the CRF family of peptides are mediated through two seven-transmembrane domain G protein-coupled receptors, CRF receptor (CRFR)1 and CRFR2. CRFR1 and CRFR2 exist in splice variant forms that are differentially expressed in the brain and periphery (6, 7). CRFR signaling has been studied predominantly in the brain (8), heart (9, 10, 11), myometrium (12), skin (13), circulatory system (14), and gut (15). CRFR activation by their cognate ligands results in GTP/GDP exchange on the G protein {alpha}-subunit, which participates in the activation of a number of signaling pathways. The ß{gamma}-subunits of the G protein complex can itself also activate a number of effectors (7).

CRFR activation has been shown to stimulate the MAPK pathway, in particular, the ERKs. ERKs belong to one subfamily of MAPKs that are composed of 42- and 44-kDa kinases, known as p42-ERK and p44-ERK, respectively (16, 17). The MAPK kinase (MEK)1/2, referred to as MEK1, mediates phosphorylation and activation of p42-ERK and p44-ERK (ERK1/2-p42, 44). ERK1/2-p42, 44 constitute a widely conserved family of serine/threonine protein kinases involved in many cellular programs such as cell proliferation, cell differentiation, cell movement, and cell survival (17). The ERK1/2-p42, 44 pathway has been shown to be regulated by several G proteins such as Gs, Gq, G12, and Gß{gamma} (7).

Activation of ERK1/2-p42, 44 by CRF ligands varies according to cell type and the type of CRFR stimulated. Activation of ERK1/2-p42, 44 by CRF ligands results in cytoprotection (9, 18), cell differentiation (19), and, possibly, myometrial contractility (12). In rat cardiac myocytes, Ucn-I activation of ERK1/2-p42, 44 at CRFR2ß results in cytoprotection against ischemia and ischemia reperfusion injury (9). In rat hippocampal neurons, Ucn-I and CRF, but not Ucn-II, promoted survival of these cells against oxidative and excitotoxic-induced cell death, mediated by CRFR1 activation (18). CRF acts as a differentiating factor in immortalized noradrenergic neuronal CATHa cells via protein kinase A (PKA) activation of ERK1-p44 (19). The function of PKA-mediated ERK1/2-p42, 44 activation by CRF in the anterior pituitary is not known (20).

There are a limited number of studies on the downstream effects of specific CRF ligand activation of select CRFRs. In some cell types, CRF ligand activation of ERK1/2-p42, 44 is mediated by PKA, [e.g. in rat hippocampal neurons that express CRFR1 (18)]. In contrast, sauvagine activation of ERK1/2-p42, 44 in Chinese hamster ovary (CHO) cells stably expressing human CRFR1 and CRFR2{alpha} is independent of PKA (21). In CRFR-expressing CHO cells, ERK1/2-p42, 44 MAPKs are partially regulated through phosphatidylinositol (PI)-3 OH kinase (PI-3 kinase), PI-specific phospholipase C (PLC), and the Ca2+-sensitive proline-rich tyrosine kinase (Pyk-2). However, the role of tyrosine kinases in activation of ERK1/2-p42, 44 was not investigated (21). In human pregnant myometrium, Ucn-I, but not CRF, activates the MEK1 ERK1/2-p42, 44 MAPK pathway (12). These cells express various CRFR1 splice variants and low levels of CRFR2ß. Interestingly, in HEK293 and CHO cells, which express different CRFR1 subtypes, CRF fails to activate ERK1/2-p42, 44 MAPK, but Ucn-I does activate MAPK via CRFR1 (12). However, we have previously shown that human (h)/rat (r) CRF binds hCRFR1 with an inhibitory constant of 0.95 nM and rUcn binds hCRFR1 with an inhibitory constant of 0.32 nM. In addition, the EC50 values for cAMP stimulation are comparable: 0.26 nM for CRF and 0.15 nM for Ucn-I (7).

The selectivity of Ucn-II and Ucn-III for CRFR2, compared with CRFR1, has been demonstrated by receptor binding affinities and cAMP activation (3, 5). However, the selectivity of Ucn-I, Ucn-II, Ucn-III, and CRF in stimulating ERK1/2-p42, 44 has not been determined. The purpose of this study was to investigate the specificity of rUcn-I, mouse (m)Ucn-II, mUcn-III, and h/rCRF in activating ERK1/2-p42, 44 in CHO cells stably expressing either cloned hCRFR1 or mCRFR2ß. ERK1/2-p42, 44 activation, in response to Ucn-I, was further studied using specific cell-permeable inhibitors. The activation of ERK1/2-p42, 44 by various CRF ligands was further examined in AtT-20, a mouse anterior pituitary cell, and immortalized mouse cholinergic neuronal cell line, CATHa, both of which express CRFR1. Studies showing activation of ERK1/2-p42, 44 by Ucn-I were extended to a CRFR2 expressing rat aortic smooth muscle cell line, A7r5. We also investigated the possible role of Gi and Go in Ucn-I activation of ERK1/2-p42, 44 activation via the CRFRs. Studies thus far have shown that CRFR mediated ERK1/2-p42, 44 activation is pertussis toxin (PTX) insensitive (12, 21, 18). We also postulate pathways for ERK1/2-p42, 44 activation by CRF/Ucn ligands. Because CRF ligand activation of ERK1/2-p42, 44 depends on cell type and CRFR expression characterization of distinct CRF ligand/receptor signaling pathways will allow for the targeted development of selective CRFR inhibitors and activators.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
The peptides, r/hCRF, rUcn, mUcn-II, and mUcn-III, were chemically synthesized (Dr. J. Rivier, Salk Institute) and dissolved in ddH2O containing 0.1% BSA at a stock concentration of 200 µM. Peptides were thawed only once. The MEK1/2 inhibitor PD98059 (PD) (Cell Signaling Technology, Beverly, MA) was used at a concentration of 0.05, 0.5, 5, and 50 µM and cultured with cells 10 min before ligand stimulation. PI-3 kinase was inhibited by a 20-min incubation of LY294002 (LY) and used at concentrations of 0.05, 0.5, 5, and 50 µM and added to cells 10 min before peptide addition. Ca2+-sensitive PLC was inhibited by U73122, which was used at concentrations of 0.1, 1, 10, and 100 µM and added to cells 10 min before peptide addition. The protein kinase C (PKC) inhibitor, H-7 dihydrochloride, was used at concentrations of 0.1, 1,10, and 100 µM and added to cells 20 min before peptide addition. Intracellular Ca2+ release was inhibited by 60 min incubation with 1'-2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester (BAPTA/AM). BAPTA/AM was used at concentrations of 0.1, 1, 10, and 100 µM. PKA was inhibited by H-89 dihydrochoride at concentrations of 0.05, 0.5, 5, and 50 µM and added to cells 10 min before ligand stimulation. A Raf-1 kinase inhibitor (R1-K1) (Calbiochem, La Jolla, CA) was used at concentrations of 0.1–100 nM and was added to the cells 30 min before ligand treatment. Genestein, an inhibitor of protein tyrosine kinases, was used at concentrations of 0.1, 1, 10, and 100 µM and added to cells 30 min before ligand stimulation. PTX was incubated with cells for 12 h at a concentration of 10 µg/ml before ligand stimulation. All cell-permeable signaling inhibitors were obtained from Calbiochem. Astressin-2B (Ast-2B), a CRFR2 selective antagonist (22), was added to CRFR2-expressing cells at concentration of 300 nM 30 min before ligand stimulation. Antalarmin, a CRFR1 selective nonpeptide antagonist (23), was preincubated with cells for 30 min at a concentration of 300 nM before ligand stimulation. In some experiments, optimal doses of cell signaling inhibitors were used, e.g. PD (50 µM), LY (50 µM), U7 (10 µM), H-7 (1 µM), BAPTAM/AM (1 µM), H-89 (5 µM), R1-KI (10 nM), and genestein (100 µM).

Detection of phosphorylated ERK1/2, p42,44 and immunoblot analysis
Full-length hCRFR1 and mCRFR2ß were stably expressed in CHO cells as previously described (3, 5). The CRFR-expressing CHO cells or CHO wild-type (WT) cells, AtT-20 (24), CATHa (25), and A7r5 (26) cells, were cultured in 12-well dishes at a density of 2 x 105 cells/well. Ribonuclease protection assays have shown that A7r5 cells express the CRFR2ß subtype, which had two isoforms differing in one codon at the junction of exons 3 and 4 (27). After an overnight culture in 750 µl of 0.1% (wt/vol) BSA/DMEM, 250 µl of 0.1% BSA/DMEM containing 4x concentrated appropriate peptide in 0.1% BSA/DMEM was added to the cells for various time periods and at various concentrations. Cells were harvested immediately in 50 µl of 1x sample treatment buffer [50 mM Tris (pH 6.8), 100 mM dithiothreitol, 2% (wt/vol) sodium dodecyl sulfate (SDS), 0.1% (wt/vol) bromophenol blue, and 10% (wt/vol) glycerol]. The samples were boiled for 5 min, and proteins were electrophoresed on a 4–12% SDS-polyacrylamide gel (Invitrogen, Carlsbad, CA). Electrophoresed proteins were subsequently transferred onto nitrocellulose membranes and then probed with antiphosphorylated ERK1/2-p42, 44 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. The membranes were washed in PBS with 0.05% (vol/vol) Tween and incubated with a 1:1000 dilution of a horseradish peroxidase-conjugated sheep antimouse IgG (Amersham Biotech Pharmacia, Little Chalfont, UK). Immunoreactive proteins were visualized using Super Signal West Pico chemiluminescent substrate (Pierce Chemical, Rockford, IL). The relative protein levels were determined using densitometry (ImageQuant 1.2, Molecular Dynamics, Sunnyvale, CA) by probing the membranes with antibody directed to total ERK2-p44 protein (Santa Cruz Biotechnology).

RT-PCR of CRFR1 and CRFR2 in cell lines
Total RNA was extracted from AtT-20, CATHa, A7r5, and mouse and rat brain using Trizol RNA isolation reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer’s recommendations. A DNase treatment for 30 min at 37 C using the RQ1 RNase-free DNase (Promega Corp., Madison, WI) was performed on the samples. RT-PCR was used to amplify the levels of CRFR1 and CRFR2. Total RNA extracted from mouse brain served as a positive control for AtT-20 and CATHa cells. Total RNA extracted from rat brain was used as a positive control for A7r5 cells. The expression of the ribosomal S-16 subunit (28) served as an internal control. The PCR conditions were: cDNA equivalent to 500 µg total RNA was amplified by PCR for 35 cycles; the annealing temperature was 60 C. The final MgCl2 concentration was 3 mM, and each reaction contained 2.5 U Taq DNA polymerase (BIO-X-ACT DNA polymerase, Bioline UK Ltd., London, UK). The following specific mouse and rat CRFR1, CRFR2, and S-16 oligonucleotide primers were used in the PCRs: mCRFR1: 5'-GGTGTGCCTTTCCCCATCATT-3' and 5'-CAACATGTAGGTGATGCCCAG-3' corresponding to nucleotides 740–760 (sense) and 998-1018 (antisense) (29), and the predicted size of the band is 279 bp. For mCRFR2: 5'-GGCAAGGAAGCTGGTGATTTG-3', and 5'-GGCGTGGTGGTCCTGCCAGCG-3' corresponding to nucleotides 957–977 (sense) and 1314–1334 (antisense) were used (30), and the predicted size of the band is 378 bp. For S-16-rib: 5'-TGCGGTGTGGAGCTCGTGCTTGT-3' and 5'-GCTACCAGGCCTTTGAGATGGA-3' corresponding to nucleotides 369–391 (sense) and 1968–1990 (antisense) primers were used (28), and the predicted size of the band is 309 bp. For rCRFR1: 5'-AAGGCGGGATCCAGGCAGTAGAGA-3', and 5'-TCCCGGTAGCCATTGTTTGTCGTG-3' corresponding to nucleotides 472–495 (sense) and 956–979 (antisense) (31), and the predicted size of the band is 508 bp. For rCRFR2: 5'CTGGTGGCTGCTTTCCTGCTTTTC-3' and 5'-ATGGGGCCCTGGTAGATGTAGTCC-3' corresponding to nucleotides 606–629 (sense) and 1007–1030 (antisense) (32), and the predicted size of the band is 425 bp.

DNA sequencing
The appropriate cDNA fragments of mouse CRFR1 and CRFR2 obtained from CATHa cells were extracted from gels using the QIAquick gel extraction kit (QIAGEN GmbH, Hilden, Germany) and subcloned into pGEM-T vector by using the pGEM-T Easy Vector System I (Promega). Nucleotide sequencing of the specific PCR bands was performed by automated direct DNA sequencing according to the manufacturer’s recommendations (model 377, PE Applied Biosystems, PerkinElmer Corp., Foster City, CA).

Preparations of neonatal mouse cardiac myocytes
Cardiac myocytes were isolated from hearts of C57BL/6 mice as described previously (9). The cardiac myocyte cell suspension was transferred to 24-well (1-cm diameter) gelatin-coated plates at a density of 105 cells/well for protein extraction experiments. Twenty-four hours after plating, the cell medium was replenished with DMEM supplemented with fetal calf serum at 1% (vol/vol) for an additional 24 h before experimentation. Within 2 d of preparation, a confluent monolayer of spontaneously beating myocytes was formed.

Statistics
All experiments were performed in triplicate and repeated at least three times. The dose response of each CRF ligand was performed on one 1.0 mm x 12-well gel (Invitrogen). Phosphorylated ERK1/2-p42, 44 and total ERK2-p44 levels were quantified using ImageQuant 1.2. Background was corrected using histogram peak, and the volumes of phosphorylated ERK1/2-p42, 44 were divided by the total ERK2-p44 values to give fold activation. Stimulation of ERK1/2-p42, 44 activation was expressed as fold increase over the vehicle-treated control (NT). The means and SEM were calculated for each treatment. Single-factor one-way ANOVA was performed for each group of treatments, and significance was assumed when P < 0.05. Differences among means were compared within the treatment groups using the Student’s t test. Because only three doses of peptide, 0.3, 3, and 30 nM, were analyzed on one gel, it was not possible to calculate Ec50 values. All dose responses for Ucn-I, Ucn-II, Ucn-III, and CRF were exposed to one large film (Kodak, Rochester, NY) to obtain comparative stimulation between the ligands.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time course of ERK1/2-p42, 44 activation by CRFR1 and CRFR2ß
To determine the optimal time for ERK1/2-p42, 44 activation, CHO cells stably expressing either CRFR1 (Fig. 1AGo) or CRFR2ß (Fig. 1BGo) were treated with 30 nM CRF, Ucn-I, Ucn-II, or Ucn-III for 1, 5, 10, 20, and 40 min. Both Ucn-II and Ucn-III failed to activate ERK1/2-p42, 44 phosphorylation at the cloned CRFR1, and CRF failed to activate ERK1/2-p42, 44 phosphorylation at CRFR2ß at all time points tested. For both receptor subtypes and all the ligands, the maximal increase in ERK1/2-p42, 44 activation was between 1 and 10 min of stimulation. To confirm that CHO-WT cells did not express any endogenous CRFRs, the CRF/Ucn-I (30 nM) were incubated with the CHO-WT cells for 1, 5, 10, 20, or 40 min. All four ligands failed to stimulate ERK1/2-p42, 44 phosphorylation in these cells, suggesting that CHO-WT cells do not express CRFRs (Fig. 1CGo). To determine whether the CRFR1 or CRFR2 ligand Ucn-I, at higher concentrations, may stimulate ERK1/2-p42, 44 activation at CHO-WT cells, 300 nM Ucn-I failed to stimulate ERK1/2-p42, 44 phosphorylation (Fig. 1DGo). Therefore, CHO-WT cells do not express any CRFR.



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  		FIG. 1. Time course of activation of ERK1/2-p42, 44 by CRF/Ucn peptides. Peptide (30 nM) was incubated with both the CRFR1- (A) and CRFR2- (B) expressing CHO cells or CHO-WT cells for 1, 5, 10, 20, and 40 min (C). The vehicle NT control is shown. ERK1/2-p42, 44 activation occurs maximally at 5 min in the CRFR1- or CRFR2-expressing cells. CRF failed to significantly activate CRFR2 at all time points, as did Ucn-II and Ucn-III at the CRFR1. The figure is representative of three individual experiments. All CRF/Ucn ligands failed to stimulate ERK1/2-p42, 44 activation in the CHO-WT cells at all time points tested. Ucn-I at 30 or 300 nM failed to stimulate ERK1/2-p42, 44 at CHO-WT cells (D), compared with the vehicle control (NT), and the bar graph is representative of three individual experiments ± SEM.

 
Ucn-I, Ucn-II, Ucn-III, and CRF activation of ERK1/2-p42, 44 in CHO cells stably expressing CRFR1 or CRFR2ß
CRFR1 expressing CHO cells were stimulated with 0.3, 3, and 30 nM Ucn-I, Ucn-II, Ucn-III, and CRF for 5 min. ERK1/2-p42, 44 were activated by Ucn-I and CRF in these cells. Ucn-I at 0.3, 3, and 30 nM produced significant increases in ERK1/2-p42, 44 phosphorylation (1.8 ± 0.12, P < 0.02, 2.25 ± 0.24, P < 0.04, and 4.2, P < 0.05 over basal, respectively; Fig. 2AGo). CRF at 0.3 nM caused an increase of 8.68 ± 0.28-fold in ERK1/2-p42, 44 phosphorylation (P < 0.001), and at 3 nM, the increase was 18.7 ± 2.7 (P < 0.03)-fold. CRF at a concentration of 30 nM produced a 67.7 ± 5.96 (P < 0.007)-fold increase in ERK1/2-p42, 44 activation over basal in the CHO-CRFR1 cells. In contrast to CRF and Ucn-I, Ucn-II and Ucn-III failed to activate ERK1/2-p42, 44 phosphorylation in these cells. The inability of Ucn-II and Ucn-III to stimulate ERK1/2-p42, 44 by CRFR1 are consistent with the low affinities of these ligands for CRFR1 (3, 5).



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  		FIG. 2. Selectivity of CRF and Ucn peptides to activate ERK1/2-p42, 44 through CRFR1 and CRFR2. Ucn-I, Ucn-II, Ucn-III, and CRF at 0.3 and 3 nM were added to equilibrated CHO cells expressing CRFR1 or CRFR2ß (CHO-CRFR1, CHO-CRFR2). After 5 min of receptor stimulation, cell lysates were harvested and subjected to SDS-PAGE immunoblot analysis using an antiphospho-ERK1/2-p42, 44 antibody and ERK2-p44 antibody. Stimulation of ERK activation was calculated by fold activation of phosphorylated ERK1/2-p42, 44/ total ERK2-p44, compared with the NT control. The experiment was repeated four times in triplicate, and the figure is representative of means of triplicates from one experiment. B, AtT-20 cells were stimulated with 0.3, 3, or 30 nM Ucn-I or CRF for 5 min. the stimulation of 0.3 and 3 nM Ucn-I resulted in 6.6 ± 0.63 (P < 0.03)- and 6.6 ± 0.67 (P < 0.03)-fold activation in phospho-ERK1/2-p42, 44, significantly greater than the NT vehicle control. No significant activation of ERK1/2 was seen when the cells were stimulated with 30 nM Ucn-I (P = 0.7). CRF significantly stimulated ERK1/2 phosphorylation at 0.3 nM (P < 0.05), 3 nM (P < 0.05), and 30 nM in which a maximal 6.2 ± 0.2 (P < 0.04)-fold stimulation of ERK1/2-p42, 44 was shown. The error bars are representative of a mean ± SEM (n = 3) fold ERK1/2-p42, 44 activation/total ERK2-p44. A representative immunoblot is shown. Significance is given by P < 0.05, compared with the NT control for each receptor subtype.

 
After 5 min of stimulation, Ucn-I, Ucn-II, and Ucn-III activated ERK1/2-p42, 44 in CHO cells expressing CRFR2ß. Ucn-I, Ucn-II, and Ucn-III at a dose of 0.3 nM produced a 2.3 (P < 0.05)-, 24.4 (P < 0.007)-, and 6.0 (P < 0.02)-fold activation of ERK1/2-p42, 44, respectively. Ucn-I, Ucn-II, and Ucn-III at 3 nM produced a 6.3 (P < 0.025)-, 24.4 (P < 0.01)-, and 6.35 (P < 0.03)-fold activation of ERK1/2-p42, 44 after 5 min of CRFR2ß stimulation, respectively. CRF at either 0.3, 3, or 30 nM failed to activate ERK1/2-p42, 44 in CHO cells expressing CRFR2ß. Five minutes of stimulation with 30 nM Ucn-I, 30 nM Ucn-II, or 30 nM Ucn-III caused a 1.4 (P < 0.05)-, 32.3 (P < 0.03)-, and 10.9 (P < 0.04)-fold activation of ERK1/2-p42, 44 over basal in these cells. Therefore, CRFR1 mediates the selective activation of ERK1/2-p42, 44 by Ucn-I or CRF, and CRFR2ß mediates the selective activation of ERK1/2, p42, 44 by Ucn-I, Ucn-II, or Ucn-III. In a separate experiment, we tested whether 300 nM Ucn-II or Ucn-III could stimulate ERK1/2-p42, 44 phosphorylation at CRFR1. Ucn-II did not phosphorylate ERK1/2 at CRFR1 because the fold stimulation was 0.75 ± 0.16. This 300-nM Ucn-II treatment was not significantly different from the NT control (P = 0.3). Similar to Ucn-II, 300 nM Ucn-III did not stimulate ERK1/2-p42, 44 phosphorylation because fold activation of ERK1/2 was 0.72 ± 0.11 and not different from the NT control (P = 0.14). CRF has been shown to have a lower affinity for CRFR2, compared with CRFR1 (7). However, 300 nM CRF failed to stimulate ERK1/2-p42, 44 activation at CRFR2 because the fold increase in ERK1/2-p42, 44 was 1.03 ± 0.4, a value that was not significantly different from the NT control (P = 0.94). Therefore, for ERK1/2-p42, 44 stimulation, even at higher concentrations of peptides, Ucn-II and Ucn-III are selective for CRFR2 and CRF is selective for CRFR1.

As shown in Fig. 2Go, in all experiments CRF gave a higher maximal fold increase in ERK1/2-p42, 44 phosphorylation, compared with Ucn-I at CHO-CRFR1, although the fold stimulation from one experiment varied, depending on the level of total ERK2-p44 and basal value of phospho-ERK1/2, p42, 44 in the NT control. The reason for CRF being stronger than Ucn-I in phosphorylating ERK1/2 is not known because both Ucn-I and CRF have similar binding affinities to CHO-CRFR1 (7). To determine whether this effect was unique to the stably transfected CHO cell line, AtT-20 cells, which express CRFR1, were treated with 0.3, 3, or 30 nM Ucn-I or CRF (Fig. 2BGo). In contrast to the CHO-CRFR1 cell line, at 0.3 and 3 nM, Ucn-I produced a greater fold activation of ERK1/2-p42, 44/ total ERK2-p44, compared with the same concentration of CRF. However, at 30 nM, CRF produced a greater fold stimulation of ERK1/2-p42, 44 activity, compared with 30 nM Ucn-I.

To demonstrate the direct involvement of CRFRs in mediating the activation of ERK1/2-p42, 44 by the CRF ligands, the effects of specific CRFR1 antagonist, antalarmin (Fig. 3AGo), or the specific CRFR2 antagonist, Ast-2B (Fig. 3BGo), were investigated. In CHO-CRFR1 cells, CRF and Ucn-I (10 nM) caused a 1.84 ± 0.1- and 4.1 ± 0.15-fold increase in ERK1/2-p42, 44 activation, compared with the NT control, respectively. Again, CRF caused a greater stimulation of phosphorylated ERK1/2-p42, 44/ total ERK2-p44 than Ucn-I, although the fold activation of ERK1/2-p42, 44 was less than that shown in Fig. 2Go. Antalarmin completely abolished Ucn-I activation of ERK1/2 at CRFR1 because the fold stimulation of ERK1/2-p42, 44 was 1.07 ± 0.08, significantly reduced from Ucn-I alone (P < 0.007). Antalarmin completely abolished CRF activation of ERK1/2 at CRFR1 because the fold stimulation of ERK1/2-p42, 44 was 1.23 ± 0.23, significantly reduced from CRF alone (P < 0.02). Antalarmin alone had no effect on ERK1/2-p42, 44 activation, compared with the control (NT) because the fold phosphorylation of ERK1/2-p42, 44 was 0.76 ± 0.33 and was not significantly different from the control. Ast-2B (300 nM) was preincubated with the cells before a 5-min stimulation with 10 nM peptide. Ucn-I, Ucn-II, and Ucn-III resulted in 2.9 ± 0.3 (P < 0.04)-, 12.6 ± 0.32 (P < 0.001)-, and 3.2 ± 0.25 (P < 0.005)-fold activation of ERK1/2-p42, 44 over the NT controls. The activation was blocked by 300 nM Ast-2B in CHO-CRFR2ß cells (Fig. 3BGo). When Ast-2B was given in combination with Ucn-I, Ucn-II, and Ucn-III, ERK1/2-p42, 44-fold stimulations decreased to 0.2 ± 0.14 for Ucn-I, 5.2 ± 3.0 for Ucn-II (P < 0.05), and 1.9 ± 0.1(P < 0.05) for Ucn-III. Ast-2B alone had no effect on ERK1/2-p42, 44 activation alone (Fig. 3BGo). Ast-2B (300 nM) did not completely abolish the Ucn-II and Ucn-III activation of ERK1/2-p42, 44, although from the statistical analysis, Ast-2B significantly blocked Ucn-II and Ucn-III activation of ERK1/2. The reason for this is not known. It is not possible that cross-talk with other endogenous CHO receptors contribute to this residual phospho-ERK1/2-p42, 44 activity because none of the CRF/Ucn-I ligands stimulated ERK1/2-p42, 44 activation in CHO-WT cells (see Fig. 1Go, C and D).



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  		FIG. 3. CRF/Ucn stimulation of ERK1/2-p42, 44 is receptor mediated. CHO cells stably expressing CRF-R1 or CRF-R2 were preincubated with 300 nM antalarmin (Ant, A) or Ast-2B (B), respectively, 30 min before 10 nM ligand stimulation. Cell lysates were harvested and subjected to ERK1/2-p42, 44 activation analysis. A representative immunoblot is shown. Significance is compared by guide bars shown (P < 0.05, n = 3).

 
Regulation of ERK1/2-p42, 44 activation through CRFR1 and CRFR2ß
To further investigate possible mediators of CRF ligand-induced ERK1/2-p42, 44 phosphorylation, the CHO-CRFR1 and CHO-CRFR2ß cell lines were stimulated for 5 min with 30 nM Ucn-I in the presence and absence of various cell-signaling inhibitors (Fig. 4Go). MEK1 inhibition by PD, PI-3 kinase inhibition by LY, and PLC inhibition by U73122 (U-7), completely suppressed ERK1/2-p42, 44 activation by Ucn-I. The PKC inhibitor H7, the intracellular Ca2+ inhibitor BAPTA/AM, and the PKA inhibitor H-89 partially inhibited ERK1/2-p42, 44 activation. The Raf-1 and tyrosine kinase inhibitors also inhibited ERK1/2-p42, 44 activation by Ucn-I via the cloned CRFR1 and CRFR2ß. There was no significant difference between the downstream regulatory mechanisms of ERK1/2-p42, 44 activation by Ucn-I, Ucn-II, and Ucn-III through cloned CRFR2ß, and no differences were found between Ucn-I or CRF activation of ERK1/2-p42, 44 through cloned CRFR1 (data not shown).



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  		FIG. 4. Upstream regulation of ERK1/2-p42,44 activation by Ucn at CRFR1 and CRFR2. Ucn-I has been shown to activate ERK1/2-p42, 44 signaling at both CRFR1 and CRFR2. To determine the intracellular signaling pathways that mediate ERK1/2-p42, 44 activation at the cloned receptors, cells were not treated (0) or treated with 30 nM Ucn-I peptide (+). Cells were pretreated with various concentrations of cell signaling inhibitors as stated before a 5-min stimulation with Ucn-I peptide. Cell lysates were harvested and subjected to immunoblot analysis for ERK1/2-p42, 44 activities. PD, LY, and U7 inhibited ERK1/2-p42, 44 activation by Ucn-I, suggesting that MEK1, PI-3 kinase, and PLC are involved in ERK1/2-p42, 44 activation by Ucn-I. The results are representative of three individual experiments.

 
ERK1/2-p42, 44 activation by CRF/Ucn ligands using cells that express endogenous CRFR1 and CRFR2
ERK1/2-p42, 44 signaling was extended to cell lines that endogenously express mCRFR1, rCRFR2, and mCRFR2. RT-PCR for CRFR1 and CRFR2 showed that AtT-20s and CATHa cells express CRFR1, and A7r5 and CATHa cells express CRFR2 (see Fig. 5Go for RT-PCR analysis of CRFR expression). Predicted mCRFR1 and mCRFR2 cDNA fragments from CATHa cells were isolated from agarose gels, subcloned into pGEM-T vector, and the nucleotide sequences determined. The sequences were compared with those in the GenBank database and were found to be identical to the known sequence of mCRFR1 and mCRFR2. At present, no CRFR2{alpha} isoform has been reported for the mouse. Therefore, we used oligonucleotide primers spanning the common sequence region for both CRFR2{alpha} and CRFR2ß and isolated the CRFR2{alpha} isoform from the mouse brain (Chen, A., unpublished observations). Using RT-PCR with specific primers for {alpha}- and ß-isoforms and cDNA obtained from the CATHa cells, we found that the dominant form of CRFR2 in these cells is CRFR2{alpha} (Chen, A., unpublished observations). Similar to CHO-CRFR1- and CRFR2-expressing cell lines, Ucn-I stimulation of ERK1/2-p42, 44 in AtT-20, A7r5, and CATHa cells was mediated by MEK1 (inhibited by PD), PI-3 kinase (inhibited by LY), and PLC (inhibited by U-7) (Fig. 6AGo). Although the A7r5 cells express high levels of CRFR2ß as reported previously (27), activation of ERK1/2-p42, 44 was weaker, compared with the CHO-CRFR2ß-expressing cells. The reason for this is probably due to the lower level of receptor expression in these cells.



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  		FIG. 5. Expression of CRFR2 and CRFR1 in AtT-20, CATHa, and A7r5 cells. Using RNA primers specific to mouse (m) or rat (r) CRFR1 or CRFR2, the RT-PCR for detection of CRFRs was performed for 35 cycles at 60 C, and the reaction was controlled by using poly (A) RNA isolated from mouse brain (B), which expresses both receptor types. The RT-PCR was controlled using primers for the S16 ribosomal protein. The following sizes of product should be detected: mCRFR1 = 279 bp, mCRFR2 = 378 bp, mS16 = 302 bp, rCRFR1 = 508 bp, and rCRFR2 425 bp.

 


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  		FIG. 6. The role of MEK1, PI-3 kinase, and PLC in Ucn-I-mediated ERK1/2-p42, 44 activation. CHO cells expressing CRFR1 or CRFR2 and the AtT-20, CATHa, and A7r5 cells were pretreated with 5 mM PD, 5 µM LY, or 10 µM U-7 for 30 min before a 5-min stimulation with Ucn-I. Cell lysates were harvested and subjected to SDS-immunoblot analysis for ERK1/2-p42, 44 activation. All three inhibitors, PD, LY, and U-7, prevent Ucn-I mediated ERK1/2-p42, 44 activation at CRFR1 and CRFR2. B, AtT-20, CATHa, A7r5, or Hc92 cells were pretreated with H-89 (PKA inhibitor), H-7 (PKC) inhibitor, BAPTA/AM (intracellular Ca2+ inhibitor) before stimulation with 30 nM Ucn-I for 5 min. Cell lysates were harvested and subjected to SDS-immunoblot analysis for ERK1/2-p42, 44 activation.

 
To determine the role of PKA, PKC, or intracellular Ca2+ in regulating ERK1/2-p42, 44 by Ucn-I, AtT-20, and CATHa cells were incubated with H-89, H-7, or BAPTA/AM before a 5-min treatment with Ucn-I (Fig. 6BGo). ERK1/2-p42, 44 activation was regulated by PKA in AtT-20 cells because H-89 suppressed ERK1/2-p42, 44 activation. Inhibition of PKC by H-7 had no effect on ERK1/2-p42, 44 activation by Ucn-I in AtT-20 or CATHa cells. The data in Fig. 6BGo show that intracellular Ca2+ may be involved in ERK1/2-p42, 44 activation in AtT-20 cells; however, because intracellular Ca2+ levels were not measured, this cannot be confirmed. To determine whether ERK1/2-p42, 44 activation by Ucn-I is mediated by Gi and Go proteins, AtT-20-, CATHa-, CHO-CRFR1-, and CHO-CRFR2ß-expressing cell lines were pretreated with PTX before stimulation with 30 nM Ucn-I. PTX reduced ERK1/2-p42, 44 activation by Ucn-I in both CHO-CRFR1 and CHO-CRFR2ß and partially inhibited ERK1/2-p42, 44 activation in AtT-20 and CATHa cells (Fig. 7Go). The Ucn-I + PTX treatment was compared with both the NT control and PTX treatment alone. For the CHO-CRFR1 cells, Ucn-1 caused a 1.56 ± 0.11-fold activation of ERK1/2-p42, 44, significantly greater than then NT control (P < 0.04). In the presence of PTX, Ucn-1 failed to stimulate ERK1/2-p42, 44 activation, compared with the NT control, because the fold induction was 0.62 ± 0.14. PTX alone resulted in a 0.54 ± 0.11-fold increase in ERK1/2-p42, 44, which was not significantly different from the Ucn-I + PTX treatment (P = 0.6). Similar to the CHO-CRFR1 cells, Ucn-I caused a 1.4-fold stimulation of ERK1/2-p42, 44 at the CHO-CRFR2 cells. In the presence of PTX, Ucn-1 failed to stimulate ERK1/2-p42, 44 activation, compared with the NT control, because the fold induction was 0.61 ± 0.1. PTX alone resulted in a 0.7 ± 0.22-fold increase in ERK1/2-p42, 44, which was not significantly different from the Ucn-I + PTX treatment.



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  		FIG. 7. ERK1/2-p42, 44 activation requires G1 and Go-proteins. PTX, 10 µg/ml, was incubated with CHO cells stably expressing CRFR1 (CHO-CRFR1) and CRFR2 (CHO-CRFR2) for 12 h before a 5-min stimulation with 30 nM Ucn-I. PTX was also incubated with AtT-20 and CATHa cells for 12 h before a 5-min stimulation with Ucn-I. Cell lysates were harvested and subjected to SDS-immunoblot analysis for ERK1/2-p42, 44 activation. Fold activation of ERK1/2-p42, 44 over total ERK is represented as bars ± SEM (n = 3). Significance is compared with the NT control for each cell type. Ucn-I caused a 2.6-, 1.4-, 12.7-, and 23.8-fold activation of ERK1/2-p42, 44 in CHO-CRFR1, CHO-CRFR2, AtT-20, and CATHa cells, respectively.

 
The studies described so far have concentrated on transformed cell lines. We therefore extended the regulation of ERK1/2-p42, 44 activation by Ucn-I to primary cultures of mouse neonatal cardiac myocytes that express CRFR2ß. To investigate the mediators of Ucn-I activation of ERK1/2-p42, 44 in cardiac myocytes, the cells were incubated with PD, LY, U-7, H-89, and H-7. PD, LY, and U-7 completely inhibited ERK1/2-p42,44 phosphorylation in these cells; however, the PKA or PKC inhibitor had no effect on Ucn-I activation of ERK1/2-p42,44 (Fig. 8Go). Ucn-I caused a 3.3 ± 0.13-fold stimulation of ERK1/2-p42, 44, a value that was significantly higher than the NT control (P < 0.004). PD completely abolished the Ucn-I-mediated ERK1/2-p42, 44 fold activation (0.91 ± 0.06), a value that was not significantly different from the NT control (P < 3.0). PD alone had no effect of ERK1/2 activity, compared with the NT control (P < 0.4). LY had similar inhibitory effects on Ucn-I-mediated ERK1/2-p42, 44 stimulation to PD. LY completely inhibited the Ucn-I stimulation of ERK1/2-p42, 44 to 0.92 ± 0.09, a value that was not significantly different from the NT control. LY alone had no effect on Ucn-I mediated ERK1/2 activity alone (P = 0.5). U-7 completely abolished the Ucn-I mediated 2.3 ± 0.32-fold stimulation of ERK1/2-p42, 44 to 0.79 ± 0.12, a value not significantly different from the NT control (P = 0.21). U-7 alone had no effect on ERK1/2-p42, 44 activity, compared with the NT control (P = 0.1). H-89 and H-7 had no effect on Ucn-I mediated ERK1/2-p42, 44 fold activation. Therefore, PI-3 kinase, MEK1, and PLC mediate ERK1/2-p42, 44 activation by Ucn-I in cardiac myocytes. This is similar to the data on CHO-CRFR2ß cells.



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  		FIG. 8. PI-3 kinase regulates ERK1/2-p42, 44 stimulation in cardiac myocytes. Primary cultures of neonatal cardiac myocytes were treated with Ucn-I for 5 min in the presence and absence of PD, LY, U-7, H-89, or H-7. PD, LY, and U-7 completely inhibited ERK1/2-p42, 44 activation by Ucn-I at this cell type that expresses CRFR2ß. This figure is representative of three experiments. The bars are representative of means ± SEM, n = 3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we investigated the specificity of Ucn-I/CRF ligands for CRFR1 and CRFR2 in stimulating ERK1/2-p42, 44 activation. We demonstrated that the peptides Ucn-II and Ucn-III selectively activate ERK1/2-p42, 44 in cells stably expressing CRFR2ß. In contrast to Ucn-II and Ucn-III, CRF is selective for CRFR1. Ucn-I activates both CRFR1 and CRFR2ß. Using specific cell-permeable inhibitors, we showed that for both receptors, the activation of ERK1/2-p42, 44 by Ucn-I requires PLC, MEK1, and PI-3 kinase because inhibition of PLC, MEK1, and PI-3 kinase inhibited ERK1/2-p42, 44 activation in the case for either cloned or endogenous receptors. We showed an essential requirement of Gi or Go proteins for CRFR-mediated stimulation of ERK1/2-p42, 44.

The selectivity of Ucn-II and Ucn-III for CRFR2 has been previously demonstrated in receptor binding studies as well as in activation of adenylate cyclase (3, 5). In those studies, mUcn-III, mUcn-II, and rUcn exhibit similar binding affinities for mCRFR2ß (7). This study extends previous characterization of the selectivity of Ucn-II and Ucn-III to CRFR2 to the case of ERK1/2-p42, 44 activation. We also show that CRF, like Ucn-I, can activate ERK1/2-p42, 44 through CRFR1. However, CRF produced a higher maximal fold increase in ERK1/2-p42, 44 activation, compared with Ucn-I, at all doses tested. In contrast to previous CRFR1 binding and cAMP studies, CRF and Ucn-I are as potent as each other. The higher-fold activation of ERK1/2-p42, 44 by CRF, compared with Ucn-I at cloned CRFR1, was not demonstrated in AtT-20 cells that express CRFR1. Ucn-I at lower doses of 0.3 or 3 nM produced a higher-fold activation of ERK1/2-p42, 44 compared with CRF. However, CRF at 30 nM activated ERK1/2-p42, 44 and Ucn-I did not (Fig. 2BGo). These data suggest that the greater activation of ERK1/2-p42, 44 by CRF at cloned CRFR1 does not extend to cell lines that endogenously express CRFR1, and variability between ERK1/2-p42, 44 stimulations by CRF and Ucn-I at CRFR1 may vary between cell type tested. In this study we show that both Ucn-II and Ucn-III are more effective than Ucn-I in activation of ERK1/2-p42, 44 at CRFR2ß. The discrepancies between cAMP stimulation, compared with fold activation of ERK1/2-p42, 44 for the different ligands via the receptors, may be dictated by ligand-specific binding to specific microdomains within CRFR1 or CRFR2, leading to different conformational changes in receptor. Conformational changes in each receptor would then facilitate coupling to G proteins and activation of intracellular signaling molecules. Therefore, if the receptor is more coupled to Gs, a stronger bias to cAMP signaling will occur. At present we do not know whether a specific ligand causes preferential binding to one G protein more than another.

In this study, we demonstrate that CRF can activate ERK1/2-p42, 44 via cloned and endogenous h/r/mCRFR1. This is in contrast to a previous study (12), whereby hCRFR1{alpha} was stably expressed in HEK293 and CHO cells, and CRF but not Ucn-I failed to activate the MAPK pathway. However, a study by Pederson et al. (18) showed that CRF activates ERK1/2-p42, 44 in primary cultures of rat hippocampal neurons that predominantly express CRFR1, which is consistent with our results.

In the present study, we demonstrate that ERK1/2-p42, 44 activation by Ucn-I involved multiple signaling effectors including PI-3 kinase, PLC, MEK1, possibly intracellular Ca2+, tyrosine kinases, and Raf-1. A putative mechanism by which the CRF/Ucn-I ligands activate ERK1/2-p42, 44 signaling is shown in Fig. 9Go. ERK1/2-p42, 44 activation is partially dependent on Gi and Go proteins because PTX inhibits ERK1/2-p42, 44 activation in the CHO-CRFR1 and CHO-CRFR2 cells. In contrast to previous studies (12, 21, 18), our data show that ERK1/2-p42, 44 activation by Ucn-I at both the CRFR1 and CRFR2 is both Gi and Go dependent. Because Gi is ubiquitously expressed in most cell types (33) and Go is expressed in higher levels in the brain and neuronal cells (34), we speculate that in the context of the CHO-CRFR2/R1 cell lines, PTX inhibits Gi protein, although we cannot rule out that PTX inhibits Go in these cells. One possible mechanism by which ERK1/2-p42,44 is activated by CRF ligands is shown (Fig. 9Go, mechanism 1). We hypothesized that after receptor activation, the Gß{gamma} subunit complex dissociates from Gi and activates MAPK in a PKC-independent but Ras-dependent manner (35). In addition, PI-3 kinase can be activated after direct interaction with Gß{gamma} subunits independent of its lipid kinase activity. PI-3 kinase can also act upstream of nonreceptor tyrosine kinases and the Shc-Grb2-Sos-Ras leading to MAPK activation. The Gß{gamma} subunit from Gi can also activate phospholipase C-ß and/or the {alpha}-subunit of Gq, which in turn results in calcium activation and Pyk-2 activation activating the Shc-Grb2-Sos Ras-Raf-1 pathway (36, 37, 38).



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  		FIG. 9. Hypothetical scheme of signal transduction pathways activated by CRFR1 and CRFR2. Based on the data from this study, activation of ERK1/2-p42, 44 by CRF/Ucn family of peptides seems to be Gi mediated via Gbg subunits involving PI-3 kinase and PLC activation of tyrosine kinases. Mechanism 1, The Gß{gamma} subunits of Gi can activate PLC-ß2 and PI-3 kinase to simultaneously activate ERK1/2-p42, 44. Mechanism 2, The Gß{gamma} subunit of Gi can activate PI-3 kinase, which in turn produces PI(3 4 5 )P3. PI(3 4 5 )P3 can in turn activate PLC{gamma}. Tyrosine kinases in turn activate the Ras Raf-1 kinase pathway, which results in MEK and hence ERK1/2-p42, 44 activation. In CATHa and AtT-20 cells, there also seems to be an involvement of PKA, which is presumably by activation of Gs (mechanism 3). Go activates PLC, which in turn mobilizes Ca2+ via inositol 1, 4, 5-triphosphate (InsP3) and DAG production (mechanism 4). Exchange protein activated by cAMP = EPAC, small G protein = Rap, growth factor receptor-bound protein 2 = Grb2, Son of sevenless, the exchange factor of Ras = SOS.

 
PLCß and PLC{gamma} subclasses have been shown to be regulated through G protein-coupled and protein tyrosine kinase-linked receptors, respectively (39, 40). The present study shows that inhibition of PLC or PI-3 kinase completely blocks ERK1/2-p42, 44 activation via the cloned CRFR1/R2 (39). This suggests that PI-3 kinase and PLC work through the same pathways. It has been reported that PLC{gamma}, the product of PI-3 kinase phosphatidylinositol, 3, 4, 5 tri-phosphate, activates purified PLC{gamma} isoenzymes by interacting with their Src homology 2 domains. Therefore, PI-3 kinase may act upstream of PLC{gamma}, as is the case for the insulin receptor (41) (Fig. 9Go, mechanism 2). In addition, PLC{gamma} can also be activated by Src tyrosine kinases (42).

The findings presented in this study support those reported by Rossant et al. (21), demonstrating that sauvagine activates MAPK in CHO cells stably expressing hCRFR1{alpha} and CRFR2{alpha}. In that study, p42, 44 MAPKs were partially regulated through PI-3K, and, possibly, through PI-specific PLC of the calcium sensitive proline rich tyrosine kinase (Pyk-2). Pyk2 can activate tyrosine kinases and MAPKs. In CRFR-expressing CHO cell activation of MAPK is independent of PKA, but possibly dependent on Ca2+. In this study, we confirm the essential role of tyrosine kinases, and possibly intracellular Ca2+, in CRFR-mediated ERK1/2-p42, 44 activation and show that PKA partially regulates ERK1/2-p42, 44 activation.

Ucn-I has been shown to protect neonatal cardiac myocytes from ischemic reoxygenation injury by activation of MEK 1 (9) and PI-3 kinase (10). We extend these observations by showing that PI-3 kinase directly regulates ERK1/2-p42, 44 phosphorylation, implicating that PI-3 kinase and ERK1/2 are in the same pathway. The PKA inhibitor H-89 had no effect on Ucn-I mediated ERK1/2-p42, 44 phosphorylation in these cells, suggesting that Gs is not essential for CRFR2 mediated ERK1/2-p42, 44 phosphorylation in these cells.

Here we show that ERK1/2,p42, 44 activation is PTX sensitive and demonstrate the role of tyrosine kinases in ERK1/2-p42, 44 activation. We show that PKA does have a role in ERK1/2-p42, 44 activation in CRFR-expressing CHO cells, in contrast to the study by Rossant et al. (21). We also show that in the cloned receptors, inhibition of PKA does have a partial inhibitory effect on ERK1/2-p42, 44 activation by Ucn-I and, furthermore, that inhibition of Raf-1 does not completely inhibit ERK1/2-p42, 44 activation (Fig. 4Go). These observations suggest that G{alpha}s activates adenylate cyclase and, therefore, cAMP, which, in turn, can activate PKA resulting in activation of Rap and B-Raf and MEK (Fig. 9Go, mechanism 3). This was evident in AtT-20 and A7r5 cells in which PKA regulated ERK1/2-p42, 44 activation in addition to regulating PI-3 kinase, calcium, and PKA-dependent pathways. CATHa cells did not show a requirement for PKA in activating ERK1/2-p42, 44, suggesting that the balance of the Gi and Gs activation of ERK1/2-p42, 44 may vary according to cell type, depending on the coupling of the receptors to specific G proteins. PTX did not completely abolish CRFR mediated ERK1/2-p42, 44 activation in CATHa and AtT-20 cells. The differences in the role of PTX to inhibit ERK activation in CRFR-overexpressing CHO cells, compared with CATHa and AtT-20 cells, may be due to the differential coupling of the receptors to G-proteins in these cells. We speculate that mechanism 1 is the predominant route for ERK1/2-p42, 44 activation in CHO cells stably expressing hCRFR1 and mCRFR2ß. However, mechanism 2 also feeds into the activation of ERK1/2-p42, 44 signaling in CHO cells. The balance between mechanism 1 and mechanism 2 varies between the CRFR cell types used in this study. For example, PTX does not completely abolish Ucn-I/ CRFR mediated ERK1/2-p42, 44 activation in AtT-20 cells.

It is also possible that Gq may play a role in ERK1/2-p42, 44 activation in some cell types such as the human myometrium (12). Because there is no ce-l permeable inhibitor of Gq, we cannot confirm the role of Gq in ERK1/2-p42, 44 activation in CRFR-expressing cells, although this study suggests that Gq is not involved. We have not discussed the possible role of Go in regulating ERK1/2-p42, 44 activity through the PTX sensitive PI pathway (43). Our studies show that a PLC pathway is essential for CRFR-mediated ERK1/2-p42, 44 stimulation. It has previously been shown that muscarinic receptors couple to PLC through Go in Xenopus oocytes. Go activates PLC and in turn inositol 1, 4, 5-triphosphate (InsP3) production. InsP3 mobilizes intracellular Ca2+ to evoke a Cl- current. This may be one mechanism (mechanism 4) by ERK1/2-p42, 44 is activated by the CRFR-mediated activation.

Here we show that independent of receptor type, Ucn-I activates ERK1/2-p42, 44 by similar regulatory mechanisms. CATHa cells express both CRFR1 and CRFR2{alpha}; these cells have previously been shown to express CRFR1 (44). The CATHa cell may be used as a model to investigate CRFR1 and CRFR2 cross-talk and interactions. CRFRs are overexpressed in a number of human cancers (45). Because MEK1 activation is involved in human cancer progression (46, 47, 48, 49, 50), inhibition of CRFR-mediated ERK1/2-p42, 44 activation may have potential therapeutic applications (51).

Some variation in mechanisms of ERK1/2-p42, 44 may occur in different cell types due to preferential coupling to other G proteins. In this paper we selected a 5-min time point to study the intracellular pathways that are involved in CRFR1- and CRFR2-mediated ERK1/2-p42, 44 stimulation. This time point was selected because ERK1/2 activation is optimal and therefore can be studied more easily than, for example, 1 or 20 min in which phosphorylation of ERK1/2 is more difficult to quantify. It is very possible that, like the ß-2-adrenergic receptor, temporal changes in receptor signaling may exist, i.e. the shift between receptor coupling from Gs to Gi (52). At present we do not have any data that might suggest temporal differences in the signaling from different CRFR subtypes or with receptors in different cellular surroundings. Detailed extensive investigations in the future will reveal whether this is so. In this study we have demonstrated ERK1/2-p42, 44 activation through cloned hCRFR1 and mCRFR2ß. It is possible that CRFR-mediated ERK1/2-p42, 44 activation may vary between the different receptor subtypes, i.e. rCRFR2{alpha} vs. mCRFR2ß. However, the pharmacology in terms of receptor binding affinities for rCRFR2{alpha} vs. mCRFR2ß has been shown to be very similar (7). In this study we report that CATHa cells express mCRFR2{alpha}, which has not been previously demonstrated or cloned (A. Chen, unpublished observations). In the future, we will investigate the selectivity of the Ucn ligands for activation of ERK1/2-p42, 44 through mCRFR2{alpha} vs. mCRFR2ß. In this study, we have shown that there are differences in ERK1/2-p42, 44 activation between cell lines that overexpress receptors, compared with cell lines endogenously expressing the receptors, and caution must be used when performing studies on cell lines overexpressing receptors. We show an essential role for tyrosine kinases in mediating ERK1/2-p42, 44 activation, suggesting that they may couple to yet-unidentified receptor tyrosine kinases. The mechanisms by which ERK1/2-p42, 44 is activated at the CRFR suggests that the CRF/Ucn ligands can activate a number of different intracellular signaling systems, which may reflect the heterogeneity of their functions.


   Acknowledgments
 
We would like to thank Dr. Louise Bilezikjian for technical assistance and Joan Vaughan, Alejandra Negro, and Chien Li for critical reading of the manuscript.


   Footnotes
 
This work was supported by a British Heart Foundation International Fellowship (to B.K.B.), NIH Grant DK 26741, The Robert J. and Helen C. Kleberg Foundation, and in part by the Foundation for Research. W.V. is a senior Foundation for Research investigator.

Abbreviations: Ast-2B, Astressin-2B; BAPTA/AM, 1'-2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester; CHO, Chinese hamster ovary; CRF, corticotropin-releasing factor; CRFR, CRF receptor; h, human; InsP3, inositol 1, 4, 5-triphosphate; LY, LY294002; m, mouse; MEK, MAPK kinase; NT, vehicle-treated control; PD, PD98059; PI, phosphatidylinositol; PI-3 kinase, PI-3 OH kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; Pyk-2, proline-rich tyrosine kinase; PTX, pertussis toxin; r, rat; SDS, sodium dodecyl sulfate; U-7, U73122; Ucn, urocortin; WT, wild-type.

Received August 8, 2003.

Accepted for publication December 5, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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