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J. Biol. Chem., Vol. 277, Issue 36, 32558-32561, September 6, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/36/32558    most recent M204964200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Assil-Kishawi, I. Articles by Abou-Samra, A. B. Search for Related Content PubMed PubMed Citation Articles by Assil-Kishawi, I. Articles by Abou-Samra, A. B. Social Bookmarking                      What's this?

Sauvagine Cross-links to the Second Extracellular Loop of the Corticotropin-releasing Factor Type 1 Receptor^*

Iman Assil-Kishawi and Abdul B. Abou-Samra

From the Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Received for publication, May 20, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radioligands [Tyr⁰,Gln¹]SVG (¹²⁵I-YQS) and [Tyr⁰,Gln¹, Leu¹⁷]SVG (¹²⁵I-YQLS) were examined. ¹²⁵I-YQLS or ¹²⁵I-YQS was cross-linked to CRFR1 using the chemical cross-linker, disuccinimidyl suberate (DSS), which cross-links the  amino groups of lysine residues that have a molecular distance of 11.4 Å. DSS specifically and efficiently cross-linked ¹²⁵I-YQLS and ¹²⁵I-YQS to CRFR1. CRFR1 contains 5 putative extracellular lysine residues (Lys¹¹⁰, Lys¹¹¹, Lys¹¹³, Lys²⁵⁷, and Lys²⁶²) that can cross-link to the 4 lysine residues (Lys¹⁶, Lys²², Lys²⁵, and Lys²⁷) of the radioligands. Identification of the CNBr-cleaved fragments of CRFR1 cross-linked to ¹²⁵I-YQLS or ¹²⁵I-YQS established that the second extracellular loop of CRFR1 cross-links to Lys¹⁶ of YQS. Additionally, site-directed mutagenesis (changing Lys to Arg in CRFR1 individually and in combination) revealed that Lys²⁵⁷ in the second extracellular loop of CRFR1 is an important cross-linking site. In conclusion, it was shown that in SVG-bound CRFR1, Lys²⁵⁷ of CRFR1 lies in close proximity (11.4 Å) to Lys¹⁶ of SVG.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Corticotropin-releasing factor (CRF),¹ sauvagine (SVG), urocortin-I (UCN-I), and urotensin-I (UTS) bind to the CRF receptors, CRFR1 and CRFR2, previously characterized from various species. Recently, two UCN-like peptides, UCN-II and UCN-III (or stresscopin and stresscopin-related peptide), which bound specifically to CRFR2 and not to CRFR1, were characterized (1-4). The CRF receptors belong to group B of the G-protein-coupled receptors, which contains 6 conserved cysteine residues and several N-linked glycosylation sites at their amino termini. Activation of the CRF receptors results in stimulation of adenylate cyclase (2, 5-8) and phospholipase C (9).

Recently, receptor mutagenesis has been extensively used to map ligand-binding sites on the CRF receptors (10-15). It has been shown that residues within the second extracellular loop and the juxtamembrane region are important for ligand binding (14). In this study we used the recently characterized oxidation-resistant SVG analog, [Tyr⁰,Gln¹,Leu¹⁷]SVG (YQLS), which binds to CRFR1 and CRFR2 with high affinity and which can be cross-linked to CRF receptors with high efficiency (16), to map cross-linked residues. The results showed that Lys¹⁶ of SVG and Lys²⁵⁷ of CRFR1 (in the second extracellular loop) are within a molecular distance of 11.4 Å of each other.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Unless indicated, all chemicals were purchased from Sigma Chemicals. Na¹²⁵I was purchased from PerkinElmer Life Sciences. The peptides, hCRF-(1-41) (CRF), [Tyr⁰,Gln¹]SVG (YQS), and [Tyr⁰,Gln¹,Leu¹⁷]SVG (YQLS) were synthesized in the Massachusetts General Hospital Biopolymer Facility. They were HPLC-purified and analyzed by mass spectroscopy, amino-terminal sequencing, and acid hydrolysis. SVG was obtained from Bachem (King of Prussia, PA) and disuccinimidyl suberate (DSS) was purchased from Pierce. Tissue culture media were from the Massachusetts General Hospital Media Facilities (Boston, MA). Centricon 30 centrifugal filters were purchased from Millipore (Bedford, MA). The molecular mass markers were purchased from Amersham Biosciences.

Cell Culture-- COS-7 cells were transiently transfected with the murine CRFR1 and various CRFR1 Lys to Arg mutants at 90% confluency using the DEAE-dextran method (15). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 200 units/ml penicillin, and 20 µg/ml streptomycin sulfate. The cells were cultured at 37 °C in a humidified atmosphere in 95% air, 5% CO₂.

Site-directed Mutagenesis-- Site-directed mutagenesis was performed as described previously (16). Single and multiple Lys to Arg mutations were introduced into CRFR1 cloned in the pcDNA1 plasmid at the following sites: 257, 262, 257 and 262, 110 and 111, and 110, 111, and 113; the resulting mutants were named K257R, K262R, K257R/K262R, K110R/K111R, and K110R/K111R/K113R; respectively. All mutations were confirmed by DNA sequencing.

Measurement of Receptor Expression on the Cell Surface-- Nine of the ten c-Myc epitope tag sequences (QKLISEEDL) was introduced within the amino-terminal domain of mCRFR1 between Glu³¹ and Ser³² (17). Stably transfected LLCPK-1 cells (90-95% confluent) and transiently transfected COS-7 cells in 24-well plates (72 h after transfection) were rinsed with phosphate-buffered saline (PBS) containing 5% heat-inactivated fetal bovine serum and incubated with the 9E10 monoclonal antibody (17) at 1:1000 dilution. The cells were incubated for 2 h at room temperature, rinsed with PBS, and incubated for 2 more hours with ¹²⁵I-labeled sheep anti-mouse immunoglobulin G (200,000 cpm/well) diluted in PBS, 5% fetal bovine serum. The supernatant was removed, and then the cells were washed and lysed with 1 N NaOH. The cell lysates were collected and counted in a Micromedic gamma counter.

Radioligand Binding to Intact COS-7 Cells-- Binding assays were performed as described previously (16, 17). Intact COS-7 cells transiently transfected with mCRFR1 or mCRFR1 mutants were plated into 24-well plates, and a binding assay was performed when cells reached 90-95% confluency. The cells were rinsed with a Tris-based binding buffer (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 2 mM CaCl₂, 5 mM KCl, 5% heat-inactivated horse serum, 0.5% heat-inactivated fetal bovine serum). ¹²⁵I-YQLS (100,000 cpm/well), prepared and purified as previously described (14), was added in the presence of increasing concentrations of the competing peptide for 2-4 h at room temperature. The cells were then rinsed three times with binding buffer and lysed with 1 N NaOH (0.25 ml, 3×). The cell lysates were collected, and the radioactivity was counted in an automated gamma counter.

Chemical Cross-linking of ¹²⁵I-YQLS or ¹²⁵I-YQS to CRFR1 and CRFR1 Mutants-- Cells expressing wild type or mutant receptors were plated in 24-well plates and allowed to reach 90-95% confluency. The cells were then rinsed with PBS. ¹²⁵I-YQLS or ¹²⁵I-YQS (1,000,000 cpm/well) was added in HEPES binding buffer (25 mM HEPES, pH 7.6, 125 mM NaCl, 5 mM KCl, 5% heat-inactivated horse serum, 0.5% heat-inactivated fetal bovine serum) for 2-4 h at room temperature. The buffer was removed, the cells were rinsed with PBS to remove bound tracer, and DSS (0.5 mM) was added to the cells in PBS (pH 8.2) for 20-30 min. The cells were then rinsed with PBS, lysed with SDS sample buffer, and analyzed on a 5-20% SDS-polyacrylamide gel. After electrophoresis the gels were dried and autoradiographed using x-ray film or a phosphorimager screen.

cAMP Stimulation Assay-- COS-7 cells were plated in 24-well plates and were allowed to reach 90-95% confluency. The cells were chilled on ice and rinsed with ice-cold PBS. The cells were then challenged with YQLS at different concentrations in Dulbecco's modified Eagle's medium containing 2 mM 3-isobutyl-1-methylxanthene (IBMX), 1 mg/ml bovine serum albumin, and 35 mM HEPES, pH 7.4 at 37 °C for 15 min. The medium was then removed, and the cells were rapidly frozen on dry ice. Intracellular cAMP was extracted by thawing the cells in 1 ml of 50 mM HCl, and cAMP was then measured using radioimmunoassay (18).

Cyanogen Bromide (CNBr) Peptide Cleavage-- After autoradiography, the radioactive protein bands were cut, and the proteins were electroeluted in 1× SDS running buffer (25 mM Tris base, 192 mM of glycine, and 0.1% SDS). The eluate was concentrated using a Centricon 30 unit (Millipore). CNBr (100 mM) was added to the sample and dissolved in 70% formic acid at room temperature for 24 h. CNBr and formic acid were removed by lyophilization (3×). The proteins were analyzed on a 5-20% gradient SDS gel or on Tricine/SDS-PAGE (12% acrylamide) according to the method of Schägger and von Jagow (19) and autoradiographed using x-ray film or a phosphorimager screen.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

DSS Cross-links ¹²⁵I-YQLS but Not ¹²⁵I-CRF to CRFR1-- ¹²⁵I-CRF and ¹²⁵I-YQLS bind to CRFR1 with total specific binding of about 6 and 13%, respectively. As reported previously (16), addition of DSS caused covalent cross-linking of ¹²⁵I-YQLS to mCRFR1. No cross-linking occurred with ¹²⁵I-CRF. In contrast, ¹²⁵I-YQLS specifically cross-linked to cells expressing CRFR1 but not to cells transfected with the pcDNA1 plasmid (Fig. 1A). The cross-linked receptor was resolved as a broad band of ~80 kDa, which is consistent with the predicted molecular mass of the glycosylated CRFR1 (Fig. 1A). Addition of increasing concentrations of unlabeled SVG buffer decreased the ¹²⁵I-YQLS-CRFR1 cross-linked band in a concentration-dependent manner (Fig. 1B). These data indicate that ¹²⁵I-YQLS but not ¹²⁵I-CRF cross-links to CRFR1 efficiently and specifically.

View larger version (71K): [in this window] [in a new window]   Fig. 1.   Chemical cross-linking of CRFR1 to 125I-YQLS in the presence of DSS (0.5 mM). A, COS-7 cells transfected with CRFR1 plasmid or with vector alone were incubated with the 125I-YQLS. B, COS-7 cells transfected with the CRFR1 cDNA were incubated with 125I-YQLS and increasing concentration of SVG. C, COS-7 cells transfected with CRFR1 and CRFR1 mutant plasmids were incubated with 125I-YQLS. The cells were incubated for 2 h at room temperature until maximal binding was achieved. Excess unbound ligand was removed, and the cells were rinsed with PBS. DSS (0.5 mM) was then added for 20-30 min at room temperature. The cells were then lysed with SDS loading buffer, and equal amounts of radioactivity were analyzed on gradient 5-20% SDS-PAGE. The gel was autoradiographed for 24 h.

Binding and Cross-linking of ¹²⁵I-YQLS to CRFR1 Bearing Lysine to Arginine Mutations-- Because DSS cross-links free amino groups lying at a molecular distance of 11.4 Å; the efficient cross-linking of ¹²⁵I-YQLS to mCRFR1 provides an opportunity to determine which lysine residues in the ligand and the receptor are in close proximity to each other. Therefore we mutated the putative extracellular Lys to Arg residues in mCRFR1 and generated the following mutants: K257R, K262R, K257R/K262R, K110R/K111R, and K110R/K111R/K113R. The mutant receptor plasmids were transiently transfected into COS-7 cells, and cell surface expression was determined using a double antibody binding assay to the epitope tag. Expression of K257R, K262R, K257R/K262R, K110R/K111R, and K110R/K111R/K113R was 90.1 ± 0.4, 81.0 ± 1.9, 72.8 ± 0.6, 90.3 ± 0.5, and 82.6 ± 1.4% of that of wild type (Table I). Specific binding of ¹²⁵I-YQLS to K262R was similar to that of CRFR1 (Table I). In contrast, specific binding of ¹²⁵I-YQLS to K110R/K111R and K110R/K111R/K113R was 245.9 ± 5.3 and 157.0 ± 2.9% of that of CRFR1 (Table I); whereas its specific binding to K257R and K257R/K262R was 56.1 ± 0.5 and 40.6 ± 2.6% of that of CRFR1, respectively (Table I). All mutants exhibited comparable apparent K_i values (Fig. 2A and Table I). YQLS increased cAMP accumulation in cells transfected with K110R/K111R, K110R/K111R/K113R, and K262R with an R_max that was 79.9 ± 17.3, 47.1 ± 1.3, and 54.4 ± 4.2% of that observed in cells transfected with the wild type CRF receptor (Fig. 2B and Table I). Additionally, YQLS increased cAMP accumulation in cells transfected with K110R/K111R, K110R/K111R/K113R, and K262R with an EC₅₀ that was similar to that observed in cells transfected with the wild type CRF receptor (Fig. 2B and Table I). In contrast, the EC₅₀ of YQLS in cells transfected with K257R and K257R/K262R was greater than that observed in cells transfected with the wild type CRF receptor (Fig. 2B and Table I), and their R_max was 31.6 ± 4.7 and 45.2 ± 5.8% of the wild type (Fig. 2B and Table I). The binding and the cAMP data suggest that the lysine residue at position 257 is an important residue for SVG interaction.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Binding and signaling characteristics of the different lysine to arginine (R) CRFR1 mutants. A, COS-7 cells transfected with the wild type or mutant CRFR1 were incubated with 125I-YQLS for 2 h at room temperature in the presence of increasing concentrations of SVG. Specific binding percent was calculated and plotted. The data are presented as mean ± S.D. of triplicates of one of two similar experiments. B, stimulation of intracellular cAMP accumulation by YQLS in COS-7 cells expressing the wild type receptor or mutant CRFR1. The cells, in 24-well plates, were incubated with increasing concentrations of the peptides at 37 °C for 15 min in the presence of IBMX (2 mM). Intracellular cAMP was extracted and measured by specific radioimmunoassay. The data are presented as mean ± S.D. of triplicates of one of two similar experiments.

The different CRFR1 mutants were then cross-linked to ¹²⁵I-YQLS using DSS; the cells were lysed, and the labeled receptors were analyzed on a gradient SDS-PAGE followed by autoradiography. The cross-linked mutant receptors appeared as diffuse bands that were similar in size to that of the wild type receptor (Fig. 1C). Cross-linking efficiency to the mutant receptors was evaluated by loading equal amounts of radioactivity of cell lysates on SDS-PAGE. The intensity of the cross-linked mutated receptors, with the exception of that of K257R, was similar to or more than that of the wild type (Fig. 1C). This indicates that none of the Lys residues at positions 110, 111, 113, and 262 participates in the cross-linking reaction. In contrast, Lys²⁵⁷ in the second extracellular loop (EC2) appears to be an important cross-linking site.

Identification of the CRFR1 Cross-linking Domain-- To confirm that the EC2 region is involved in the cross-linking reaction, CNBr-cleavage of wild type and mutant receptors was used. CRFR1 contains several methionine residues in its backbone; among them Met²³⁰ and Met²⁷⁶ flank the predicted cross-linking site, Lys²⁵⁷. CNBr cleavage is predicted to generate a 46-residue fragment from CRFR1 cross-linked to the 41-residue radioligand, ¹²⁵I-YQLS. The predicted size of the cross-linked fragment (87 residues) is ~9 kDa (the CNBr-cleaved fragments are schematically represented in Fig. 3C). To resolve the small CNBr-cleaved fragments, equal amounts of radioactivity of the cross-linked receptor were loaded on high resolution Tricine/SDS-PAGE (19). As predicted, CNBr cleavage of ¹²⁵I-YQLS-cross-linked CRFR1 produced a major ~ 9-kDa band and a less intense 11-kDa band (Fig. 3A, CRFR1). The intensity of the 11-kDa band was variable from experiment to experiment and represents incomplete CNBr cleavage. The intensity of the ~ 9-kDa band decreased dramatically in K257R as compared with the wild type (Fig. 3A, R257), confirming that Lys²⁵⁷ is an important cross-linking site. Additionally, the ~9-kDa fragment in K262R had a similar intensity to that of the wild type (Fig. 3A, R262), indicating that Lys²⁶² is not an important cross-linking site. However the ~9-kDa band disappeared completely in K257R/K262R indicating that Lys²⁶² participates in the cross-linking reaction only when Lys²⁵⁷ is mutated (Fig. 3A, R257/R262). These data identify the EC2 region as a cross-linking site.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   CNBr cleavage of CRFR1 mutants cross-linked to 125I-YQLS or 125I-YQS. A, Tricine-PAGE analysis of CNBr-cleaved wild type CRFR1 and CRFR1 bearing Lys to Arg mutations cross-linked to 125I-YQLS. Cross-linking and SDS-PAGE were performed as described in the legend to Fig 1; the ~80-kDa band was cut from an SDS-PAGE gel, electroeluted, and CNBr-cleaved. The cleaved products were lyophilized and reconstituted in the SDS sample buffer containing 6% -mercaptoethanol. Equal amounts of radioactivity were analyzed on Tricine-PAGE followed by autoradiography. The molecular size markers are (from bottom up): insulin chain A (2.5 kDa), insulin chain B (3.5 kDa), aprotinin (6.5 kDa), lysozyme (14.3 kDa), trypsin inhibitor (20 kDa), and carbonic anhydrase (30 kDa). The positions of the ~9- and ~11-kDa CNBr fragments and the free ligand are shown on the right side. B, Tricine-PAGE analysis of CNBr-cleaved wild type CRFR1 and CRFR1 cross-linked to 125I-YQLS or 125I-YQS. The conditions are similar to those of panel A. Note that the presence of the methionine residue at position 17 in YQS decreased the size of the CNBr-cleaved receptor fragment from ~9 to ~6 kDa. Similarly the size of the free ligand decreased from ~5 to less than ~2 kDa. C, scheme showing the predicted CNBr-cleaved fragments and the location of the extracellular lysine (K) residues in the murine CRFR1. D, scheme of CNBr cleavage of 125I-YQS and 125I-YQLS showing the positions of the label (125I), methionine residue in YQS (M17) and the lysine (K) residues in both peptides (K16, K22, K25, and K27).

To positively identify the 9-kDa band a M230L mutant was constructed by changing the methionine residue at position 230 to leucine. The M230L mutant had an expression level and specific binding that were 85.3 ± 5.2 and 167.4 ± 0.9% of the wild type values, respectively (Table I). The apparent binding affinity was also similar to that of the wild type receptor (Table I). Additionally, YQLS increased cAMP accumulation in cells transfected with M230L with an EC₅₀ that was similar to that observed in cells transfected with the wild type CRF receptor (Table I). M230L was found to cross-link to ¹²⁵I-YQLS efficiently (Fig. 1C). The Leu²³⁰ mutation increased the size of the cross-linked CNBr-cleaved fragment from ~9 to ~11 kDa. The size of the CNBr fragment in M230L is identical to the less intense ~11-kDa band seen in the wild type, consistent with an increase in the size of the fragment by 25 residues (CNBr cleavage occurs at Met²⁰⁵ instead of Met²³⁰; Fig. 3C, M²³⁰).

Identification of the Sauvagine Cross-linking Residue-- To identify the Lys residue within sauvagine that cross-links to the second extracellular domain of CRFR1, ¹²⁵I-YQS was used as the radioligand. ¹²⁵I-YQS contains a native methionine residue at position 17 that could be cleaved by CNBr (Fig. 3D). The ¹²⁵I-YQS radioligand is radiolabeled on Tyr⁰ (at position zero). CNBr cleavage of ¹²⁵I-YQS is predicted to result in a 17-amino acid fragment bearing the ¹²⁵I label on Tyr⁰ and containing one lysine residue (Fig. 3D, K¹⁶). If Lys¹⁶ is cross-linked to CRFR1, a labeled receptor fragment of 63 residues is predicted (17 residues from YQS and 46 residues from CRFR1). In contrast, if cross-linking occurs through the other lysine residues of SVG (Lys²², Lys²⁵, Lys²⁷) then the receptor fragment is not labeled with ¹²⁵I. Analysis of the CNBr-cleaved, ¹²⁵I-YQS-cross-linked CRFR1 with high resolution Tricine-PAGE revealed a labeled band with a size of ~6 kDa (Fig. 3B). This band is smaller than the ~9-kDa band produced from CRFR1 cross-linked to ¹²⁵I-YQLS (Fig. 3A). The difference in size between the ~6- and ~9-kDa bands reflects CNBr cleavage of SVG at Met¹⁷; this identifies Lys¹⁶, the only Lys residue in the cleaved fragment, as the cross-linking residue.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Photoaffinity cross-linking of radiolabeled [Tyr⁰]ovine CRF to rat CRFR1 was previously reported; however, the cross-linked residues were not characterized (20). We also reported that the oxidation-resistant sauvagine radioligand, ¹²⁵I-YQLS, which exhibits high affinity binding to the CRF receptors, cross-links efficiently to mCRFR1 through 1 (or more) of the 4 lysine residues of the ligand (positions 16, 22, 25, and 26) to 1 (or more) of the lysine residues of the receptor clustered at the juxtamembrane region (positions 110, 112, and 113) and/or the second extracellular loop (positions 257 and 262) (16). The data presented in this study indicate that Lys¹⁶ of the ligand preferentially cross-links to Lys²⁵⁷ on the EC2 of mCRFR1. When Lys²⁵⁷ is mutated to Arg, the other Lys residue in EC2, Lys²⁶², substitutes for Lys²⁵⁷, indicating that ligand binding to CRFR1 is not rigid and that residue 16 of CRFR1-bound YQS is within 11.4 Å from Lys²⁵⁷ more often than from Lys²⁶² of CRFR1.

It is interesting to note that the single mutation of Lys²⁵⁷ to Arg and the double mutation of Lys²⁵⁷ and Lys²⁶² to Arg result in mutant receptors with decreased binding activity and decreased cAMP accumulation efficiency. This suggests that Lys²⁵⁷ may be important for ligand-receptor interaction. In contrast, none of the other mutants has a decreased binding activity. The decreased binding of Lys²⁵⁷ is consistent with data from human CRFR1 and CRFR2 hybrids showing important regions for SVG interaction in the EC2 (Asp²⁵⁴) and at the junction of the EC2 with the fifth transmembrane domain (12, 13, 16).

The nature of bimolecular interactions of polypeptide ligands with their cognate receptors from group B of G-protein-coupled receptors has been recently investigated. Polypeptide ligands, such as CRF and PTH, exhibit strong helical tertiary structures (21, 22). Important binding determinants, both in the amino terminus as well as the carboxyl terminus of the ligands, have been characterized. In general, cross-linking studies identified residues within the carboxyl terminus of the ligands that lie within close proximity to the amino terminus of their respective receptors and residues within the amino terminus of the ligands that lie within close proximity to the extracellular loops and the transmembrane domains of their respective receptors. However, differences were uncovered in some receptors. For instance, residues 1 and 13 of PTH-(1-34) cross-linked to Met⁴²⁵ in the third extracellular domain and Arg¹⁸⁶ in the juxtamembrane region that is amino-terminal to transmembrane 1 of the PTH receptor type 1, respectively (23, 24). Surprisingly, residues 6, 22, and 26 of secretin cross-linked to the most distal part of the amino terminus of the secretin receptor at positions Val⁴, Leu¹⁷, and Leu³⁶, respectively (25), and residue 23 of PTH cross-linked to a region between residues 23 and 40 of the PTH receptor type 1 (26). Our data that show that residue 16 of SVG and residue 257 of CRFR1 are in close proximity to each other indicate that the residues in close proximity in ligand-bound CRFR1 are different from those found in the other members of this family of G-protein-coupled receptors. These results should facilitate modeling of ligand-receptor interaction in CRF receptors.

	FOOTNOTES

^* This work was supported by Grant DK45020 from the NIDDK, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Endocrine Unit, Massachusetts General Hospital, Boston, MA 02114. Tel.: 617-726-6723; Fax: 617-726-1703; E-mail: samra@helix.mgh.harvard.edu.

Published, JBC Papers in Press, May 23, 2002, DOI 10.1074/jbc.M204964200

	ABBREVIATIONS

The abbreviations used are: CRF, corticotropin-releasing factor; SVG, sauvagine; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PTH, parathyroid hormone; DSS, disuccinimidyl suberate; IBMX, 3-isobutyl-1-methylxanthene; m, mouse.

	REFERENCES

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