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The First Extracellular Domain of Corticotropin Releasing Factor-R1 Contains Major Binding Determinants for Urocortin and Astressin¹

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037-1099

Address all correspondence and requests for reprints to: Marilyn Perrin, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037-1099. E-mail: perrin{at}salk.edu

	Abstract

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The CRF receptors are members of a 7-transmembrane receptor family that includes GH-releasing hormone (GRF), calcitonin, vasoactive intestinal peptide (VIP), secretin, and PTH receptors. To determine the structural features of the CRF receptor that may influence ligand recognition, a series of mutant receptors was analyzed for binding to astressin, a CRF antagonist, and to urocortin, a CRF agonist. Mutant receptors included chimeras between the CRF-R1 and GRF-R or Activin IIB-R, a single membrane spanning receptor serine/threonine kinase. Binding to the mutant receptors was assessed using ¹²⁵I-[DTyr¹] astressin (Ast*) and ¹²⁵I-[Tyr⁰]-rat urocortin (Ucn*). There was no binding to a chimeric receptor in which the first extracellular domain (E1_c) (i.e. the N-terminal region) of the CRF-R1 was replaced by that of the GRF-R. The complementary chimera in which E1 domain of the GRF-R was replaced by that of the CRF-R1 bound astressin and urocortin with K_i values approximately 10 nM, compared with inhibitory binding dissociation constant (K_i) values of approximately 2–4 nM for the wild-type CRF-R1. The chimera in which E1 of the activin IIB receptor was replaced by E1 of the CRF-R1 bound astressin with a K_i approximately 4 nM. A chimera in which both the first and fourth extracellular domains of the CRF-R1 replaced the corresponding domains of the GRF-R bound astressin with K_i approximately 4 nM and urocortin with a K_i approximately 2 nM. A chimera in which all four extracellular domains of the CRF receptor replaced those of the GRF-R bound astressin and urocortin with K_i values approximately 4 nM and approximately 1 nM, respectively. In conclusion, the major determinants for high affinity binding of CRF agonists and antagonists to CRF-R1 are found in the first extracellular domain of the receptor.

	Introduction

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IN ADDITION to its major role in the regulation of the hypothalamic-pituitary-adrenal axis, CRF displays many effects in the central nervous, reproductive, immune, and cardiovascular systems. The actions of CRF are initiated by binding and activation of receptors on its target tissues. The first CRF receptor gene to be identified encodes CRF-R1, a receptor that is expressed in the pituitary (1, 2, 3), the gonads (2, 4), and the central nervous system (5, 6). A second CRF receptor gene was cloned that encodes two isoforms CRF-R2 (7) and CRF-R2ß (8, 9, 10). In the rat, CRF-R2 is found mainly in the central nervous system (6), whereas CRF-R2ß is expressed predominantly in peripheral tissues such as the heart, muscle, epididymis, and the gastrointestinal tract (8, 9, 10). Both CRF receptors are members of a 7-transmembrane domain receptor family that includes receptors for calcitonin, vasoactive intestinal peptide, secretin, and GH-releasing factor (GRF).

Overall, CRF-R1 and CRF-R2 are approximately 68% homologous but approximately 80% homologous in the transmembrane domains and identical in the third intracellular loop that is assumed to be important for interactions with G proteins. The significant differences in the sequences between the two receptor types, as well as between the two splice variants of CRF-R2, occur in their extracellular domains.

An important question deals with the structural features that may be involved in ligand binding and subsequent signaling of these receptors. There are extensive data on the structural requirements for binding and signaling of other peptide as well as nonpeptide hormone receptors, including those that are characterized by 7-transmembrane domains and are coupled to G proteins. For the small nonpeptide hormones such as adrenaline and related agents as well as for the small peptide hormone, TRF, the binding domains are proposed to be in the transmembrane domains (11, 12). The receptor’s extracellular domains are important for binding ligands like acetylcholine and GABA (13) and the large glycoprotein hormones such as follicle stimulating hormone (14). More relevant to the CRF-R are the GRF, calcitonin, PTH, VIP, and secretin receptors that appear to contain major binding determinants in their extracelullar domains (15, 16, 17, 18).

Another question to be considered is whether agonists and antagonists bind to different regions of the receptor. For the tachykinin receptors, a difference has been found between the binding domains for agonists and antagonists (19). Another example is that of the opioid receptors, for which the fourth extracellular domain was found to determine the selectivity of the -opioid agonists (20).

Functional analyses of mutant receptors have provided insight into the structural requirements for receptor-ligand interactions. The goal of the work presented here was to use mutational analysis as a tool to investigate the roles of the various domains of the CRF-R in binding CRF ligands. We created chimeric receptors between the CRF-R and GRF or activin receptors to study the effect of the receptor’s extracellular domains on the binding CRF ligands.

	Materials and Methods

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Mutagenesis
Chimeric mutant receptors were created using PCR. The choice of transmembrane domains and extracellular loops was based on the published sequences. When exchanging the N-terminal domains of the CRF and GRF receptors a small portion of the first transmembrane domain was also included [residues 1–127 from rCRF-R1 (21) or 1–137 from the rGRF-R (22)]. For the chimera involving the CRF-R and activin type IIB-R, residues 1–118 of the former were joined to residues 111–494 of the latter (23). The extracellular domains 2, 3, and 4 were taken to be residues 172–189, 250–266, and 335–347, respectively, for rCRF-R (21) and 182–203, 264–280, and 350–360, respectively for the rGRF-R (22).

For example, to create the E1_c/GRF-R chimera, an upstream primer (I) complementary to nucleotides encoding amino acids nos. 1–7 of the rCRF-R and a downstream primer (II) whose 3'-end was complementary to nucleotides coding for amino acids 121–125 of the rCRF-R and whose 5' end was complementary to nucleotides encoding amino acids 136–141 of the rGRF-R were used to amplify the rCRF-R in a first round of PCR. The second round of PCR used the amplified product from the first round together with a downstream primer (III) complementary to nucleotides encoding amino acids 418-stop of the rGRF-R to amplify the rGRF-R. The final PCR product was ligated into pcDNA3 (Invitrogen) using BamHI and XhoI restriction sites which were included in primers (I) and (III), respectively.

Chimeras in which the other extracellular domains were exchanged were created by using PCR and E1_c/GRF-R as the template. For example, for the E1_c/E2_c/GRF-R, the first round of PCR used the pair of primers: (I) and (IV) to amplify E1_c/GRF-R and the pair of primers (III) and (V) to amplify the rGRF-R.

The sequences of the primers were:

(I): 5'-GATCGGATCCATGGGACGGCGCCCGCAGCTC

(II): 5'-CAATGGAGATGCTGTGGCCCAGGTAGTTGATGATG

(III): 5'-GATCCTCGAGCTAGCACTCAGAGGTGAGCAC

(IV): 5'-TTGCTCTGGTGCACCTCGGGGCTCACGGTGAGCTGGA-CCAGGAACACAGCACTGG

(V): 5'-GCCCCGAGGTGCACCAGAGCAATGTGGCCTGGTGTAG-GGTCTCTGTGGCCGTCTC

The two PCR products were then annealed and amplified using primers (I) and (III), and the product of this reaction was finally ligated into pcDNA3. Sequences were confirmed by dideoxy sequencing.

Membrane fractions
Crude membrane fractions were prepared from transiently transfected COSM6 cells as previously described (1, 24) and stored in 10% sucrose at -80 C until use. Protein concentrations were determined with the Biorad assay kit using -globulin as standard.

Peptide radioiodination
The peptides, [DTyr¹]Astressin (cyclo(30–33)[yHLLREVLEXARAEQLAQEAHKNRKLXEII-amide) (where X = Nle and y = DTyr) and [Tyr⁰]rUcn (YDDPPLSIDLTFHLLRTLLELARTQSQRERAEQNRIIFDSV) were synthesized as described in (25), radioiodinated using the chloramine-T (Sigma Chemical Co., St. Louis, MO) method and purified by HPLC as previously described (24, 25, 26).

Receptor binding assays
The RRAs were performed in a manner similar to that previously described (24). Crude membrane fractions (10 µg-200 µg protein) were combined with 50,000–120,000 cpm Ast¹ or Ucn¹ and peptide competitors in assay buffer (10 mM MgSO₄, 0.075% BSA, 7.5% Sucrose, and 1.75 mM EGTA) for 2 h at 20 C. Reactions were performed in 96-well MultiScreen plates (Millipore, Bedford, MA) with GF/C filters, prewetted with 0.1% polyethyleneimine, for Ast¹ binding or with 0.22 µ Durapore filters, prewetted with assay buffer for Ucn¹ binding. The reaction was terminated by aspiration through the plate, followed by 0.2 ml wash with assay buffer. Inhibitory binding dissociation constants (K_i) values and their 95% confidence limits were determined from at least three independent homologous competitive displacement assays. The data from the experiments were pooled and analyzed with the LIGAND computer program (27).

	Results

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To assess the characteristics of the chimeric receptors, the binding of both astressin (CRF antagonist) and urocortin (CRF agonist) was measured. The K_i values were estimated from homologous competitive displacement assays using either Ast¹ or Ucn¹ as the radioligands. In Fig. 1, we show an example of the homologous competitive displacement of Ast¹ bound to the wild-type and selected chimeric receptors. The results for all the mutants are listed in Table 1.

View larger version (14K): [in this window] [in a new window]   Figure 1. Competitive displacement by astressin of Ast* bound to COSM6 cells transfected with either (A) wild-type rCRF-R or (B) chimeric receptors. Data are from a representative assay replicated at least twice. B = cpm bound; B0 = cpm bound in absence of competing peptide. Wild-type receptor (); E1c/GRF-R (); E1c/E2c/GRF-R (•); E1c/E4c/GRF-R (); E1c/E2c/E4c/GRF-R (); E1c/E2c/E3c/E4c/GRF-R ().

The effects of the other extracellular domains on CRF binding were investigated by creating another series of chimeras. One chimera, E1_g/CRF-R, was made by replacing the N-terminal domain of the CRF-R by that of the GRF-R; this chimera lacked the first extracellular domain of the CRF-R but retained extracellular domains 2–4 as well as its transmembrane and intracellular regions. Other chimeras were constructed so that the second to fourth extracellular domains in the E1_c/GRF-R chimera were successively replaced by the corresponding domains of the CRF-R. Extracellular domains 2–4 were substituted separately (e.g. E1_c/E2_c/GRF-R), in pairs (e.g. E1_c/E2_c/E3_c/GRF-R), or all three together (E1_c/E2_c/E3_c/E4_c/GRF-R).

There was no detectable specific binding of either Ast¹ or Ucn¹ to E1_g/CRF-R. There was specific, displaceable binding of Ast¹ to all the other chimeras and binding of Ucn¹ to most of them (Table 1). In chimeras containing E3_c, the specific binding/mg protein was lower for Ucn¹ than for Ast¹; in two cases, E1_c/E3_c/GRF-R and E1_c/E2_c/E3_c/GRF-R, the specific binding of Ucn¹ was so small that it was not possible to determine, accurately, the K_i for urocortin using Ucn¹.

Inclusion of the second extracellular domain, (E1_c/E2_c/GRF-R), did not affect the K_i values of astressin or urocortin compared with their values for E1_c/GRF-R itself. Expression of E3_c did not change appreciably the K_i for astressin. The chimera that included both the first and fourth extracellular domains, (E1_c/E4_c/GRF-R), exhibited lower K_i values of both analogs (K_i = 4.2 nM for astressin and K_i = 1.6 nM for urocortin) compared with the K_i values for E1_c/GRF-R (10 nM).

The expression of E1_c together with pairs of other extracellular domains yielded the following results: the combination of extracellular domains 2 and 4 or 3 and 4 resulted in K_i values for both astressin and urocortin that were not significantly different from K_i values for E1_c/GRF-R alone. The chimera containing the pair of domains 2 and 3, (E1_c/E2_c/E3_c/GRF-R) bound astressin with a K_i = 5.4 nM, a value that was slightly lower than that for E2_c or E3_c separately. Although this chimera showed no usable specific binding of Ucn¹, urocortin was able to displace Ast¹.

A chimera expressing all four extracellular domains of the CRF-R, E1_c/E2_c/E3_c/E4_c/GRF-R, bound both Ast¹ and Ucn¹, and the K_i values for were 4.3 nM and 1.3 nM for astressin and urocortin, respectively. These values were practically the same as those for the chimera E1_c/E4_c/GRF-R.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The family of receptors to which the CRF-R belongs includes those for GRF, secretin, calcitonin, VIP, and PTH. The ligands for this family are comparatively large peptides (e.g. 41 amino acids for CRF); their cognate receptors span the membrane seven times and possess relatively large extracellular domains, containing eight conserved cysteine residues, six of which are in the N-terminal domain. The comparatively large size of both the ligands and the extracellular domains suggests that the extracellular regions of the receptor contain determinants for ligand-receptor interactions. Indeed, there is a growing body of data in support of this idea (15, 16, 17, 18). To investigate the roles of the extracellular domains of the CRF-R1 in binding CRF ligands, we have created a series of chimeras between the CRF-R1 and either the GRF-R or the activin receptor and have studied their binding to a CRF agonist, urocortin and to a CRF antagonist, astressin.

In this study, we divided the receptor into relatively large domains, namely the N-terminal portion and the extracellular loops. To investigate the contribution to the binding of CRF ligands of the first extracellular domain, E1_c, (i.e. the N-terminus) we created two chimeras: in one, E1_c took the place of the corresponding region of the GRF-R (E1_c/GRF-R) so that the GRF-R contributed extracellular domains 2–4 with which E1_c might interact. To eliminate these interactions we created another chimera in which no such interactions were possible, namely E1_c/ActIIB-R, in which E1_c of the CRF-R replaced the extracellular domain of the activin IIB receptor. The activin receptor is a serine/threonine kinase that has only one transmembrane and intracellular domain (23).

The data from E1_c/GRF-R and E1_c/ActIIB-R suggested that the first extracellular domain of the CRF-R contained major binding determinants for both the CRF agonist and antagonist. There was significant specific binding of astressin and urocortin to both E1_c-expressing chimeras. Indeed, the observation that the K_i for astressin binding to E1_c/ActIIB-R was no higher than that for binding to E1_c/GRF-R suggested that the extracellular loops of the GRF-R that were present in the latter did not contribute significantly to the ligand recognition. Using a similar approach, Osuga et al. (18) fused the E1 of the LH or FSH receptors to the single transmembrane domain of the CD8 receptor and obtained high affinity ligand binding (18).

The roles of the other extracellular domains of the receptor were explored by creating mutants in which E2_c-E4_c were successively substituted for the corresponding domains of the GRF-R that were present in the E1_c/GRF-R chimera. Inclusion of extracellular domains 2 or 3, or both together, produced chimeras that bound astressin and urocortin with K_i values approximately 5–10 nM, i.e. not much different from the corresponding K_i values obtained for the E1_c/GRF-R chimera in which there were no other CRF extracellular regions. Inclusion of E4_c resulted in K_i values for both astressin and urocortin that were lower than those for E1_c/GRF-R, although the difference was not statistically significant in the case of urocortin.

Our data may be compared with those obtained from the secretin/VIP chimeric receptors (17, 28). For high affinity secretin binding, the first extracellular domain alone was not sufficient but required both the first and second extracellular domains, whereas the first extracellular domain of the VIP receptor sufficed to produce characteristic VIP responses. Studies with calcitonin/glucagon chimeras suggested that the N-terminal domain was important for binding calcitonin (29). The data for the PTH receptor were similar to those for CRF-R in that the first and fourth extracellular domains were found to be critical for ligand binding (16).

In conclusion, we have shown that for CRF-R1 the extracellular domains, specifically the N-terminus, contained important binding determinants for both CRF antagonists and agonists.

	Acknowledgments

 
We wish to thank Dr. K. Mayo for the GRF-R cDNA. We wish to acknowledge helpful discussions with Dr. A. Hsueh, technical assistance by L. Cervini, R. Kaiser, C. Miller, and J. Vaughan, and manuscript preparation by D. Dalton, S. Guerra, and D. Johns.

	Footnotes

 
¹ This work was supported by NIH Grant DK-26741, the Foundation for Medical Research, Inc. (to M.P. and W.W.V.), and the Foundation for Research (to D.B. and W.W.V.).

² FMR, Inc., and FFR senior principal investigator.

Received October 1, 1997.

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