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. Author manuscript; available in PMC: 2011 May 26.
Published in final edited form as: J Neurochem. 2007 Nov 1;104(4):926–936. doi: 10.1111/j.1471-4159.2007.05052.x

Expression, binding, and signaling properties of CRF2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage–dependent regulation of functional receptor

Eric Gutknecht *,†,1, Richard L Hauger ‡,1, Ile Van der Linden *, Georges Vauquelin †, Frank M Dautzenberg *
PMCID: PMC3102762  NIHMSID: NIHMS92950  PMID: 17976162

Abstract

Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF1-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of ~100 pmoles/105 cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of ~25–30 pmoles/105 cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the ~300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 ± 1.6 nmol/L), whereas the CRF1-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 μmol/L. When the CRF1-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 ± 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 ± 0.2 μmol/L). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2(a) receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture.

Keywords: corticotropin-releasing factor receptor desensitization, corticotropin-releasing factor receptor signaling, cyclic AMP, ligand binding, mRNA expression

Corticotropin-releasing factor (CRF) and its structurally related family members urocortin 1–3 (UCN1–3) potently modulate neuroendocrine, autonomic, and behavioral responses to stress by activating two CRF receptors: CRF1 and CRF2 (Dautzenberg and Hauger 2002; Bale and Vale 2004; Grigoriadis 2005; Hauger et al. 2006; Steckler and Dautzenberg 2006). Both receptor subtypes are highly homologous (~70%) and belong to the class B1 subfamily of G protein-coupled receptors (GPCRs) (Dautzenberg et al. 2001b; Harmar 2001). Three biologically active splice variants, CRF2(a-c), have been identified for the CRF2 receptor, whereas only one high affinity variant of the CRF1 receptor has been established to be a fully functional GPCR (Hauger et al. 2003a).

Corticotropin-releasing factor type 1 and 2 receptors differ strongly in terms of their agonist and antagonist binding preferences. Binding and functional studies in cell lines recombinantly or endogenously expressing CRF1 receptors revealed a distinct ligand-selective profile: CRF, UCN1, and the non-mammalian CRF agonists fish urotensin I, and frog sauvagine bind with high affinity to the mammalian CRF1 receptor and stimulate cAMP and calcium signaling pathways (Donaldson et al. 1996; Dautzenberg et al. 1997, 2001a, 2004b). In contrast, UCN2 and UCN3 do not bind to or activate CRF1 receptors at physiologically relevant concentrations (Hsu and Hsueh 2001; Lewis et al. 2001; Reyes et al. 2001; Dautzenberg et al. 2004a,b; Grigoriadis 2005). Pharmacological characterization of the CRF2 receptor splice variants revealed no major differences between CRF2(a), CRF2(b), and CRF2(c) receptors (Donaldson et al. 1996; Kostich et al. 1998; Palchaudhuri et al. 1999; Dautzenberg et al. 2004b). However, the binding profiles of these three CRF2 receptors markedly diverge from the binding profile of the CRF1 receptor (Donaldson et al. 1996; Perrin et al. 1999; Dautzenberg et al. 2001b; Hsu and Hsueh 2001; Lewis et al. 2001; Reyes et al. 2001). Urotensin I, sauvagine, and UCN1–3 bind with up to 1000-fold higher affinities to the CRF2 receptor than species homologs of CRF (see Hauger et al. 2003a). In agreement with the binding data, a similar rank order of potency is typically observed when these five agonists are used to stimulate cAMP stimulatory G protein (Gs)-coupled cAMP signaling (Donaldson et al. 1996; Dautzenberg et al. 2001b; Hsu and Hsueh 2001; Lewis et al. 2001; Reyes et al. 2001) or phospholipase C-mediated transient mobilization of intracellular calcium stores (Dautzenberg et al. 2004a). Therefore, UCN2 and UCN3 are generally considered to represent endogenous ligands for mammalian CRF2 receptor variants, whereas UCN1 is thought to be an endogenous ligand for both CRF receptors.

Pharmacological characterization of CRF1 and CRF2 receptors has mainly been completed using recombinant receptor expression systems (Perrin and Vale 2002; Grigoriadis 2005; Hauger et al. 2006). However, in a recombinant setting the imbalanced receptor-G protein stoichiometry may strongly influence the receptor signaling properties (see Kenakin 1997). Thus, confirming recombinant GPCR data in an endogenous cellular setting is of high importance. Another aspect of scientific interest is whether or not CRF1 and CRF2(a) receptors engage in crosstalk or are co-regulated in the CNS. Therefore, identification of cell lines endogenously expressing CRF2(a) receptors alone or together with CRF1 receptors is critical for gaining further insight into the regulation of CRF receptor signaling.

A large number of brain-derived or neuroendocrine cell lines, including Y79 retinoblastoma, IMR-32 neuroblastoma, CATH.a cathecholaminergic, AtT-20 pituitary, PC12 pheochromocytoma, and small lung cell carcinoma NCI-H82 cells endogenously express CRF1 receptors (Vita et al. 1993; Dieterich and DeSouza 1996; Iredale et al. 1996; Hauger et al. 1997; Kiang et al. 1998; Dautzenberg and Hauger 2002; Dermitzaki et al. 2007). Despite the widespread expression of CRF2 receptors in the CNS and the periphery, few cell lines have been found to express endogenously one of the three CRF2 receptor isoforms (Kiang et al. 1998; Hsu and Hsueh 2001; Brar et al. 2004; Nemoto et al. 2005). The human pancreatic carcinoid BON cell line only expresses the CRF2 receptor but the splice variant has not been determined (von Mentzer et al. 2007). The rat aortic smooth muscle A7r5 cell line exclusively expresses the CRF2(b) receptor that couple to the Gs protein (Hsu and Hsueh 2001; Hoare et al. 2005). Although rodent CATH.a catecholaminergic cells express CRF2(a) receptors (Brar et al. 2004) and pheochromocytoma PC12 cells express CRF2(b) receptors (Dermitzaki et al. 2007), in addition to CRF1 receptors, molecular mechanisms regulating CRF receptor signaling have not been characterized in these two cell lines. However, we have extensively studied regulation of the CRF1 receptor endogenously expressed in human retinoblastoma Y79 cells and found that this cell line provides a valuable system for studying CRF receptor regulation in an endogenous setting (Hauger et al. 1997, 2003b; Dautzenberg et al. 2001a, 2002a).

In the present study, we demonstrate that CRF2(a) receptors are endogenously expressed in Y79 cells, and CRF2(a) receptors can be up-regulated with increasing duration of cell culture (> 20 passages). We also established functional Gs-coupling and cAMP signaling when retinoblastoma CRF2(a) receptors are activated by their selective agonists, UCN2 and UCN3. CRF1 and CRF2 receptor signal transduction in Y79 cells could be functionally separated using selective ligands and antagonists. Finally, we provide the first evidence that CRF2(a) receptor function is rapidly regulated by a homologous desensitization mechanism.

Materials and methods

Materials, peptides, reagents, and radiochemicals

All cell culture media and reagents were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). All peptides (purity > 95) were obtained from Bachem Corporation (Bubendorf, Switzerland) and NBI27914 was obtained from Tocris (Bristol, UK). 125I-antisauvagine and 125I-sauvagine (both 2000 Ci/mmol) were purchased from Amersham (GE Healthcare, Little Chalfont, UK).

Cell culture

Y79 retinoblastoma cells (American Type Culture Collection (Manassas, VA, USA) No. HTB-18) were grown as suspension cultures in RPMI 1640 medium as described previously (Hauger et al. 1997, 2003b; Dautzenberg et al. 2001a). Cells were grown at densities ranging from 5 × 107 to 2 × 108 cells/flask in Falcon F-175 flasks and used between passages 5 and 60, depending on the experimental procedure.

cAMP assays

Y79 cells were plated at 80 000 cells/well in assay buffer containing 3-isobutyl-1-methylxanthine (Hank's buffered salt solution supplemented with 3-isobutyl-1-methylxanthine 1 mmol/L, MgCl2 10 mmol/L, HEPES 5 mmol/L, and 0.1% bovine serum albumin) in 96 wells black plates (Costar-BD Biocoat, Erembodegem, Belgium). Cells were incubated for 15 min at 37°C with the agonists and lysed following the homogenous time-resolved fluorescence cAMP Dynamic kit two step protocol (Cisbio International, Bagnols/Cèze, France) to determine the production of cAMP. Fluorescence resonance energy transfer was measured in the Discovery reader (Perkin Elmer, Boston, MA, USA). In the desensitization intracellular cAMP levels were measured in nonacetylated cell lysates using a double-antibody radioimmunoassay kit (cAMP[125I] assay system, RPA 509; Amersham International, Little Chalfont, UK), as previously described (Dautzenberg et al. 2001b; Hauger et al. 2003b).

Radioreceptor binding experiments

The preparation of Y79 membrane particulates was essentially as reported previously (Hauger et al. 1997). Y79 were harvested and precipitated at 150 g for 10 min. All subsequent steps were performed at 4°C. Cells were washed with ice-cold phosphate-buffered saline and recentrifuged. The pellet was resuspended in ice-cold membrane buffer (50 mmol/L Tris–HCl, pH 7.4) containing 5 mmol/L MgCl2, 2 mmol/L EGTA, and 100 kIU/mL aprotinin and homogenized with a Polytron (Kinematica, setting 5 for 10 strokes). The nuclei were precipitated for 5 min at 600 g, the supernatant was removed and stored away, and the pellet was re-extracted as described above. After combining both supernatants they were precipitated at 13 000 g for 30 min.

In siliconized polypropylene tubes (Sigma-Aldrich, Bornem, Belgium) CRF2(a) receptor binding was studied using Y79 membranes (~250 μg of membrane protein for CRF2(a) receptor binding and ~100 μg for CRF1 receptor binding) using a competitive binding assay between 0.15 nmol/L 125I-antisauvagine or 125Isauvagine and increasing concentrations of unlabeled CRF agonists or antagonists (0–10)−5 mol/L). After incubation at 22°C for 120 min, the tubes were centrifuged at 14 000 g, washed twice (Ruhmann et al. 1996) and the radioactivity was counted in a γ-counter (Wallac, Turku, Finland).

RNA isolation and cDNA synthesis

Total RNA was isolated and purified from Y79 cells using the RNeasy kit (Qiagen/Westburg, Leusden, The Netherlands) with Dnase I treatment on the column. First strand cDNA was synthesized from 1 μg total RNA for 1 h at 42°C using Random Hexamer-primers and Superscript II reverse transcriptase (Invitrogen Life Technologies).

Semiquantitative RT-PCR

The three splice variants of the hCRF2 receptor [CRF2(a), CRF2(b), and CRF2(c)] were amplified with the following primer combinations: CRF2afor (5′-GAGCTGCTCTTGGACGGCTGGGGGC-3′) and CRF2arev (5′-CTGCCACAGATACGCAGT-3′); CRF2bfor (5′-CAGGCTCCAGTCCCTAAC-3′) and CRF2brev (5′-CAGGTAGTTGACGACAAGG-3′); and CRF2cfor (5′-CTGTGCTCAAGCAATCTGCCT-3′) and CRF2crev (5′-CAAAATGGGCTCACACTGTGAG-3′). Primers were designed based on the mRNA sequence find under Ensemble database CRF2(a), ENST222836 (nucleotides 61–389, 328 bp); CRF2(b) ENST348438 (nucleotides 36–518, 482 bp); and CRF2(c) ENST341843 (nucleotides 1–416, 416 bp). Amplification was performed for 35 cycles (94°C for 10 s, 60°C for 20 s, and 72°C for 1 min) with 1 U Taq DNA polymerase and a cDNA equivalent of ~50 ng RNA. The calculated sizes for the amplification products were as follows: CRF2(a) = 310 bp, CRF2(b) = 410 bp, and CRF2(c) = 276 bp. In control amplifications using cloned DNA these sizes were confirmed (not shown). The cDNA for glyceraldehye-3-phosphate dehydrogenase (GAPDH) (NM_002046, nucleotides 208–602, 395 bp) was amplified for 20 cycles with the different cDNAs using the following primer pairs: GAPDHfwd (5′-CCTTCATTGACCTCAACTAC-3′) and GAPDHrev (5′-TGTCATGGATGACCTTGG-3′). The different fragments were sequenced for confirmation of the specific amplification of the various CRF2 receptor splice variants.

Quantitative RT-PCR

Quantitative RT-PCR (Q-PCR)was performed using an ABIPrism 7700 cycler (Applied Biosystem, Foster City, CA, USA) using qPCR™ Core Kit w/o dUTP (Eurogentec, Seraing, Belgium). Serial dilutions of the cDNAs were used to generate standard curve for the threshold cycles for hCRF1, hCRF2, GAPDH, and β-actin. Samples were diluted 10-fold prior the experiment to ensure that amplification was in the linear part of the standard cDNA curve. Pre-developed Taqman assay reagents from ABI (Applied Biosystem, Warrington, UK) were used for human β-actin and GAPDH. Primers and probes for human CRF1 and human CRF2(a) were designed with Primer Express software v2.0 (Applied Biosystem, Foster City, CA, USA). The sequences for the CRF1 and CRF2 receptor primers used for Q-PCR are given in Table 1. A linear regression line calculated from the standard curves allowed the determination of transcript levels in RNA samples from of Y79 cells on the different passages. Primers and probes sets for hCRF1 and hCRF2 showed very weak [hCRF2(a)] or no (hCRF1) cross-reactivity for plasmid DNA.

Table 1.

Sequence of Q-PCR primers for the amplification of CRF1 and CRF2 receptors

Primer CRF1 NM_004382 (1131–1188) CRF2 NM_004382 (292–331)
Forward 5′-CACGTCTGAGACCATTCA-3′ 5′-TGTGAGCCCATTTTGGATGA-3′
Reverse 5′-GGGCAGCAGCACCAGAGT-3′ 5′-AAGGGCGATGCGGTAGTG-3′
Probe 5′-ACAGGAAGGCTGTGAAA-3′ 5′-AAGCAGAGGAAGTATGACC-3′
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CRF1, CRF type 1 receptor; CRF2, CRF type 2 receptor.

Data reduction and statistical analyses

Data reduction for the binding and cAMP experiments was performed using a log-logit program. IC50, EC50, and maximum values were calculated from the full concentration–response curves for binding, agonist stimulation, and antagonist inhibition of cAMP accumulation using the prism™, version 4.0 (GraphPad Software, Inc., San Diego, CA, USA). Statistical significance was determined by anova across experimental groups using prism™, version 4.0. 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.

Results

Functional expression of CRF2(a) receptors in late passage Y79 cells

We began our study by observing substantial differences in the stimulation of cAMP formation by the CRF1 receptor-preferring agonist ovine CRF (oCRF) and the CRF2 receptor-selective agonist UCN3 during the sequence of a profiling of freshly frozen Y79 cells versus cells that had been in culture for more than 30 passages. While the cAMP response to oCRF remained stable (99.6 ± 4.8 pmol/105 cells in passage 4 vs. 95.4 ± 6.1 pmol/105 cells in passage 36), the cAMP response to UCN3 increased from a very low response (2.2 ± 0.8 pmol/105 cells in passage 4; with a basal response of 1.2 ± 0.6 pmol/105 cells) to ~25% of the oCRF responses in passage 36 (24.5 ± 1.9 pmol/105 cells).

Next, RNA was isolated from Y79 cells in passages 5 and 40. After cDNA was synthesized, a semiquantitative RT-PCR analysis was performed in order to identify the CRF2 receptor splice variant expressed in Y79 cells. No positive PCR amplification for any CRF2 receptor splice variant was obtained from Y79 cell cDNA isolated in passage 5 (not shown). However, hCRF2(a) cDNA was amplified from cDNA isolated from passage 40 (Fig. 1). Neither hCRF2(b) nor hCRF2(c) cDNA was amplified, whereas using cDNAs isolated from hippocampal or amygdalar RNA revealed successful amplification of both splice variants. Thus, at late passages the higher cAMP response to UCN3 was mediated exclusively by the hCRF2(a) receptor.

Fig. 1.

Fig. 1

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Semiquantitative RT-PCR amplification of cDNAs encoding different CRF2 receptor splice variants from human brain tissue and Y79 cells. Amplification for 35 cycles was performed using different primer sets (see Materials and methods). In control reactions, GAPDH cDNA was amplified from all tissues for 20 cycles; L, DNA standard; H, hippocampus; A, amygdala; NC, negative control (H2O).

Up–regulation of CRF2(a) receptor mRNA and functional responses during long–term cultivation of Y79 cells

We next determined if CRF2(a) receptor expression increased as a function of cell culture duration. After total RNA was isolated from Y79 cells at different passages ranging from passage 5 to 60, Q-PCR analysis was performed. Cells from identical batches were also analyzed for stimulation of cAMP formation by oCRF, UCN3, and the non-selective CRF receptor agonist sauvagine utilizing the homogenous time-resolved fluorescence readout, which is a simple mixand-measure protocol providing a rapid quantification of cAMP in cellular extracts (http://www.htrf.com/products/gpcr/camp). As the preliminary functional studies suggested CRF2(a) receptor expression becomes up-regulated while CRF1 receptor levels remain constant, we determined if expression of CRF2(a) mRNA progressively increases in Y79 cells during longer periods of cell culture. To this end, RNA was isolated from Y79 cells, first strand cDNA synthesis was completed, and the relative mRNA levels of CRF1 and CRF2(a) receptor were quantified by Q-PCR versus two internal standards, GAPDH and β-actin, which revealed similar results. Q-PCR analyses revealed constant CRF1 mRNA levels throughout increasing culturing of Y79 cells (Fig. 2b) whereas CRF2(a) mRNA increased from nearly undetectable levels at passage 5 to maximal levels between passages 26 and 30 (Fig. 2c).

Fig. 2.

Fig. 2

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Stimulation of cAMP production by sauvagine, oCRF and UCN3 (a) and Q-PCR for CRF1 (b) and CRF2 (c) mRNA in Y79 cells at increasing cell culture passages. (a) Y79 cells in different culture passages (80 000 per well) were incubated with increasing concentrations of sauvagine, oCRF, and UCN3 (300 nmol/L) for the indicated time. By anova, there were significant differences across the groups (F = 396.3, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: ap < 0.05 versus passages 12 and 15; bp < 0.0001 versus passage 5; cp < 0.002 versus passage 20; dp < 0.0001 versus passages 5, 12, and 15; ep < 0.0001 versus passage 20. (b) Q-PCR amplification of CRF1 cDNA from first strand cDNA synthesized from total RNA isolated from Y79 at different culture passages. (c) Q-PCR amplification of CRF2 cDNA from first strand cDNA synthesized from total RNA isolated from Y79 at different culture passages. The results are representatives of three independent experiments performed in quadruplicate and normalized against GAPDH. Similar results were obtained with β-actin cDNA as internal standard (not shown). By anova, there were significant differences across the groups (F = 86.39, p < 0.0001). The following post hoc differences were found to be statistically significant between groups: fp < 0.0001 versus passage 5; gp < 0.0005 versus passage 12; hp < 0.0001 versus passage 12; ip < 0.002 versus passage 15; jp < 0.0001 versus passage 15; kp < 0.0001 versus passage 20.

In the cAMP stimulation experiments, oCRF and sauvagine produced similar maximal responses (~100 pmoles/well) in all passages of Y79 cells (Fig. 2a).~ In contrast, UCN3-mediated cAMP stimulation was significantly increased with greater number of cell passages. At early passages, UCN3-induced CRF2(a) receptor cAMP signaling was minimal based on maximal responses of 2.75 ± 0.23 and 3.28 ± 0.19 pmoles/well at passages 5 and 12, respectively. cAMP responses to UCN3 first became up-regulated at passage 15 (7.34 ± 0.18 pmoles/well), followed by further increases at passages 20 (13.61 ± 0.49 pmoles/well) and 26 (20.53 ± 2.07 pmoles/well). By passage 30, the UCN3-stimulated cAMP accumulation reached a plateau of 25 pmoles/well (Fig. 2a) with no further up-regulation of the cAMP response at passages 55–60 (data not shown).

Functional separation of CRF1 and CRF2(a) receptors in Y79 cells

Because we observed that endogenous CRF2(a) receptors exhibited maximal cAMP responses in Y79 cells beginning at passage 25, all subsequent pharmacological studies in Y79 cells were performed between passages 25 and 45. To more fully characterize CRF1 and CRF2(a) receptor signaling, we generated agonist concentration-response curves for a panel of published mammalian and amphibian CRF and UCN peptides. The CRF1/CRF2-activating peptides stimulated cAMP responses to a maximum of ~100 pmoles/well (Fig. 3, Table 1), whereas the potencies of those peptides was either close to the values reported for CRF1 receptors (oCRF and human/rat CRF) or did not allow to discriminate between CRF1 and CRF2 receptors (human UCN1 and sauvagine). In contrast to cAMP responses to CRF, UCN1, and sauvagine, significantly smaller stimulation of cAMP accumulation was observed for the CRF2 receptor-selective agonists UCN2, UCN3 and their N-terminally extended versions stresscopin [SCP; extended version of UCN3, which contains an additional Thr-Lys dipeptide sequence at its N-terminus (see Hsu and Hsueh 2001)] and stresscopin-related peptide [SRP; extended version of UCN2, which contains five additional N-termainal amino acids, His-Pro-Gly-Ser-Arg (see Hsu and Hsueh 2001) (Fig. 3, Table 2)]. These latter four peptides stimulated cAMP production in Y79 cells to a maximum of 25–28 pmoles/well (Fig. 3, Table 2) with potencies in the range reported for recombinant CRF2 receptors. Therefore, CRF2-selective agonists exclusively activated CRF2 receptors in the Y79 cells.

Fig. 3.

Fig. 3

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Stimulation of cAMP production in Y79 cells by various CRF receptor agonists. Cells (80 000 per well) were incubated with increasing agonist concentrations (0.01 nmo/L to 1 μmol/L) for 15 min at 25°C and cAMP accumulation was measured by homogenous time-resolved fluorescence readout as described in the Materials and methods section. Data are representative of eight independent stimulations performed in quadruplicate.

Table 2.

Potencies of various CRF peptides to stimulate cAMP accumulation in Y79 retinoblastoma cells

Peptide EC50 (nmol/L) Emax (pmol over basal)
oCRF 1.61 ± 0.59a 102.8 ± 8.8e
h/rCRF 4.25 ± 1.09b 105.2 ± 9.6e
hUCN1 2.67 ± 0.77c 101.2 ± 6.42e
hUCN2 15.5 ± 3.1d 26.6 ± 3.2
SRP 9.69 ± 2.13d 28.6 ± 5.1
hUCN3 39.7 ± 4.1 24.6 ± 4.8
mUCN3 37.5 ± 12.3 25.6 ± 4.4
SCP 52.1 ± 8.3 28.6 ± 5.6
Sauvagine 1.57 ± 0.28a 105.8 ± 7.8e
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The data are means ± SEM of four to seven independent stimulation experiments performed in quadruplicate. Statistical differences

a

p < 0.0001 versus hUCN2, hUCN3, mUCN3, SCP, and SRP

b

p < 0.02 versus hUCN2, hUCN3, mUCN3, SCP, and SRP

c

p < 0.002 versus hUCN2, hUCN3, mUCN3, SCP, and SRP

d

p < 0.001 versus hUCN3, mUCN3, and SCP

e

p < 0.0001 versus hUCN2, hUCN3, mUCN3, SCP, and SRP.

CRF, corticotropin-releasing factor

oCRF, ovine CRF

h/rCRF, human/rat CRF

UCN, urocortin

hUCN, human UCN

SCP, stresscopin.

To further confirm the agonist findings, the inhibitory potencies of CRF receptor antagonists on agonist-induced activation of CRF1 or CRF2 receptors were assessed. The following three antagonists were chosen: (i) the CRF1/2 non-selective antagonist astressin (Gulyas et al. 1995); (ii) the CRF1-specific small molecule antagonist NBI27914 (Chen et al. 1996); and (iii) the CRF2-selective antagonist anti-sauvagine (Ruhmann et al. 1998). An oCRF concentration of 10 nmol/L, which is submaximal (EC80) for CRF1 receptors but only minimally activating CRF2 receptors (Dautzenberg et al. 2001b), was chosen for the CRF1 receptor antagonism experiments. An EC80 concentration (100 nmol/L) of UCN3, the most selective CRF2 agonist, was chosen for the CRF2(a) receptor antagonism experiments.

Using oCRF as agonist, astressin and NBI27914 almost equipotently inhibited cAMP production in the nanomolar range (IC50 ~40 nmol/L), whereas only micromolar antisauvagine concentrations showed inhibitory activities (Fig. 4, Table 3). Conversely, using UCN3 as agonist, NBI27914 failed to exert any appreciable antagonist potency. Under these conditions, astressin and antisauvagine inhibited UCN3-mediated cAMP accumulation with IC50 values below 10 nmol/L (Fig. 4, Table 3). Similar antagonist potencies were obtained with UCN2 as agonist (not shown).

Fig. 4.

Fig. 4

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Antagonist-mediated inhibition of CRF1 or CRF2(a) receptor-mediated stimulation of cAMP accumulation in Y79 cells. Y79 cells were incubated with increasing concentrations of astressin, NBI27914 and antisauvagine (0.1 nmol/L to 10 μlmol/L each) in the presence of 10 nmol/L oCRF (CRF1) or 100 nmol/L UCN3 [CRF2(a)]. The results are representatives of five independent antagonist experiments performed in quadruplicate.

Table 3.

Antagonist potencies of antisauvagine, astressin, and NBI27914 on cAMP accumulation in Y79 cells stimulated with either a CRF1-selective (oCRF, 10 nmol/L) or a CRF2-selective (UCN3, 100 nmol/L) agonist

Antagonist CRF1 (oCRF)
IC50 (nmol/L)		 CRF2 (UCN3)
IC50 (nmol/L)
Antisauvagine 2040 ± 179 6.46 ± 1.62
Astressin 36.7 ± 9.2 5.47 ± 2.01
NBI27914 38.4 ± 3.6 > 10 000
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The data are means ± SEM of three to six independent stimulation experiments performed in quadruplicate. CRF, corticotropin-releasing factor; CRF1, CRF type 1 receptor; CRF2, CRF type 2 receptor; UCN, urocortin.

CRF2(a) receptor binding in Y79 cells

To further assess the utility of Y79 cells as an endogenous setting for CRF2(a) receptor function, CRF receptor binding studies were completed. 125I-antisauvagine was used in these experiments based on our previous data establishing that this radiolabeled ligand is a valuable tool for studying CRF2 receptors (Higelin et al. 2001). Because our cAMP studies indicated that a low number of CRF2(a) receptor binding sites may be expressed in Y79 cells, we used a centrifugation binding method (Dautzenberg et al. 1997; Hauger et al. 1997). Preliminary binding experiments confirmed that the CRF2(a) receptor protein levels in early passages were too low to obtain appreciable receptor labeling, while in later passages specific receptor binding was observed (not shown). In saturation binding experiments, 125I-antisauvagine specifically labeled a maximum of 21.45 ± 0.89 fmoles of CRF2 receptors per mg protein in membranes prepared from Y79 cells prepared in passages 35 (Fig. 5). The calculated Kd of 220 ± 22 pmol/L (Fig. 5) was close to the Kd value obtained for recombinant CRF2(a) receptors (Dautzenberg et al. 2001b; Higelin et al., 2001).

Fig. 5.

Fig. 5

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Saturation binding of 125I-antisauvagine to membranes prepared from Y79 cells. Membrane proteins (250 μg per data point) were incubated with increasing concentrations of 125I-antisauvagine (1 pmol/L to 2 nmol/L) for 2 h at 22°C. Bound radiolabel was separated from the free radioactivity by rapid centrifugation in a table top centrifuge at 4°C and two washing steps. Non-specific binding was determined by a large molar access of unlabeled UCN3 (10 μmol/L). The results are representative of three independent experiments performed in triplicate.

For inhibition binding experiments, either 125I-antisauvagine or the CRF1/2 non-selective radiolabel 125I-sauvagine was used as the radioligand for assessing the competitive binding potencies of the four endogenous CRF1 or CRF2 receptor ligands: oCRF and human UCN1–3. When CRF2 receptors were labeled with 125I-antisauvagine, the four peptides competed with a rank order typical for CRF2 receptors: UCN1 > UCN2 > UCN3 > oCRF (Fig. 6). At high concentrations, the four peptides completely inhibited 125I-antisauvagine binding. In contrast, substantial differences were observed when 125I-sauvagine was employed as radiolabeled ligand. UCN1 and oCRF were equipotent in inhibiting 125I-sauvagine binding (Fig. 6). Moreover, oCRF inhibited 125I-sauvagine binding with ~30-fold higher affinity compared with that for 125I-antisauvagine. Thus, 125I-sauvagine was predominantly labeling CRF1 receptors in Y79 membranes. In agreement with this observation, UCN2 and UCN3 inhibited less than 25% of 125I-sauvagine binding to Y79 cell membranes. The measured Ki values for UCN2 (Ki = 21.2 ± 3.6 nmol/L) and UCN3 (Ki = 21.2 ± 3.6 nmol/L) were close to their agonist potencies in the cAMP experiments.

Fig. 6.

Fig. 6

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Inhibition of 125I-antisauvagine and 125I-sauvagine binding to membranes of Y79 cells by various natural CRF agonists. Membranes were incubated at 22°C for 2 h with the two radiolabels and increasing concentrations of various CRF agonists (0.1 nmol/L to 10 μmol/L). The results are representative of four independent experiments performed in triplicate.

Desensitization of CRF2(a) receptors in Y79 cells by UCN2 and UCN3

Because we have previously shown that CRF1 receptors become homologously desensitized via a G protein-coupled receptor kinase 3 (GRK3) mechanism in Y79 cells exposed to oCRF (Hauger et al. 1997, 2003b; Oakley et al. 2007), we were interested in determining if endogenous retinoblastoma CRF2(a) receptors are also regulated by rapid homologous desensitization. Y79 cells were pre-incubated with near saturating concentrations of UCN2 and UCN3 for 5–60 min, extensively washed to remove bound ligand, and then re-stimulated with 100 nmol/L UCN2. CRF2 receptor agonist pre-treatment only minimally increased basal cAMP levels in Y79 cells. However, pre-treatment with either UCN2 or UCN3 resulted in a progressive and substantial reduction in cAMP responsiveness to UCN2 re-stimulation. While 100 nmol/L UCN3 pre-treatment gradually decreased maximal CRF2(a) receptor-mediated cAMP responses over a 60-min period to a maximum desensitization of ~70% (Fig. 7b), UCN2 was a more potent desensitizing agonist for retinoblastoma CRF2(a) receptors. A ~80% desensitization of CRF2(a) receptors was observed after a 5-min pre-treatment with 100 nmol/L UCN2 while no CRF2-receptor-mediated cAMP signal was measured following a 30-min UCN2 pre-incubation (Fig. 7a).

Fig. 7.

Fig. 7

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Comparison of time course for homologous CRF2 receptor desensitization induced by UCN2 or 3. After pre-treatment with 100 nmol/L UCN2 (a) or UCN3 (b) for 5 min to 1 h was completed, Y79 cells were extensively washed and maximally re-stimulated with 100 nmol/L UCN2 for 15 min. Data are mean ± SEM of values expressed as picomoles of cAMP generated by 106 cells that was collected in five independent experiments (n = 10 replicates per group). By anova, there were significant differences across the groups (F = 48.58, p < 0.0001). The following post hoc differences were found to be statistically significant between UCN2-stimulated cAMP responses in individual groups: ap < 0.001 versus control; bp < 0.001 versus 5 min UCN3; cp < 0.01 versus 15 min UCN3; dp < 0.001 versus 15 min UCN3; ep < 0.05 versus 5 min UCN2.

Discussion

The data reported herein indicate that human retinoblastoma Y79 cells endogenously co-express CRF1 and CRF2(a) receptors. Because we have shown that this cell line replicates co-expression of both CRF receptors in brain neurons modulating anxiety-like, defensive behavior, and stress responses including the bed nucleus of the stria terminalis, medial, and cortical amygdaloid nuclei, the entorhinal area of the hippocampus, and the ventral tegmental area (Chalmers et al. 1996; Sanchez et al. 1999; Sahuque et al. 2006), we have now established retinoblastoma cells as a well-controlled cellular setting for studying GRK, arrestin, and protein kinase C regulation of signal transduction by CRF2(a) as well as CRF1 receptors. In our previous research, we have demonstrated that retinoblastoma CRF1 receptor function is controlled by GRK3 and protein kinase C mechanisms in Y79 cells (Hauger et al. 1997, 2003b; Dautzenberg et al. 2001a, 2002a,b).

Until now, we had not observed endogenous expression of CRF2 receptors in this cell line. However, our molecular analyses were only performed using Y79 cells at earlier passages (Dautzenberg et al. 2000). When we investigated CRF receptor signaling at very late stage passages (≥ 35 passages in culture) in this study, we detected both CRF1 and CRF2 receptor responses in Y79 cells. This finding prompted us to determine if CRF2 receptor expression levels were initially very low and thus were not detectable in our earlier analyses. Our previous Y79 cell experiments were also performed before the two CRF2 receptor-specific agonists UCN2 and UCN3 were identified (Hsu and Hsueh 2001; Lewis et al. 2001; Reyes et al. 2001). Our hypothesis that very low expression of retinoblastoma CRF2 receptors at early passages would progressively increase during cell culture was supported by our successful amplification using semiquantitative RT-PCR of CRF2(a) cDNA but not of CRF2(b) and CRF2(c) cDNA isolated from late passages (> 40) but not at passage 5. Our detailed functional and QPCR experiments then identified a substantial up-regulation of CRF2(a) receptor mRNA and Gs-coupled cAMP signaling, which initially was close to the detection limit (i.e. ~2% of the total CRF receptor-mediated cAMP responses), slowly increased between passage 10 and 25, and then reached a maximum of ~25% of the total CRF receptor-mediated cAMP responses beginning at passage 30. Similarly, a gradual increase in CRF2(a) receptor mRNA expression was observed from low levels at passage ~5- to 10-fold higher levels at passage 30. The up-regulation of CRF2(a) receptor mRNA levels corresponded well with the observed ~10-fold increases in CRF2(a) receptor-mediated cAMP responses. One study recently failed to detect increases in cAMP levels in Y79 cells incubated with SCP and SRP (0–100 nmol/L) (Hsu and Hsueh 2001) while another study did not observe any stimulation of cAMP accumulation in Y79 cells exposed to 1 nmol/L UCN2 or UCN3 (Radulovic et al. 2003). Using Y79 cells at early culture passages when CRF2(a) receptor expression is very low and/or employing insufficient agonist concentration to stimulate CRF2(a) receptor cAMP signaling most likely explains these discrepancies.

In contrast to the CRF2(a) receptor findings, CRF1 receptor mRNA expression and cAMP signaling remained unaltered from early to prolonged Y79 cell culturing. These findings were unexpected as CRF1 and CRF2 receptor gene expression has been reported to be regulated by identical cAMP and calcium signaling cascades, glucocorticoids, and transcription factors (Iredale et al. 1996; Iredale and Duman 1997; Xu et al. 2001; Nanda et al. 2004; Parham et al. 2004). Thus, the transcriptional and translational mechanisms governing CRF2(a) receptor expression and its neurobiological significance may differ from those regulating CRF1 receptor expression. In addition, no effects on cAMP responses were observed when Y79 cells of different passages were challenged with isoproterenol, a β-adrenoceptor agonist (Hauger et al. 1997), pituitary adenlyate cyclase-activating polypeptide-38, a potent activator for the pituitary adenlyate cyclase-activating polypeptide type 1 (PAC1) receptor (Dautzenberg et al. 1999), or forskolin to directly activate adenylate cyclase function (our unpublished observations). The up-regulation of retinoblastoma CRF2(a) receptors during long-term cell culturing thus, may model an important neurobiological process whereby brain neurons may be induced to express higher levels of CRF2(a) receptors during synaptic plasticity.

Having established optimal conditions for promoting CRF2(a) receptor expression in Y79 cells, we completed a careful pharmacological characterization of the two endogenous CRF receptors. Maximal cAMP responses stimulated by mixed CRF agonists (i.e. CRF, UCN1, and sauvagine) and the CRF2 receptor-selective agonists (i.e. UCN2, UCN3, and the related N-terminally extended peptides SRP and SCP) were clearly separable. Stimulation of cAMP accumulation by mixed CRF receptor agonists was approximately fourfold more efficacious than the CRF2 receptor-selective ligands. Further confirmation that UCN3 and UCN2 selectively activated endogenous retinoblastoma CRF2(a) receptors was obtained in the functional antagonist experiments. While the non-selective peptide antagonist astressin (Gulyas et al. 1995) and the CRF1-selective antagonist NBI27914 (Chen et al. 1996) equipotently inhibited CRF-stimulated cAMP accumulation, the CRF2-selective antagonist antisauvagine (Ruhmann et al. 1998) displayed only a low micromolar antagonist potency. In contrast, antisauvagine and astressin blocked UCN3-stimulated cAMP production in Y79 cells with low nanomolar potencies comparable with their antagonist activity at recombinant CRF2(a) receptors (Dautzenberg et al. 2002b). Furthermore, NBI27914 failed to antagonize UCN3-stimulated cAMP accumulation. In complementary saturation binding experiments, 125I-antisauvagine bound to CRF2(a) receptors in late passage Y79 cell membranes with an affinity similar to that observed for recombinant CRF2(a) receptors (Higelin et al., 2001). In addition, UCN2, UCN3, and non-selective CRF receptor agonists competed for 125I-antisauvagine binding with almost identical affinities at retinoblastoma (see Fig. 6) and recombinant (Dautzenberg et al. 2001b) CRF2(a) receptors. UCN1 and CRF fully displaced 125I-sauvagine binding with a CRF1 receptor-like profile while UCN2 and UCN3 only partially competed (~20–25% displacement) for 125I-sauvagine binding. Because the UCN2 and UCN3 IC50 values for competition with 125I-sauvagine were close to the values obtained for UCN2 and UCN3 displacement of 125I-antisauvagine binding, a CRF2(a) receptor-selective binding profile was confirmed. Thus, we conclude that CRF1 and CRF2(a) receptors endogenously expressed in Y79 cells are fully functional and can be separated from each other by pharmacological tools.

We also provide the first evidence that the human CRF2(a) receptor can be homologously desensitized by exposure to its two selective agonists in a time-dependent manner. CRF2(a) receptor desensitization induced by UCN2 was more rapid in development and greater in magnitude than that caused by UCN3 in accordance with their agonist potencies. These findings are consistent with the established principle that the rate and magnitude of homologous GPCR desensitization and internalization is positively correlated with agonist potency (Clark et al. 1999). The maximal level of cAMP accumulation generated by forskolin-induced activation of the adenylyl cyclase was similar in Y79 cells treated with UCN2 and in control cells not exposed to this ligand (data not shown), indicating that CRF2 receptor desensitization observed in Y79 cells was an homologous, agonist-dependent process. It will be important to determine if homologous desensitization mechanisms regulating CRF1 and CRF2(a) receptors differ, and if differential molecular regulation of CRF1 and CRF2(a) receptor signaling modulates anxiety-like defensive behavior.

In conclusion we have characterized endogenous expressed CRF2(a) receptors in human Y79 retinoblastoma cells. CRF2(a) receptors are markedly and progressively up-regulated during prolonged cell culture to ~25% of the total CRF receptor population in Y79 cells. To our knowledge, our study is the first molecular and pharmacological of a cell line endogenously co-expressing both human CRF1 and CRF2(a) receptors. Y79 cells represent an informative cell model for studying crosstalk between CRF1 and CRF2(a) receptors and co-regulation of the two CRF receptors. In current studies, we are determining if mechanisms governing homologous CRF2(a) receptor desensitization, internalization, and recycling differ from the GRK3- and β-arrestin2-mediated mechanisms that we have found to regulate CRF1 receptors (Dautzenberg et al. 2001a; Oakley et al. 2007). Because limbic brain neurons implicated in anxiety, depressive, and stress disorders located in the bed nucleus of the stria terminalis, medial and cortical amygdaloid nuclei, the entorhinal area of the hippocampus, and the ventral tegmental area (Hauger et al. 2006) co-express both CRF receptors, understanding coordinate and differential regulation of CRF1 and CRF2(a) receptor signal transduction will provide important insight into the pathophysiology of affective illnesses.

Acknowledgements

Dr. RLH received the support from a Department of Veterans Affairs Merit Review Grant; the VA Center of Excellence for Stress and Mental Health (CESAMH); the VA Mental Illness Research, Education and Clinical Center (MIRECC) of VISN22; NIH/NIA (AG022982) and NIH/NIMH (MH074697) RO1 Grants. We also gratefully acknowledge Sandra Braun for performing the CRF2(a) receptor desensitization experiments and Alan Turken and Tina Smets for completing cAMP assays.

Abbreviations used

CRF

corticotropin-releasing factor

CRF1

CRF type 1 receptor

CRF2

CRF type 2 receptor

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GPCR

G protein-coupled receptor

GRK

G protein-coupled receptor kinase

Gs

cAMP stimulatory G protein

oCRF

ovine CRF

Q-PCR

quantitative RT-PCR

SCP

stresscopin

SRP

stresscopin-related peptide

UCN

urocortin

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