Expression, purification, and characterization of a soluble form of the first extracellular domain of the human type 1 corticotropin releasing factor receptor.

The first extracellular domain (ECD-1) of the corticotropin releasing factor (CRF) type 1 receptor, (CRFR1), is important for binding of CRF ligands. A soluble protein, mNT-CRFR1, produced by COS M6 cells transfected with a cDNA encoding amino acids 1--119 of human CRFR1 and modified to include epitope tags, binds a CRF antagonist, astressin, in a radioreceptor assay using [(125)I-d-Tyr(0)]astressin. N-terminal sequencing of mNT-CRFR1 showed the absence of the first 23 amino acids of human CRFR1. This result suggests that the CRFR1 protein is processed to cleave a putative signal peptide corresponding to amino acids 1--23. A cDNA encoding amino acids 24--119 followed by a FLAG tag, was expressed as a thioredoxin fusion protein in Escherichia coli. Following thrombin cleavage, the purified protein (bNT-CRFR1) binds astressin and the agonist urocortin with high affinity. Reduced, alkylated bNT-CRFR1 does not bind [(125)I-D-Tyr(0)]astressin. Mass spectrometric analysis of photoaffinity labeled bNT-CRFR1 yielded a 1:1 complex with ligand. Analysis of the disulfide arrangement of bNT-CRFR1 revealed bonds between Cys(30) and Cys(54), Cys(44) and Cys(87), and Cys(68) and Cys(102). This arrangement is similar to that of the ECD-1 of the parathyroid hormone receptor (PTHR), suggesting a conserved structural motif in the N-terminal domain of this family of receptors.

Corticotropin releasing factor (CRF), 1 a 41-amino acid peptide, was identified initially on the basis of its primary role in the activation of the hypothalamic-pituitary-adrenal in response to stress. The CRF family of ligands includes sauvagine from frog and urotensin from fish as well as the additional mammalian family members, urocortin (1,2), urocortin II (3,4), and urocortin III (4,5). Broader roles for CRF and its ligand family now involve effects on the cardiovascular, reproductive, gastrointestinal, immune, and central nervous systems (6 -8).
The action of CRF and related ligands is initiated by binding to their receptors, which transduce an increase in intracellular cAMP. Thus far, two receptors, CRFR1 and CRFR2, have been cloned in mammals (9 -15). Homologous receptors have been identified in chicken (16), fish (17), and Xenopus (18) and a third receptor, CRFR3, with high levels of sequence identity to CRFR1, has recently been cloned in catfish (17). Both CRFR1 and CRFR2 exist as multiple splice variants and belong to the type B 7-transmembrane receptor family that includes receptors for growth hormone releasing factor, secretin, calcitonin, vasoactive intestinal peptide, glucagon, glucagon-like peptide, and parathyroid hormone (PTH). Receptors for the CRF ligand family have been characterized in the central nervous system, pituitary, gastrointestinal tract, epididymis, heart, gonads, and adrenals (6).
The affinities of CRF and urocortin in binding to CRFR1 are nearly the same but in binding to CRFR2, urocortin is ϳ10 times more potent than CRF (19). Both urocortins II and III are highly selective in binding and activating CRFR2 compared with CRFR1 (4,5). A synthetic peptide antagonist, astressin, binds with equally high affinity to CRFR1 and CRFR2 (19,20).
The CRF receptor family consists of proteins with a relatively large first extracellular domain (ECD-1). Mutagenesis studies have identified regions of the CRF receptors that are implicated in differential recognition of agonists and antagonists, as well as in governing the ligand selectivity of the two types of receptors (21)(22)(23)(24)(25). We showed that a chimeric receptor in which the ECD-1 of CRFR1 replaced the ECD of the activin receptor, a single transmembrane receptor kinase (26), was capable of high affinity binding to both astressin and urocortin (21). The mode of receptor activation was explored by our study of a tethered peptide-receptor chimera in which the first 16 amino acids of CRF were substituted for the ECD-1 of CRFR1 (CRF (1-16)/R1 ⌬N ) (27). This chimera displayed continual signaling suggesting that the N-terminal third of CRF, when presented in proximity to the receptor, is able to cause activation.

mNT-CRFR1 Expression in COS M6 Cells
The cDNA encoding amino acids 1-119 of human CRFR1 (9) was modified by polymerase chain reaction to insert both a Myc-epitope following amino acid 31 and a FLAG-epitope following amino acid 119. This cDNA was subcloned into pSecTag2 HygroA (Invitrogen) (pSec-ECD-1). The integrity of the construct was confirmed by automated sequencing. Transfection of pSec-ECD-1 (ϳ10 g) into COS M6 (ϳ10 ϫ 10 6 cells/10-cm dish) was accomplished using the DEAE-dextran method (9). At 1, 3, and 5 days post-transfection, medium containing mNT-CRFR1 was collected and subjected to Western analysis using a FLAG antibody (Sigma). Medium from transfected COS M6 cells was enriched using FLAG-agarose (Sigma) immunoaffinity chromatography.

bNT-CRFR1 Expression in E. coli
A cDNA encoding amino acids 24 -119 of human CRFR1 (9) was modified by polymerase chain reaction to insert a FLAG epitope following amino acid 119. The cDNA was inserted into pET-32a(ϩ) (Novagen) with KpnI and XhoI (pET-ECD-1) and its integrity confirmed by automated sequencing. E. coli strain, Origami trxB gor (DE3)pLysS (Novagen) was chemically transformed according to manufacturers directions using pET-ECD-1. Bacteria were grown to an A 600 of 0.6 -1.0 and induced with isopropyl-1-thio-␤-D-galactopyranoside (1 mM) for 2 h at 37°C. The bacterial pellet was solubilized and sonicated in 10 mM Tris-HCl, pH 8 (10 ml/g weight pellet), yielding soluble bNT-CRFR1 as a thioredoxin fusion protein in the supernatant. This fusion protein was subjected to thrombin cleavage and enriched by affinity chromatography with S-protein-agarose (Novagen) prior to purification by reversedphase HPLC with a Vydac C 4 column (10 ϫ 250 mm, 5-m particle size, 300-Å pore size, number 214TP510) run in 0.05% trifluoroacetic acid with increasing concentrations of CH 3 CN as the mobile phase. After thrombin cleavage, bNT-CRFR1 contains 24 amino acids at the N terminus composed, in part, of an affinity epitope tag (S-tag) and 8 amino acids at the C terminus corresponding to the FLAG tag. The sequence of bNT-CRFR1 is GSGMKETAAAKFERQHMDSPDLGT[human CRFR1 24 -119 ]DYKDDDDK (the S-and FLAG-tags are underlined); the additional amino acids, GSG and DLGT, are part of the thrombin cleavage site and the multiple cloning site in the vector, respectively. In the HPLC purification, bNT-CRFR1 eluted at ϳ35% CH 3 CN.

Western Blot
Samples were reduced, electrophoresed through 15% SDS-polyacrylamide gels, and blotted onto polyvinylidene difluoride membranes. Antibody to the FLAG epitope (Sigma) was used to visualize the proteins.

Protein Sequence
Purified mNT-CRFR1 was deglycosylated (N-glycanase) before electrophoresis on 15% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride membrane. Bands detected via Western blot analysis in a parallel experiment were excised and subjected to automated Edman degradation.

Size Exclusion Chromatography
Lyophilized bNT-CRFR1 was dissolved in 25 mM NaHEPES, 0.1 M NaCl, pH 7.5, at 0.150 mg/ml and filtered by microcentrifugation through a microporous membrane (0.45-m Durapore ultrafree-MC Millipore/Amicon). The sample (ϳ100 l) was injected onto a TSK-GEL G2000SWXL column (7.8-mm inner diameter ϫ 30 cm, Toso Haas) equilibrated in the above buffer and eluted at a flow rate of 0.5 ml/min.

Disulfide Arrangement
Analysis of the disulfide arrangement was carried out employing procedures described in Ref. 36. In a typical reaction, 30 g of bNT-CRFR1 was dissolved in 100 l of 50 mM MES, pH 6.2. To this was added 1 g of enzyme (trypsin or endoproteinase Asp-N) and digestion was allowed to proceed at 37°C for 16 h. Peptide fragments were resolved on reversed-phase HPLC and analyzed by chemical sequence analysis on a PE-Applied Biosystems Procise 494 Protein Sequencer and by MALDI-MS on a Bruker Reflex time-of-flight instrument. The mass accuracy of this instrument is typically 1000 ppm.

Reduction and Alkylation of bNT-CRFR1
Purified bNT-CRFR1 (30 g) was dissolved in 20 l of 0.25 M Tris-HCl, pH 7.5, containing 6 M guanidinium hydrochloride and 2 mM EDTA. To the solution, 2 l of 10% aqueous 2-mercaptoethanol was added and reduction was allowed to proceed at 37°C for 30 min. To the reaction mixture was added 5 l of 4-vinylpyridine (20% in ethanol) and alkylation was carried out at 37°C in the dark for 45 min. The alkylated product was isolated after reversed-phase HPLC. Completeness of conversion to the alkylated product was verified by MALDI-MS analysis.

Radioreceptor Assays
Binding to Soluble Proteins-Partially purified mNT-CRFR1 was incubated in triplicate tubes with [ 125 I-D-Tyr 0 ]astressin (ϳ200,000 cpm) and increasing concentrations of unlabeled peptide in 0.2 ml of HDB (137 mM NaCl, 5 mM KCl, 0.7 mM NaH 2 PO 4 , 25 mM NaHEPES, pH 7.4), 0.1% bovine serum albumin overnight at room temperature. The ligandreceptor complex was precipitated with a monoclonal antibody to the Myc epitope in the presence of goat anti-mouse antibody, normal mouse serum, and polyethylene glycol as described (37), and the radioactivity was quantitated by ␥-counting. Purified bNT-CRFR1 was incubated in triplicate wells with [ 125 I-D-Tyr 0 ]astressin (ϳ200,000 cpm) together with increasing concentrations of unlabeled peptides in 0.2 ml of HDB, 0.1% bovine serum albumin for 90 min at room temperature in MAGV microtiter plates (Millipore) precoated with 0.1% polyethylenimine. The mixture was aspirated under vacuum and the plates were washed twice with HDB, 0.1% bovine serum albumin and counts bound in the wells were quantitated by ␥-counting.
Binding to Membrane Fractions-Transfection and binding to membrane fractions were performed as described (21). Background ccounts/ min (counts bound in absence of receptor) were subtracted from all counts/min. The K i for astressin and the B max values were determined by the LIGAND computer program; the K i for the photoactive analog was calculated by the PRISM (GraphPad) fitting program. All assays were performed at least three times.

Photoaffinity Labeling
Following incubation of the purified bNT-CRFR1 (100 g) with [D-Tyr-Gly 0 -Bpa 1 ]astressin (300 g) in 0.25 ml of 50 mM NaHEPES, pH 7.4, for 90 min at room temperature, the solution was irradiated at 365 nm for 30 min at room temperature. The products were then purified on HPLC and analyzed by MALDI-TOF and ESI on an ion trap instrument.

RESULTS
Initial experiments were directed at determining if a soluble form of the first extracellular domain of CRFR1 was capable of binding CRF family ligands. Because post-translational modifications, such as glycosylation, might influence protein folding and/or ligand recognition, we sought to express a soluble form of the ECD-1 in mammalian cells. A soluble protein, mNT-CRFR1, secreted by COS M6 cells was found to be glycosylated (data not shown) and bind astressin with K i ϭ 30 Ϯ 10 nM (n ϭ 3) (Fig. 1). The agonist urocortin displaces [ 125 I-D-Tyr 0 ]astressin bound to mNT-CRFR1 but another agonist, sauvagine, does not displace the bound radiolabeled antagonist. Based on N-terminal sequencing, mNT-CRFR1 was found to begin with residues SLQDQHCE. This sequence is C-terminal to the predicted signal peptide cleavage site (9 -11) indicating that amino acids 1-23 are proteolytically processed during maturation.
For further studies, another soluble form of the ECD-1 was expressed in a bacterial system. We used the information on the N-terminal sequence of mNT-CRFR1 to construct a cDNA which would encode a protein whose N terminus begins with Ser 24 . We also inserted a FLAG epitope after Ala 119 . This cDNA, cloned into the pET-32a(ϩ) vector to yield a thioredoxin fusion protein, was used to transform an E. coli strain which contains mutations in both thioredoxin reductase (trxB) and glutathione reductase (gor), thereby enhancing disulfide bond formation in the cytoplasm. After induction with isopropyl-1thio-␤-D-galactopyranoside and cell lysis, the protein, found as a fusion protein in the soluble fraction, was cleaved by thrombin, enriched by S-protein immunoaffinity chromatography and purified to near homogeneity by reversed-phase HPLC. The purified protein, bNT-CRFR1, visualized with Coomassie Blue stain following SDS-polyacrylamide gel electrophoresis, was found to migrate according to the predicted molecular size of ϳ14 kDa.
In order to characterize the purified protein, we analyzed its structure and behavior in solution by both size exclusion chromatography and circular dichroism and determined its arrangement of disulfides. Following lyophilization from trifluoroacetic acid/CH 3 CN, bNT-CRFR1 was readily solubilized either in water or in a near physiological buffer (25 mM Na-HEPES, 0.1 M NaCl, pH 7.5) at relatively high concentration (5 mg/ml). The UV absorption spectra (240 -340 nm) of dilutions of the samples showed no evidence of light scattering, i.e. absorption at wavelengths longer than 310 nm (data not shown). In addition, the protein samples were quantitatively recovered after being subjected to filtration through a 0.45-m microporous membrane which retains large protein aggregates. Most striking were the results of size exclusion chromatography. A single symmetrical peak with retention time consistent with those of other disulfide-containing proteins of similar masses was observed for bNT-CRFR1 (data not shown). No higher molecular weight species (aggregates) of the protein were found to elute prior to the monomeric form. Together, these observations indicate that the bacterially produced receptor is a relatively compact, folded protein that is soluble and monomeric under physiological conditions.
The UV circular dichroism spectra of bNT-CRFR1 ( Fig. 2A) at low or physiologic ionic strength are dominated by the random coil, with a negative ellipticity extreme at 199 nm with mean residue ellipticity of Ϫ18,000 degrees cm 2 /dmol and a positive ellipticity peak at 227 nm with mean residue ellipticity of ϩ2500 degrees cm 2 /dmol. Spectral deconvolution by the method of Bohm et al. (35) gives helical, antiparallel, parallel, ␤-turn, and random coil contributions of 7.8, 9.2, 3.1, 30.0, and 54.5%, respectively. Addition of TFE at 50% (v/v) to the low ionic strength bNT-CRFR1 solution resulted in the appearance of negative extrema at 223 and 208 nm and a positive peak at 192 nm. By inspection, this TFE-induced spectrum is highly helical; deconvolution gives helical, antiparallel, parallel, ␤-turn, and random coil contributions of 42.0, 2.3, 8.2, 22.1, and 38.5%, respectively. In order to determine if ligand facilitates the formation of secondary structural features of bNT-CRFR1, the protein was incubated with astressin at approximately equimolar concentrations, and the resulting CD spectrum (Fig.  2B) was compared with the arithmetic sum of the component spectra. The resulting difference CD spectrum (Fig. 2B, inset) clearly shows the formation of CD spectral features at 223, 208, and 198 nm which correspond to the n* and exciton-split * absorptions characterizing ␣-helix, respectively. Under the reasonable assumption that the micromolar concentrations used in the experiment in Fig. 2B ensure that all the receptor and ligand are complexed, the data are presented in the figure in units of mean residue ellipticity. Because the number of residues of bNT-CRFR1 is 132 and that of astressin is 32, at equimolar concentrations of bNT-CRFR1 and ligand ϳ80% of the observed CD signal is contributed by bNT-CRFR1. Undoubtedly, a small contribution to the change in secondary structure shown in Fig. 2B (inset) arises from changes in the structure of astressin upon complex formation but, because it is not possible to determine the individual contributions of receptor and ligand to the difference CD spectrum, the data in Fig.  2B (inset) are a strong qualitative indication of the effect of ligand binding on the structure of bNT-CRFR1.
In order to determine the disulfide pattern, bNT-CRFR1 was digested with trypsin under slightly acidic conditions to minimize disulfide exchange, and resulting fragments were separated by reversed-phase HPLC. We noticed that nonstandard cleavage (between Leu and Ser) occurred during subsequent Asp-N digestion which was probably due to the high enzyme to substrate ratio employed. Two major cystine-containing frag-

FIG. 1. Competitive displacement by astressin of [ 125 I-D-
Tyr 0 ]astressin bound to mNT-CRFR1. The data are from a representative assay. Background counts were subtracted from total counts bound. ments were identified (Fig. 3). One fragment, termed T1, was found to consist of two peptides that contained one cysteine each (Cys 68 and Cys 102 ). N-terminal sequence analysis yielded two sequences corresponding with CRFR1 58 -76 and CRFR1 97-110. The calculated mass of the disulfide-connected peptides was 3759.85 Da, in agreement with the observed mass (m/z ϭ 3757.4). We conclude that Cys 68 and Cys 102 are connected in the expressed protein. Another fragment (T2) was found to contain four cysteine residues. This fragment was further digested with endoproteinase Asp-N, an enzyme that proteolyzes peptide bonds N-terminal to Asp residues. The resulting fragments were again analyzed by N-terminal sequence analysis and MALDI-MS. Two pairs of disulfide-connected peptides were identified from this digest. One peptide corresponded with residues CRFR1 27-33 and CRFR1 49 - (Fig. 3).
By radioreceptor assay, the K i for astressin bound to bNT-CRFR1 is 50 Ϯ 6 nM (n ϭ 9). The binding affinity before thrombin cleavage and before HPLC purification is similar to that of the purified protein (data not shown). In displacing [ 125 I-D-Tyr 0 ]astressin bound to bNT-CRFR1, urocortin is less potent than astressin (Fig. 4A) and neither r/hCRF nor sauvagine displaces the bound astressin (Fig. 4B). The number of binding sites (B max ) derived from the radioreceptor assay is approximately equal to the number of sites calculated from the estimated quantity of purified protein and from a binding stoichiometry of 1:1 (B max /number of binding sites ϭ 140 Ϯ 30%, n ϭ 8). These data suggest that nearly all of the expressed protein is able to bind astressin.
The role of the six cysteines was explored by measuring the binding of the bNT-CRFR1 following reduction and alkylation. There is no significant binding of [ 125 I-D-Tyr 0 ]astressin to the reduced, alkylated bNT-CRFR1 (Fig. 5A). A mutant CRFR1 in which the first 40 amino acids are deleted (CRFR1 ⌬(1-40) ) binds astressin with an affinity approximately equal to that of the wild type receptor (Fig. 5B).
The binding of the photoactive analog [D-Tyr-Gly 0 -Bpa 1 ]astressin to bNT-CRFR1 is shown in Fig. 6 from which it is seen that the analog displays ϳ5 times greater affinity (K i ϭ 10 Ϯ 5 nM) than astressin. The affinity of [D-Tyr-Gly 0 -Bpa 1 ]astressin for the wild type receptor is nearly equal to that of astressin (data not shown). Upon light activation, there is a specific labeling of the bNT-CRFR1 as shown by SDS-polyacrylamide gel electrophoresis and mass spectrometry following HPLC purification (Fig. 7). The stoichiometry of the interaction of the photoactive astressin with bNT-CRFR1 is found to be 1:1 by mass spectrometry. DISCUSSION The receptors for CRF and related ligands belong to the type B family of 7-transmembrane G-protein-coupled receptors (GPCRs). These represent the largest number of proteins that recognize and transduce extracellular signals, resulting in responses as diverse as vision and embryonic development (38). Obtaining structural information on these complex proteins has presented such formidable problems that, at present, the only three-dimensional structure of a complete GPCR is that of rhodopsin (39). Information on the binding sites and structure of other GPCRs has been derived from analyses of the functional effects of mutations and also from molecular modeling using the rhodopsin structure as the starting point (40).
Using mutational analyses, residues that affect binding of peptide or non-peptide antagonists as well as the selectivity of various CRF agonists have been identified in the extracellular and transmembrane domains of CRF receptors (22)(23)(24)(25)(41)(42)(43). All of the extracellular domains contribute, in some degree, to the binding of the CRF peptide ligands. Interestingly, the ECD-1 of the Xenopus CRFR1 suffices to determine ligand specificity (23). Of special relevance for this work was our demonstration that a chimeric receptor in which the ECD-1 of CRFR1 replaced the ECD of the single transmembrane domain activin receptor displays high affinity binding for both a CRF antagonist and agonist (21). This observation prompted us to initiate a structural characterization of the isolated ECD-1 of CRFR1 with the goal of providing information on this important receptor-binding region.
Most GPCRs lack an N-terminal signal peptide sequence (44) and are classified as type IIIb integral membrane proteins (45). Our N-terminal sequence data on mNT-CRFR1, the ECD-1 expressed by mammalian cells, shows that the CRFR1 protein has a cleavable N-terminal signal sequence, as predicted from its the primary structure (9), and thus, is correctly classified as a type IIIa integral membrane protein.
In order to obtain a greater amount of soluble protein more economically, we expressed the ECD-1 of CRFR1 in E. coli (bNT-CRFR1), and found binding characteristics similar to those of the protein expressed by the mammalian cells. In both cases, the K i values of astressin and urocortin for the bNT- CRFR are ϳ5-10-fold lower than those for the wild type receptor and also than those for the CRFR/activin-R chimera (21). The reduced affinity of astressin and urocortin for bNT-CRFR1 may be due to the absence of ligand interactions with the plasma membrane (46). In addition, it is possible that a higher entropic price is required for binding of two soluble proteins compared with that for binding a protein anchored in the membrane. This has been suggested, for example, for the interaction of a soluble portion of CCR5 with gp 120 (47), for which the affinity is reduced by a factor of 10 3 -10 4 . Consistent with our data, another study of a soluble ECD-1 of CRFR1 expressed in E. coli reported only low affinity binding to ovine CRF (43). The fact that both sauvagine and r/hCRF do not compete with astressin for binding to the bNT-CRFR1 may be due to the fact that binding of these two agonists requires coupling of the receptor to G-proteins. Since astressin is an antagonist, such interactions with G-proteins are not necessary for its high affinity binding. The fact that urocortin, an agonist, binds to bNT-CRFR1 is consistent with the observation that urocortin displays some antagonist binding characteristics. For example, guanyl nucleotides do not modulate the binding of urocortin to the wild type receptor (19,48), as they do the binding of CRF (49). In addition, ligands like sauvagine and CRF may require other extracellular or transmembrane domains for their high affinity binding.
The observation that the CD spectra of bNT-CRFR1 at both low and physiological ionic strength near neutrality is effectively that of a random coil suggests either denaturation under the conditions of the spectropolarimetric data collection or a highly mobile structure capable of large shifts with relatively low energy barriers between conformers. The latter suggestion is much more consistent with the observation of the induction of helical structure upon the addition of TFE or ligand and rules out irreversible denaturation. Presumably, bNT-CRFR1 is structured or perhaps samples a small number of related structures as it complexes with ligand during the binding reaction. Of necessity, the solution composition of the radioreceptor assay precludes its use directly for the CD spectral work. The incubation of bNT-CRFR1 with astressin in high ionic strength CD buffer results in the formation of the ␣-helical structure in the protein (Fig. 2B, inset). Another example of a conformational change following hormone binding was deduced from the CD spectrum of follicle stimulating hormone bound to a soluble form of the ECD of the follicle stimulating hormone receptor (28). The functional importance of the six conserved cysteine residues in the CRF receptor family was inferred from the disruption of binding and signaling by receptors in which those residues were mutated (50). Abolition of observable astressin binding following reduction and alkylation of the disulfide bonds in bNT-CRFR1, yet maintenance of binding following deletion of amino acids 1-40 in the wild type CRFR1, suggests that the disulfide bridge between Cys 30 and Cys 54 is not required, but that the remaining disulfides are required for binding astressin. The disulfide bonds restrict the number of conformations that bNT-CRFR1 can assume while allowing a degree of flexibility of the protein as evidenced by the absence of secondary structure under low-salt conditions in the absence of ligand or TFE. The CD spectra measured in the presence of ligand or TFE indicate that the flexible portions of the molecule become more ordered under these conditions. The connectivity pattern we have proposed, namely bonds between the pairs of cysteines 30 and 54, 44 and 87, and 68 and 102, differs from that deduced from mutational analysis (50), but the pattern we propose is identical to that described for the ECD-1 of the PTH receptor (31).
Other examples in which the N-terminal domain of a GPCR binds ligand are the metabotropic glutamate receptor (33,34,51), the luteinizing hormone/human chorionic gonadotropin receptor (29), the follicle stimulating hormone receptor (28), the pituitary adenylate cyclase-activating polypeptide receptor (52), the PTH receptor (31), and the glucagon-like peptide-1 receptor (32), the last two also being members of the receptor subfamily to which the CRF receptor belongs. The glucagonlike peptide-1R, expressed and purified as a soluble protein in Chinese hamster lymphoblastoma cells, exhibited lower binding affinity compared with the native receptor, and lost activity after reduction of the disulfide bonds (32). The ECD-1 of the PTHR was expressed in inclusion bodies in E. coli from which the protein was isolated and refolded. The binding affinity of the purified soluble ECD-1 of the PTHR was reduced by ϳ1000fold compared with that of the full-length receptor (31). Our system, using a thioredoxin reductase and glutathione reductase double mutant E. coli strain, eliminated the necessity of refolding the protein.
In the case of the metabotropic glutamate receptor, it was found that the ECD-1 forms a disulfide linked dimer, but the dimer persists even after reduction of the disulfide bond (34,51). The 1:1 stoichiometry for astressin binding to the bNT-CRFR1 is the same as that reported for the PTH binding to the ECD-1 of the PTHR determined by isothermal titration calorimetry (31). In our system, all of the expressed protein appears to be functional since ϳ100% is capable of binding the ligand.
In conclusion, we have shown that the disulfide arrangement of the cysteines in the ECD-1 of CRFR1 is identical to that of the PTHR, another member of the receptor subfamily, supporting the hypothesis that the N-terminal extracellular domain of the proteins in this family share a common topology. Furthermore, the binding of an antagonist induces a conformational change in the soluble ECD-1. Previously we demonstrated that a tethered peptide-receptor chimera, CRF (1-16)/R1 ⌬N , is constitutively activated (27), and in this work we have found that the isolated ECD-1 binds a CRF analog lacking 11 N-terminal residues. These observations are consistent with a model for CRFR1 activation in which the ECD-1 first captures a CRF ligand by binding its C-terminal residues. Being spatially constrained in a suitable proximity to the receptor, the ligand is then able to initiate signaling by presentation of its activating N-terminal residues to the receptor.