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Originally published In Press as doi:10.1074/jbc.M412914200 on January 14, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11560-11568, March 25, 2005
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Distinct Conformations of the Corticotropin Releasing Factor Type 1 Receptor Adopted following Agonist and Antagonist Binding Are Differentially Regulated*

Stephen J. Perry{ddagger}§, Sachiko Junger{ddagger}, Trudy A. Kohout¶, Sam R. J. Hoare||, R. Scott Struthers¶, Dimitri E. Grigoriadis¶, and Richard A. Maki{ddagger}

From the Departments of {ddagger}Molecular Biology, Endocrinology, and ||Pharmacology, Neurocrine Biosciences Inc., San Diego, California 92130

Received for publication, November 15, 2004 , and in revised form, January 12, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit {beta}-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or {beta}-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The 41-amino acid neuropeptide corticotropin releasing factor (CRF)1 is the principal regulator of the hypothalamic-pituitary-adrenal axis, and as such plays a critical role in mediating the response to stress in the body (1, 2). In mammals, CRF and the related urocortins 1, 2, and 3 bind to and activate two distinct G protein-coupled receptors (GPCRs), termed CRF1 and CRF2 (3). The CRF1 receptor is expressed mainly in the pituitary and central nervous system, where it is responsible for most of the central functions of CRF and urocortin 1, including integration of endocrine, autonomic and behavioral responses to stress, and adrenocorticotrophic hormone (ACTH) release from corticotrope cells of the anterior pituitary (4). Furthermore, there is strong evidence that alterations in the CRF1 receptor system occur in many anxiety and depressive disorders (5–7). CRF2 receptors bind all three urocortins with high affinity and CRF with lower affinity (3). These receptors exist as three independent isoforms (CRF2(a), CRF2(b), and CRF2(c)) and are expressed both in the central nervous system and the periphery, including in the heart, skeletal muscle, gastrointestinal tract, and epididymis (3). The functions performed by the various isoforms of the CRF2 receptor are currently being elucidated (8).

Both CRF1 and CRF2 receptors belong to the Class B family of G protein-coupled receptors, which includes (but is not limited to) the receptors for glucagon, parathyroid hormone (PTH), secretin, and vasoactive intestinal peptide. All class B receptors possess a large extracellular domain (ECD) with which they bind with high affinity to the carboxyl-terminal regions of their peptide ligands (9). This interaction alone is not sufficient to stimulate coupling of the receptor to G proteins, however, and a second interaction must occur between the juxtamembrane domain of the receptor (the transmembrane helices and intervening loops) and the first few residues within the amino-terminal portion of the peptide ligand (7, 10). Because discrete regions of class B ligands perform high affinity binding and receptor stimulation, truncating the endogenous peptides at their amino termini produces high affinity competitive antagonists for class B receptors. Further modifications made to CRF truncated in this manner have produced a number of different antagonist peptides including astressin (cyclo(30–33)-[D-Phe12,Nle21,38,Glu30,Lys33]CRF-(12–41)), a high affinity antagonist for CRF1 receptors that also possesses enhanced biological stability, allowing its extensive use in vivo to dissect the functions of the CRF system (11, 12). Astressin has no detectable agonist activity at the CRF1 receptor and thus it is believed to act as a neutral competitive antagonist. In addition to binding to the ECD of CRF1 receptors, a recent report has suggested that astressin may form a second low affinity contact with the juxtamembrane domain because astressin retains the ability to inhibit CRF activation of a CRF1 receptor fragment that lacks the ECD (13).

Following activation by agonists, almost all GPCRs undergo a series of modifications to prevent continuous signaling of the receptor, and to enable the cells on which they are expressed to regulate their sensitivity to future exposures to agonist. This is achieved first by preventing the activated receptors from further interacting with G proteins (desensitization), and then by internalizing the receptors into intracellular compartments (also called sequestration or endocytosis) (14, 15). Desensitization occurs through phosphorylation of intracellular domains of the receptor by GPCR kinases (GRKs) that specifically recognize agonist-occupied receptor molecules, followed by the recruitment and binding of {beta}-arrestins, which sterically hinder further receptor-G protein coupling. The subsequent internalization of the receptors can occur through multiple pathways, the most common of which utilize clathrin-coated pits and caveolae, although some less well defined pathways have also been described, including those that use non-coated vesicles and macropinosomes (16). Internalization can result in either short or long term reductions in sensitivity to further agonist stimulation depending on whether the receptors become resensitized and recycle back to the cell surface, or are targeted for degradation (down-regulated) (17).

A few examples of GPCRs undergoing regulation by antagonists have also been described, including internalization and down-regulation of the 5-hydroxytryptamine type 2A receptor by several atypical antipsychotics (18, 19); down-regulation of the gonadotropin releasing hormone receptor in pituitary gonadotrophs by the gonadotropin releasing hormone analogue cetrorelix (20); and phosphorylation and internalization of angiotensin II type 1A receptor by several antagonist peptide analogs of angiotensin II (21, 22). Furthermore, it has recently been reported that the class B PTH1 receptor also undergoes internalization following binding to the truncated antagonist peptide PTH-(7–34), a process that is independent of receptor activation (23). In light of these discoveries, we investigated whether this phenomenon of peptide antagonist-induced internalization also occurs with CRF1 receptors, and to probe the mechanism(s) underlying this process.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Peptides were synthesized by solid-phase methodology on a Beckman Coulter 990 peptide synthesizer (Fulton, CA) and purified as previously described (13). All chemicals were purchased from Sigma unless otherwise stated. Tissue culture medium and reagents were from Mediatech (Herndon, VA), except fetal bovine serum was from HyClone (Logan, UT) and horse serum from Invitrogen. Renilla mulleri GFP was licensed from Prolume Inc. (Pinetop, AZ). Membranes prepared from Ltk- cells expressing human CRF1 (hCRF1) receptors and from human embryonic kidney (HEK-293) cells expressing hCRF1, rat CRF1 (rCRF1), and rCRF1-ECD/activin IIB chimera receptors have been described previously (13, 24).

Mammalian Expression Constructs—Construction of hCRF1 receptor tagged with the hemagglutinin signal sequence and FLAG epitope (HA-FL-CRF1) in pcDNA5/FRT/V5-His®TOPO®, its stable expression in CHO-K1 Flp-In cells (designated CHO-CRF1 cells), and its indistinguishable pharmacology from the wild-type hCRF1 receptor are described previously (25). Complementary DNAs for dynamin 1, caveolin 1, and {beta}-arrestin2 were amplified from a human brain cDNA library, and inserted into the pcDNA3.1/V5-His®TOPO® vector following the manufacturer's instructions. Mutations were made in dynamin 1 (K44A) and caveolin 1 (S80A, S80E) (26) using the QuikChange® site-directed mutagenesis kit following the manufacturer's instructions (Stratagene, La Jolla, CA). The {beta}-arrestin2-R. mulleri GFP construct was made by adding EcoRI restriction sites to the {beta}-arrestin2 and R. mulleri GFP coding sequences by PCR with the primer pairs: 5'-AAAGAATTCACCATGGGGGAGAAACCC-3' and 5'-AAAGAATTCGCAGAGTTGATCATCATAGTC-3'; and 5'-AGAATTCGGAAGCAAGCAGATCCTGAAGAAC-3' and 5'-TCACGATGCGGCCGCTACA-3', respectively. Both products were cloned into pcDNA3.1/V5-His®TOPO® and subsequently digested with EcoRI. The released {beta}-arrestin2 fragment was purified and subsequently ligated into the linearized R. mulleri GFP construct. All plasmid DNA constructs were amplified in Escherichia coli using standard molecular biology procedures, harvested using Qiagen® DNA preparation kits, and their correct sequences confirmed by DNA sequence analysis using an ABI377 automated DNA sequencer and Big-DyeTM Terminator version 3.0 sequencing kits (Applied Biosystems, Foster City, CA).

Cell Culture and Transfection—HEK-293 cells, Chinese hamster ovary Flp-In (CHO-K1 Flp-In) cells, and mouse pituitary corticotrope adenoma AtT-20/D16v-F2 cells (AtT20, purchased from ATCC) were maintained at 37 °C and 5% CO2, in Dulbecco's modified Eagle's medium supplemented with 10 mM HEPES, pH 7.4, 0.2 mM glutamine, 1 mM sodium pyruvate, and penicillin-streptomycin (50 IU/ml and 50 µg/ml, respectively), and either 10% (v/v) heat-inactivated fetal bovine serum for HEK-293 and CHO-K1 Flp-In, or 10% (v/v) heat-inactivated horse serum for AtT20. Stable expression of receptor in CHO-CRF1 cells was maintained by selection with 500 µg/ml hygromycin B. Transient transfections into HEK-293 cells were performed using FuGENE 6® (Roche Diagnostics) at a ratio of 3 µl FuGENE 6® to every 1 µg of plasmid DNA. Experiments were performed on these cells 48 h after transfection. Overexpressed caveolin 1 mutants and dynamin 1 K44A were detected in cell lysates separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-caveolin 1 and anti-dynamin 1 antibodies (Upstate, Charlottesville, VA), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) and detection by chemiluminescence (Pierce, Rockford, IL).

Measurement of Receptor Internalization by Flow Cytometry—Cells transiently or stably expressing FL-CRF1 receptor were seeded at 4 x 105 cells/well in poly-D-lysine-coated 6-well dishes (BIOCOATTM, Fort Washington, PA). The following day the cells were subjected to the appropriate drug treatments, washed twice with ice-cold internalization medium (Dulbecco's modified Eagle's medium containing 25 mM HEPES, pH 7.4, 0.2 mM glutamine, 1 mM sodium pyruvate, 50 IU/ml penicillin, and 50 µg/ml streptomycin) and then incubated for 1 h at 4 °C with anti-FLAG M2 antibody (Sigma) diluted 1:500 in internalization medium. The cells were then washed three times with ice-cold internalization medium and incubated at 4 °C in the dark for a further 30 min with goat anti-mouse IgG antibody conjugated to Alexa FluorTM 488 dye (Molecular Probes, Eugene, OR) diluted to 1:250 in internalization medium. Cells were subsequently washed with ice-cold PBS three times, detached from the dishes with PBS containing 5 mM EDTA, and fixed by the addition of formaldehyde to 0.8% (w/v). The fluorescence intensity of 104 cells from each well was then measured on a FACScanTM flow cytometer (BD Biosciences). Concanavalin A (Sigma) was added to the cells at 0.25 mg/ml 1 h prior to stimulation. Hypertonic medium treatment, potassium depletion, and disruption of caveolae with filipin III were carried out using previously described methods (27–29).

Examination of Receptor Internalization by Fluorescence Microscopy—CRF1 receptor internalization was visualized using a previously described method with a minor modification (30). Briefly, transiently transfected AtT20 or HEK-293 cells expressing FL-CRF1 receptor with or without {beta}-arrestin2-R. mulleri GFP were grown on Nunc® Lab-Tek® II CC2® multichamber glass slides. Surface receptors on AtT20 cells were labeled (20 min at 37 °C) with Alexa Fluor 488-conjugated M1 anti-FLAG antibody, or on HEK-293 cells with M1 anti-FLAG-Cy3 conjugate (prepared according to the manufacturer's instructions using either Alexa Fluor 488 Monoclonal Antibody Labeling Kit, (Molecular Probes), or Cy3 mono-Reactive Dye Pack (Amersham Biosciences)). Cells were washed once and exposed to 10 µM CRF or 10 µM astressin (2 h at 37 °C) to induce internalization. Cells were washed twice with PBS and immediately fixed with 4% (w/v) paraformaldehyde in PBS for 15 min at room temperature. Cells were washed three times at 10-min intervals with PBS before mounting using ProLong® Gold antifade reagent (Molecular Probes). Fluorescence was visualized on an Olympus IX70 inverted microscope equipped with the CARV confocal module (Kinetic Imaging, Nottingham, UK) using appropriate dichroic filter sets. Images were acquired with a MicroMAX cooled CCD camera (Princeton Instruments, Trenton, NJ) and processed using the Meta-Morph® Imaging System (Universal Imaging Corporation, Downingtown, PA).

[32P]Orthophosphate (32Pi) Labeling and Receptor Immunoprecipitation—CHO-CRF1 or CHO-K1 Flp-In cells were serum starved for 1 h in phosphate-free Dulbecco's modified Eagle's medium supplemented with 2 mM L-glutamine and 25 mM HEPES, pH 7.4. Cells were labeled with 100 µCi/ml of 32Pi (PerkinElmer Life Sciences) for 1 h, then 100 nM microcystin-LF (Calbiochem, San Diego, CA) was added and the cells were incubated for a further 15 min before stimulation with 100 nM astressin or CRF at 37 °C, or left untreated. Cells were washed twice with PBS and extracts were prepared by lysing cells in 500 µl of glycerol lysis buffer (50 mM HEPES, pH 7.4, 0.5% (v/v) Nonidet P-40, 250 mM NaCl, 2 mM EDTA, 10% (v/v) glycerol, 100 µM Na3VO4, 10 mM NaF, 100 nM microcystin-LF, and Complete® EDTA-free protease inhibitor mixture tablet (Roche)). The samples were clarified by centrifugation, and FL-CRF1 receptor was immunoprecipitated from equal amounts of cell lysate with 30 µl of M2 anti-FLAG-agarose conjugate (Sigma) for 16 h with constant mixing. Immunoprecipitates were washed 5 times with glycerol buffer and eluted with 40 µl of 2 x SDS sample buffer (Invitrogen) supplemented with 200 mM DL-1,4-dithiothreitol. Half the volume (20 µl) of the immunoprecipitates was resolved on a 4–20% Tris glycine SDS-polyacrylamide gel (Invitrogen). The gel was dried and incorporation of 32Pi measured using the VersaDoc3000 phosphorimager (Bio-Rad).

Measurement of Receptor Down-regulation by ELISA—CHO-CRF1 cells were seeded at 7.5 x 104 cells/well in poly-D-lysine-coated 96-well dishes (BIOCOAT). The following day the cells were subjected to the appropriate treatments, washed once with ice-cold PBS and lysed in 200 µl/well of ice-cold lysis buffer (1% (v/v) Nonidet P-40 in PBS supplemented with Complete EDTA-free protease inhibitor mixture, Roche) for 30 min at 4 °C with constant agitation. Detergent-insoluble fractions were sedimented by 10 min centrifugation at 2,000 x g, and the amount of CRF1 receptor present in the clarified cell lysates was quantified by ELISA. Briefly, protein concentration was measured using the BCA protein assay method (Pierce). Equal amounts of protein (12 µg/well) were transferred to anti-FLAG M2-coated 96-well plates (Sigma) and incubated overnight at 4 °C. Each well was washed 4 times with ELISA wash buffer (0.05% (v/v) Tween 20 in PBS) and incubated for 2 h at room temperature with the previously described anti-CRF1 receptor antiserum 4467a-CRF1 (31) diluted 1:10,000 in antibody dilution buffer (PBS containing 1% (w/v) bovine serum albumin). The plates were washed, incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Biosciences) diluted 1:2,000 in antibody dilution buffer, followed by a final four washes. Plates were incubated with 200 µl/well of ready-to-use horseradish peroxidase substrate 3,3',5,5'-tetramethylbenzidine (Sigma) for 30 min at room temperature, followed by addition of 100 µl/well 0.5 M H2SO4 to stop the reaction. The optical density of each well was read at 450 nm using an EMax microplate reader (Molecular Devices, Sunnyvale, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To determine whether CRF and the antagonist astressin are both capable of inducing the internalization of the CRF1 receptor, CHO-CRF1 cells (CHO-K1 cells stably expressing the hemagglutinin-FLAG-tagged hCRF1 receptor) were treated with 100 nM CRF or astressin for 30 min to 24 h, and the loss of receptors from the cell surface was measured by flow cytometry. Time-matched vehicle-treated controls were also performed to allow all data points to be normalized to the appropriate level of cell surface expression. Fig. 1A shows that both CRF and astressin induced substantial internalization over this period, however, the total amount of internalization following 24 h stimulation with CRF was greater than with astressin (71 ± 7.4 and 51 ± 8.1%, respectively). This difference was the result of a higher rate of receptor sequestration by CRF in the first hour of stimulation (63% with CRF, 17% with astressin), after which sequestration by both peptides proceeded at similar rates (evident from the similar slopes of the graphs between 2 and 24 h in Fig. 1A). Full dose-response relationships were then produced for CRF and astressin to allow the EC50 values to be calculated (Fig. 1B). Both peptides internalized CRF1 receptor in a dose-dependent manner, however, despite the fact that astressin possessed only partial efficacy in the internalization assay, astressin and CRF promoted internalization with almost identical potencies (EC50 = 7.6 and 7.8 nM, respectively). Taken together, these data indicate that the agonist CRF possesses high efficacy for internalizing CRF1 receptors, whereas the antagonist astressin appears to possess only partial efficacy. Despite this difference in internalization efficacy, astressin and CRF were equipotent for this effect.



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  		FIG. 1.
Measurement of agonist and antagonist-induced internalization of CRF1. A, time-dependent internalization of CRF1 receptors. CHO-K1 cells expressing FLAG-hCRF1 receptors were stimulated with 100 nM CRF ({blacksquare}) or 100 nM astressin ({circ}) or vehicle (x) for times between 30 min and 24 h and the amount of receptor remaining at the cell surface was detected using anti-FLAG M2 and anti-mouse Alexa Fluor 488-conjugated antibodies, and measured by flow cytometry. The graph shows mean ± S.E. values for % receptor remaining at the cell surface normalized to time-matched vehicle-treated controls of six independent experiments. B, dose-dependent internalization of CRF1 receptors measured after 24 h stimulation with CRF ({blacksquare}) or astressin ({circ}). The graph shows mean ± S.E. values for % receptor remaining at the cell surface compared with non-stimulated cells (NS) of 10–14 independent experiments.

 
We next determined whether the phenomenon of astressin-induced internalization also occurred in a cell line that expresses the CRF1 receptor endogenously. Corticotropes of the anterior pituitary are major sites of CRF1 receptor expression, where it stimulates the secretion of ACTH into the blood in response to CRF released from the hypothalamus (2, 32). The mouse pituitary corticotrope adenoma AtT20 cell line expresses CRF1 receptors (~100 fmol/mg of membrane protein, data not shown), produces cyclic AMP (25), and secretes ACTH (33) when challenged with CRF peptide. To measure CRF1 receptor internalization, AtT20 cells were transfected with FLAG-tagged CRF1 receptor to allow its movement to be tracked both by immunocytochemistry and flow cytometry (Fig. 2). Prior to treatment, immunostaining of live AtT20 cells with anti-FLAG M1 antibody conjugated to Alexa Fluor 488 dye revealed substantial cell surface expression and no visible staining of intracellular receptors (not shown). The cells were then treated for 2 h with vehicle, or with a maximal dose of CRF or astressin (10 µM), and the redistribution of the immunofluorescence-stained receptors was monitored. No internalization of CRF1 receptors was observed in cells treated with vehicle for 2 h (Fig. 2A, i), demonstrating that the conjugated M1 antibody alone did not induce receptor internalization. Following treatment with CRF, however, many of the labeled receptor molecules had redistributed from the cell surface into compartments within the cytosol (Fig. 2A, ii). A less robust but similar pattern of CRF1 receptor redistribution to intracellular compartments was observed following astressin treatment, with substantial levels of receptor remaining at the cell surface (Fig. 2A, iii). This reduced level of receptor redistribution with astressin correlated well with the amount of CRF1 receptor internalization measured in AtT20 cells using flow cytometry (Fig. 2B), where CRF induced internalization of 39% after 2 h, increasing to 45% at 4 h, while astressin internalized 9% after 2 h and 16% after 4 h.



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  		FIG. 2.
Detection of CRF1 receptor internalization in mouse corticotrope AtT20 cells. A, cell surface FLAG-tagged hCRF1 receptors transiently expressed in AtT20 cells was stained with anti-FLAG M1 antibody conjugated to Alexa Fluor 488 and images of representative cells from three separate experiments were collected by fluorescence microscopy following 2 h treatment with vehicle (NT; panel A, i), 10 µM CRF (panel A, ii), or 10 µM astressin (panel A, iii). B, quantification of FLAG-CRF1 receptor internalization in AtT20 cells by flow cytometry following stimulation with 10 µM CRF ({blacksquare}) or 10 µM astressin ({circ}). Values are mean ± S.E. of five or six experiments; *, p < 0.05; **, p < 0.01 compared with time = 0.

 
The majority of GPCRs are internalized via clathrin-mediated endocytosis (16). This process normally requires the receptors to be bound to agonist, phosphorylated by GRKs, and to recruit cytosolic arrestins, however, clathrin-mediated internalization following antagonist binding has also been reported (34). Furthermore, some peptide antagonists are known to cause receptor phosphorylation without activating G protein coupling (21, 22, 34). To determine whether such mechanisms could underlie astressin-induced CRF1 receptor internalization, we compared the ability of astressin and CRF to promote CRF1 receptor phosphorylation and the recruitment of {beta}-arrestin2 (Fig. 3, A and B). CHO-CRF1 cells were metabolically labeled with 32Pi, stimulated with 100 nM astressin or CRF between 5 min and 1 h, and the amount of radioactivity incorporated into the CRF1 receptor was assessed by autoradiography of receptor immunoprecipitates. Fig. 3A shows that stimulation of cells with astressin caused no phosphorylation of the receptor at any of the time points tested. In contrast, stimulation with CRF induced robust phosphorylation of CRF1 receptors by 5 min (4.27 ± 0.28-fold over basal), peaking at 10 min (4.46 ± 0.54-fold over basal), and then slowly diminishing to 2.08 ± 0.54-fold after 1 h. Next, we tested whether stimulation with astressin or CRF led to recruitment of cytosolic {beta}-arrestin2 to CRF1 receptors. Cells were first transfected with expression constructs for {beta}-arrestin2-R. mulleri GFP and FL-CRF1 receptor. Forty-eight hours later, receptors expressed at the cell surface were visualized by immunostaining with anti-FLAG M1 antibody conjugated to Cy3, then the cells were stimulated with 100 nM astressin or CRF for 5 or 15 min, and the distributions of receptor and {beta}-arrestin2 were determined by fluorescence microscopy (Fig. 3B). Prior to stimulation, the CRF1 receptor was only detected on the cell surface, whereas {beta}-arrestin2-R. mulleri GFP was evenly distributed throughout the cytosol and excluded from the nucleus. After 5 min of stimulation with CRF, the receptors were still present at the cell surface, however, the majority of the {beta}-arrestin2-R. mulleri GFP had localized to the cell membrane and displayed a distribution that almost completely overlapped with the CRF1 receptor. After 15 min, much of the receptor and {beta}-arrestin2-R. mulleri GFP had redistributed from the cell surface into punctate structures within the cytosol, again showing almost complete overlap of their distributions. This was entirely different from what was observed following astressin stimulation: no relocalization of {beta}-arrestin2-R. mulleri GFP was observed after 5 or 15 min stimulation, whereas a small amount of receptor internalization could be observed after 15 min in some cells (Fig. 3B). Taken together, these data demonstrate that CRF binding to the CRF1 receptor triggers phosphorylation of the receptor, recruitment of {beta}-arrestin2, and internalization of receptor-{beta}-arrestin2 complexes, whereas astressin treatment induces neither phosphorylation nor {beta}-arrestin2 recruitment, but still induces receptor internalization.



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  		FIG. 3.
Comparison of CRF1 receptor regulation and internalization by CRF and astressin. A, CHO-K1 cells and CHO-CRF1 cells were metabolically labeled with 32Pi for 1 h before stimulation with 100 nM astressin or CRF for various times (t). Receptor molecules were immunoprecipitated from lysates made from equal numbers of cells, separated by gel electrophoresis, and radiolabeled receptors were quantified and visualized by phosphoimaging. Autoradiograph shown is representative of three independent experiments. B, HEK-293 cells were co-transfected with FLAG-CRF1 receptor and {beta}-arrestin2-R. mulleri GFP and stained with M1 anti-FLAG antibody-Cy3 conjugate before stimulation with 100 nM astressin or CRF for 5 or 15 min. Distributions of receptor (red) and {beta}-arrestin2-R. mulleri GFP (green), and overlays (yellow) were visualized by fluorescence microscopy. C–E, CHO-CRF1 cells pretreated with medium containing 0.5 M sucrose (C), or subjected to K+ ion depletion with or without replacement of K+ (D), or treated with filipin III (E), were stimulated with 100 nM astressin or CRF for 1 h, and internalized receptor was measured by flow cytometry. F, HEK-293 cells co-transfected with FLAG-CRF1 receptor, empty vector (V), dynamin 1 K44A, caveolin 1 S80E, or caveolin 1 S80A were treated with 100 nM astressin or CRF for 2 h and the internalized receptor was measured by flow cytometry. Data are mean ± S.E. of four to six experiments. Insets are representative Western blots demonstrating expression of mutants of dynamin 1 and caveolin 1 in cell lysates.

 
Next, we sought to identify the mechanisms by which CRF and astressin induced CRF1 receptor endocytosis by testing their sensitivities to known inhibitors of internalization pathways. Clathrin-mediated endocytosis is sensitive to both treatment with hypertonic medium (Dulbecco's modified Eagle's medium containing 0.5 M sucrose (27)), and to potassium ion depletion (28). CHO-CRF1 cells were subjected to both of these treatments prior to the addition of 100 nM CRF or astressin for 1 h, and internalization was measured by flow cytometry (Fig. 3, C and D). CRF-induced receptor internalization was strongly inhibited by both sucrose (83%), and potassium depletion (97%), the latter effect being largely reversed when potassium was replaced in the depletion buffer (compare 34.5 ± 4.7% with potassium replacement to 47.4 ± 2.4% when cells were maintained in medium). In contrast, neither of the treatments significantly affected astressin-induced internalization indicating that, whereas CRF-driven endocytosis is likely a clathrin-mediated process, astressin utilizes an entirely different endocytic pathway. Several GPCRs have been reported to internalize through caveolae, cup-like cholesterol-rich membrane structures that contain large quantities of the membrane-associated protein caveolin 1 and, like clathrin-coated pits, require dynamin 1 to internalize receptors (35, 36). Three approaches were taken to determine whether caveolae were utilized by astressin or CRF to internalize CRF1 receptors. First, CHO-CRF1 cells were treated with the cholesterol-depleting agent filipin III under conditions previously demonstrated to cause disruption of most of the caveolae in cells (1 µg/ml filipin III for 1 h (29)), and then receptor internalization was measured after 1 h of stimulation with 100 nM astressin or CRF. Filipin III failed to inhibit internalization induced by either peptide, suggesting that caveolae were not involved (Fig. 3E). Second, overexpression of the dominant negative mutant dynamin 1 K44A, which is required for both caveolae- and clathrin-mediated endocytosis, failed to inhibit astressin-induced internalization but, as was expected, did significantly reduce internalization by CRF (Fig. 3F). Third, coexpression of CRF1 receptors with the dominant negative S80E mutant of caveolin 1 (or the phenotypically neutral S80A mutant) (26) failed to disrupt either astressin- or CRF-induced receptor internalization (Fig. 3F). Taken together, these data indicate that cells treated with astressin utilize neither caveolae nor clathrin-coated pits to internalize CRF1 receptors, whereas CRF-induced internalization likely occurs through clathrin-coated pits, as demonstrated by its sensitivity to potassium depletion, hypertonic sucrose solution, and K44A dynamin expression.

Following internalization many GPCRs are down-regulated by trafficking to lysosomes for degradation, whereas others remain sequestered within intracellular compartments (17). We tested whether the use of different internalization pathways following CRF and astressin stimulation also resulted in alternative trafficking and processing of the CRF1 receptor. An ELISA detection method was developed to measure loss of receptor protein in cell lysates at between 1 and 24 h following stimulation with either 100 nM astressin or CRF. Fig. 4A shows that there was no reduction in total CRF1 receptor protein following astressin stimulation at any of the time points tested, whereas stimulation with CRF showed a gradual loss of the receptor in the cells, from 83% remaining after 1 h, dropping to 38% after 24 h. Despite the kinetics of receptor down-regulation being markedly different from those we observed for receptor internalization (Fig. 1A, and shown on Fig. 4A for comparison), the down-regulation is dependent on CRF1 receptor internalization because cross-linking of the receptor on the cell surface with the lectin concanavalin A prior to stimulation suppressed internalization by both astressin (53% inhibited) and CRF (55% inhibited) and also inhibited CRF-stimulated down-regulation of the receptor by 53% (Fig. 4B).



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  		FIG. 4.
Determination of CRF1 receptor down-regulation following treatment with CRF and astressin. A, CHO-CRF1 cells were treated with 100 nM CRF ({blacksquare}) or astressin ({circ}) for times between 1 and 24 h, and the amount of receptor protein remaining in the cell lysates was detected by ELISA (see "Experimental Procedures"). Data are mean ± S.E. from three experiments. The equivalent time course of CRF-induced internalization is included from Fig. 1A for comparison ({square}). B, CHO-CRF1 cells were treated with 0.25 mg/ml concanavalin A prior to stimulation for 24 h with 100 nM astressin or CRF, and internalization and down-regulation of the receptor were measured. Data are mean ± S.E. from four experiments.

 
We next investigated the nature of the interactions between astressin and the receptor that are required for receptor internalization. Peptide interaction with CRF1 receptors proceeds according to a two-domain model in which the carboxyl-terminal portion of the ligand binds the ECD, and the amino-terminal portion binds the juxtamembrane domain. In addition to the high affinity interaction with the ECD, it was recently reported that astressin might also interact with low affinity with the juxtamembrane domain (13). For example, astressin bound with higher affinity to the full-length receptor than to a chimera of the ECD and the single transmembrane domain of the activin IIB receptor, suggesting a second astressin binding site within the juxtamembrane domain. This raises the possibility that an interaction of astressin with the juxtamembrane domain is involved in receptor internalization. This hypothesis was tested using 12-residue carboxyl-terminal astressin analogs that have been reported to bind CRF1 receptors with high affinity (Yamada number 19 and Yamada number 20 (37)). These peptides lack 18 amino-terminal residues of astressin and so would be predicted to bind only the ECD, according to the two-domain model described above. Indeed, both peptides bound with similar, if not slightly higher, affinity to the rat CRF1-ECD/activin IIB chimera than to the full-length receptor, consistent with these peptides binding predominantly, if not exclusively, to the ECD (Table I; because our previous experiments were conducted using hCRF1 receptors, we also confirmed that the affinities of all three peptides were similar for both rat and human forms). In contrast astressin bound with 6-fold higher affinity to the full-length receptor than the CRF1-ECD/activin IIB chimera, suggesting that binding is stabilized by interaction with the juxtamembrane domain (Table I, consistent with previous data (13)). Yamada 19 and Yamada 20 bound with high affinity to hCRF1 receptors (Fig. 5A, Table I, Ki = 1.3 and 1.0 nM, respectively, in agreement with published values (37)). Full internalization dose-response relationships of both peptides were then compared with those for CRF and astressin and the EC50 values were calculated (Fig. 5B). Interestingly, whereas CRF and astressin potently induced internalization of the CRF1 receptor (EC50 of 5 and 1 nM, respectively), neither of the Yamada peptides showed any activity in this assay at concentrations up to 10 µM (Fig. 5B). These findings suggest that interaction with the ECD alone is insufficient to induce internalization. Furthermore, the structure-activity relationship of these ligands suggests that internalization involves interaction of the amino-terminal region of astressin with the juxtamembrane domain of the receptor.


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  		TABLE I
Inhibition of [125I]astressin binding to various CRF receptors by astressin analogs Astressin analog binding was measured by displacement of [125I]astressin binding to HEK-293 cell membranes in the presence of 30 µM GTP{gamma}S, for the following receptors: rCRF1, rCRF1-ECD (ECD of rCRF1 receptor attached to the transmembrane domain of the activin IIB receptor), and hCRF1 receptors. Displacement data were fitted to a single-site competition equation to determine Ki, using a Kd value for [125I]astressin of 26, 220, and 65 pM for rCRF1, rCRF1-ECD, and hCRF1 receptors, respectively. Data are mean ± S.E. (n = 3–9). Statistical significance of the difference of pKi value between rCRF1-NT and rCRF1 was tested by two-tailed Student's t test.

 



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  		FIG. 5.
Comparison of CRF1 receptor internalization by CRF, astressin, and short astressin analogs. A, binding affinities of astressin (Ki = 0.38 nM) and two 12-residue peptides (Yamada number 19, Ki = 1.3 nM and Yamada number 20, Ki = 1 nM) were measured in competition binding assays against [125I]sauvagine on membranes prepared from Ltk- cells expressing the hCRF1 receptor. The graph shows a representative set of data from one of three independent experiments. B, dose-response curves for internalization of CRF1 receptors in CHO-CRF1 cells treated for 16 h with CRF, astressin, and Yamada numbers 19 or 20. Values are mean ± S.E. from four to seven experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
It is now widely recognized that most GPCRs are internalized following their stimulation with ligand, a process that prevents their persistent signaling and allows cells to regulate receptor number, and thus sensitivity to subsequent challenges with agonist. Internalization, coupled with receptor desensitization, is also an important process in the pathogenesis of several diseases, the development of tolerance to certain drugs, and may contribute to the efficacy of other therapeutics. For example, loss of {beta}-adrenergic receptors from cardiac myocytes leads to heart disease (38), whereas desensitization of opioid receptors is believed to underlie tolerance to opiates (39), and at least part of the therapeutic activity of cetrorelix is a result of its ability to down-regulate the gonadotropin releasing hormone receptor (20). The first two examples represent the well studied "classic" mechanism of GPCR regulation following stimulation (or overstimulation) with a native or synthetic agonist. The last example, however, is more interesting because it results from the binding of an antagonist to the receptor, causing the receptor to become down-regulated without it ever being activated. Because current models of receptor regulation involve a priori activation of the receptor, the existence of such antagonist-mediated mechanisms has profound implications for our present understanding of how GPCRs function and are regulated, and possibly for interpreting the in vivo activities of these compounds.

In this study we describe a previously unrecognized mechanism by which cells regulate CRF1 receptors following binding to the antagonist peptide astressin, a process that promotes endocytosis of the receptor molecules and their subsequent sequestration into intracellular compartments. The mechanism by which astressin achieves this differs greatly from both the classic sequestration that follows stimulation with CRF, and from the mechanism used to endocytose PTH1 following binding to PTH-(7–34), the only other example of a class B receptor antagonist known to induce internalization (23). We show here that the CRF1 receptor undergoes rapid phosphorylation following CRF binding, probably by GRKs because GRK3 has been implicated in the regulation of these receptors (40, 41). Similarly, phosphorylation of PTH1 is observed when either PTH or PTH-(7–34) are bound, although the kinases responsible appear to be GRK2 and protein kinase C (34, 42). Astressin binding to CRF1 receptors, however, does not induce any detectable level of phosphorylation, indicating that the receptor does not adopt a conformation that is recognized as a substrate by GRKs. Furthermore, CRF and PTH both induce the recruitment of cytosolic {beta}-arrestins to their receptors, whereas neither PTH-(7–34) nor astressin are capable of this (34, 42, 43). Thus, it appears that both CRF1 and PTH1 receptors adopt distinct conformations following binding to their agonists or antagonists: the agonists (CRF and PTH) place both receptors into conformations that are recognized as kinase substrates and as {beta}-arrestin binding sites, and presumably become desensitized in the process; PTH-(7–34) appears to be capable of placing PTH1 into a conformation that is phosphorylated but not recognized as a binding partner by {beta}-arrestins, whereas astressin-bound CRF1 receptor is neither a kinase substrate nor a {beta}-arrestin binding partner. This model of multiple receptor conformations possessing distinct signaling and regulatory properties is supported by a number of studies that demonstrate regulation and activation of GPCRs are completely separable events. For example, introduction of a zinc ion bridge between transmembrane helices 3 and 6 of PTH1 constrains the receptor in a conformation that cannot couple to G proteins in response to agonist, but which still permits its phosphorylation and internalization (42). Furthermore, several GPCRs for which multiple agonists or antagonists have been described undergo distinct signaling and regulatory events in a ligand-specific manner. Examples of these include the angiotensin II type 1A receptor, for which there exist synthetic angiotensin II analogs capable of promoting both signaling and endocytosis, whereas other analogs only induce internalization (21, 22); the µ opioid receptor, where morphine and etorphine both potently activate signaling, but only etorphine induces receptor internalization (44, 45); and the chemokine receptor CCR7, where binding of only one of its two endogenous ligands promotes receptor phosphorylation and desensitization, while both fully activate signaling (46).

Most cells sequester GPCRs through clathrin-coated pits, although other pathways can also be used, for example, caveolae mediate the internalization of both endothelin A and B receptors and possibly also vasoactive intestinal peptide receptors (16). Our study shows that CRF-induced receptor internalization is likely a clathrin-mediated process, because it is blocked by known inhibitors of clathrin-coated pit function (hypertonic sucrose solution (27) and potassium ion depletion (28)) and by overexpression of the GTPase-deficient K44A mutant of dynamin 1, whose activity is required for detachment of clathrin-coated vesicles from the plasma membrane (47). Whereas hypertonic sucrose and K44A dynamin are not specific inhibitors of clathrin-mediated endocytosis, sensitivity to both treatments and to potassium depletion indicate that CRF-driven internalization is likely a clathrin-mediated event. In contrast, astressin-mediated internalization is not affected by these treatments, indicating that an entirely different pathway is utilized. Because dynamin activity is also required for caveolae-mediated internalization (35, 36), the lack of any effect of overexpression of K44A dynamin suggests that this is not the alternative pathway. We confirmed this by showing that neither the disruption of caveolae with filipin III (29), nor overexpression of the caveolin 1 S80E dominant negative mutant has any effect on astressin-induced endocytosis of the receptor (26). This differs from the pattern observed for PTH1, where clathrin-mediated endocytosis is used following both PTH and PTH-(7–34) binding (23, 34). However, the use of distinct endocytic pathways for the same receptor in response to multiple ligands has been described for other GPCRs, including CXCR3, where two of the three agonists (CXCL9 and CXCL10) induce internalization through a dynamin-dependent mechanism, whereas the third (CXCL11) does not; the nature of the alternative pathway was not investigated further (48).

In addition to clathrin-coated pits and caveolae, cells utilize several other pathways to endocytose membrane-bound receptors, including the well described processes of phagocytosis and fluid-phase internalization through pinocytosis, as well as some dynamin-independent mechanisms for which there are as yet no tools available to define their exact nature (49). Furthermore, several of these ill-defined mechanisms have been implicated in the endocytosis of GPCRs, including the bradykinin type 2, N-formyl peptide and M2 muscarinic receptors (16). It is interesting to note that the class B family receptor for secretin also internalizes in a dynamin-independent manner, raising the possibility that the CRF1 receptor internalizes through the same mechanism, and that it may represent a general mechanism for class B receptor internalization (50).

In addition to utilizing different paths of CRF1 receptor internalization, we also demonstrated that astressin and CRF binding determine distinct fates for the receptor, because only CRF-bound receptors were subsequently targeted for down-regulation. This process requires the receptors to be internalized first because trapping the receptors on the cell surface with the cross-linking agent concanavalin A prior to treatment with CRF inhibited their down-regulation. Thus, astressin and CRF target the receptors to alternative intracellular compartments where they either remain sequestered or are down-regulated. Several studies of GPCR trafficking have identified interactions between the extreme carboxyl-terminal tails of GPCRs with PDZ domain-containing proteins (named after the first three proteins in which they were characterized: PSD-95/Dlg and ZO-1) as critical for determining receptor fate following internalization (17, 30, 51, 52). The tails of both CRF1 and CRF2 receptors contain putative PDZ-binding motifs and therefore might also be regulated by PDZ proteins. It is interesting to note that an interaction between PTH1 and the PDZ protein Na+/H+ exchanger regulatory factor 2 has been shown to inhibit PTH-(7–34) internalization in descending convoluted tubule cells (23). Deletion of the PDZ-binding motif from PTH1 relieves this inhibition and allows PTH-(7–34) to induce a level of receptor internalization equivalent to that observed with PTH. Whereas we cannot discount that such a mechanism of regulation exists for CRF1 receptors, preliminary experiments in which we truncated the COOH-terminal tail, in effect ablating the PDZ-binding motif, did not enhance astressin-induced internalization in either CHO-K1 or HEK-293 cells expressing the CRF1 receptor (data not shown). However, because the identities of PDZ proteins that bind CRF1 receptors are currently unknown, their effects on internalization or trafficking cannot be tested directly.

Finally, we have also investigated the basis of the interaction between astressin and the CRF1 receptor responsible for inducing internalization. The existing explanation of how the CRF1 receptor binds to peptide agonists is described by a two-domain model in which the amino terminus of the receptor binds with high affinity to the carboxyl-terminal portion of the agonist, substantially increasing the local concentration of agonist and so allowing the second weak interaction to occur between the amino-terminal region of the peptide and the juxtamembrane domain of the receptor (7, 10). The exact site of this second interaction remains controversial, although contacts between peptide ligands and the second and third extracellular loops of both CRF1 and CRF2 receptors have been reported (53–56). Previous data suggest that astressin binding is stabilized by an interaction with the juxtamembrane domain of the CRF1 receptor. In addition, analysis of chimeric receptors suggests strong binding of antagonists to the juxtamembrane domain of CRF2 receptors (25). In this study we confirmed that astressin binds to the full-length CRF1 protein with 6-fold higher affinity than to the CRF1-ECD/activin IIB receptor construct (Table I). In contrast, two high affinity 12-residue carboxyl-terminal astressin analogs (the Yamada peptide numbers 19 and 20) bind with similar affinities to both proteins. We propose that this difference in the affinities observed for astressin is the result of an additional interaction between the juxtamembrane domain of the receptor and the extra 18 amino acids present in astressin that are absent in the Yamada peptides. Furthermore, this putative interaction appears necessary to induce the conformational change in the receptor required for its internalization, because the short Yamada analogs neither make this contact nor induce internalization.

In summary, we have demonstrated that the CRF1 receptor is subjected to ligand-specific modes of internalization and trafficking following binding of peptide agonists and antagonists. Furthermore, we present evidence that for antagonist binding to promote internalization it must contact both the amino terminus and juxtamembrane domain of the receptor. These findings may have important consequences for the design of CRF1 receptor antagonists for the treatment of anxiety disorders and depression, because they show that the receptor adopts a distinct conformation when bound to antagonists, which could be exploited to further suppress receptor signaling by inducing its internalization.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130. Tel.: 858-617-7595; Fax: 858-617-7696; E-mail: sperry{at}neurocrine.com.

1 The abbreviations used are: CRF, corticotropin releasing factor; ACTH, adrenocorticotrophic hormone; Nle, norleucine; HEK, human embryonic kidney; CHO, Chinese hamster ovary; ECD, extracellular domain; GPCR, G protein-coupled receptor; PTH, parathyroid hormone; GRK, GPCR kinase; GFP, green fluorescent protein; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; CCR, C-C chemokine receptor; PDZ, PSD-95/Dlg/ZO-1; CXCL, C-X-C chemokine ligand; GTP{gamma}S, guanosine 5'-O-(3-thiotriphosphate). Back


   ACKNOWLEDGMENTS
 
We thank Nick Ling for synthesis and purification of peptides, and Khamkeo Khongsaly, Tiffany M. Flynn, and Shelby L. Reijmers for additional technical support. We also thank Marilyn Perrin and Wylie Vale at the Salk Institute (La Jolla, CA) for the gift of the CRF1-ECD-activin IIB receptor chimera construct.



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 DISCUSSION
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