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Molecular Endocrinology 11 (13): 2048-2053
Copyright © 1997 by The Endocrine Society

Localization of Agonist- and Antagonist-Binding Domains of Human Corticotropin-Releasing Factor Receptors

Chen W. Liaw, Dimitri E. Grigoriadis, Marge T. Lorang, Errol B. De Souza and Richard A. Maki

Departments of Molecular Neurobiology and Neuroscience Neurocrine Biosciences, Inc. San Diego, California 92121


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The CRF receptors, CRFR1 and CRFR2, are members of the G protein-coupled receptor superfamily. Despite their considerable sequence similarity, CRFR1 and CRFR2 have quite different affinities for the peptide ligand rat/human CRF. Previous studies using chimeric receptors between human CRFR1 and CRFR2 have identified three potentially important regions in the second and third extracellular domains of CRF receptor for the binding of rat/human CRF. The present report further demonstrates that these same three regions also affect the binding of urocortin and sauvagine, two other members of the CRF peptide family, albeit to different extents. We also show that a fourth region in the third extracellular domain, Asp254, has been identified to be important for sauvagine but not CRF or urocortin binding. Thus, the three peptide ligands not only interact with a different set of regions on CRFR1 and CRFR2 but also differentially interact with some of the same regions. These data could, at least in part, account for the much higher affinity of CRFR2 for urocortin and sauvagine compared with rat/human CRF. We have also identified two amino acid residues, His199 in the third transmembrane domain and Met276 in the fifth transmembrane domain, that are important for binding the non-peptide high-affinity CRFR1 antagonist NBI 27914. Mutations of His199 and Met276 to the corresponding amino acids in CRFR2 each decreased the binding affinity of NBI 27914 for CRFR1 by 40- and 200-fold, respectively. This suggests that the transmembrane regions are critically important in forming the binding pocket for the non-peptide antagonist.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRF is the principal regulator that integrates the body’s endocrine, autonomic, and behavioral responses to stress (1, 2, 3). The CRF peptide family consists of CRF itself (4), fish urotensin I (5), frog sauvagine (6), and the recently characterized mammalian urocortin (7, 8). CRF mediates its actions by binding to high-affinity membrane receptors that are coupled to Gs and transduces the signal through an increase in the intracellular level of cAMP. Two CRF receptors (CRFR1 and CRFR2) with approximately 70% sequence identity and distinct tissue distribution pattern and pharmacology have been cloned and characterized (9, 10, 11, 12, 13, 14, 15, 16, 17). Radioligand binding and second messenger studies show that CRFR1 has comparable high affinity for CRF, urocortin, and sauvagine, while CRFR2 binds urocortin and sauvagine with much higher affinity than CRF (8).

By taking advantage of the large difference in the affinity of CRF for the two CRF receptors, we previously generated and analyzed a series of chimeric receptors between human CRFR1 and CRFR2. In these studies, we identified three regions that are potentially important for rat/human CRF (r/hCRF) binding (18). Here we have further examined the roles of these regions in binding sauvagine and urocortin and demonstrated that they have different relative contributions in binding the three peptide ligands. We have also identified a fourth region in the third extracellular domain, Asp254, which is important for sauvagine binding yet has little effect on r/hCRF or urocortin binding when mutated to Glu, the corresponding amino acid (aa) in CRFR2.

In situ hybridization studies have shown that CRFR1 and CRFR2 mRNA each have a distinct distribution pattern in the brain (19), suggesting the two receptor subtypes might have distinct functional roles. This differentiation of functions by distinct receptor subtypes allows the development of subtype-specific non-peptide ligands that have the therapeutic potential to target different aspects of CRF-mediated disorders with minimal side effects. To aid in the design of such subtype-specific non-peptide ligands, it is important to understand the molecular interactions between receptors and the ligands. Recently, we have described the synthesis of a series of non-peptide antagonists for the CRFR1 receptor (20). These antagonists are highly selective for the CRFR1 receptor subtype and have no affinity for the CRFR2 subtype. In the present study, we have identified two aa within the transmembrane domains (TMs) that are important for binding one of these antagonists termed NBI 27914 (see compound 3b in Ref.20), suggesting that TMs are important in forming at least part of the binding pocket for the non-peptide ligands.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have previously identified three regions in the human CRF receptor that are potentially important for binding the peptide ligand r/hCRF (18). One is at the junction of the third extracellular domain and fifth TM involving three consecutive aa, Val266, Tyr267, and Thr268; the other comprises two regions mapped to the second extracellular domain involving aa 175–178 and His189 residue (Fig. 1Go). Since the other two members of the CRF peptide family, urocortin and sauvagine, have only modest sequence identity to r/hCRF (~45%) (7) and have much higher affinity for CRFR2 than r/hCRF, we thought it was important to examine whether the three regions described above were also important for the binding of urocortin and sauvagine.



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  		Figure 1. Schematic Model of the hCRF Receptor

Sequence alignment of CRFR1 and CRFR2 are shown between EC2 and TM5 with the aa of CRFR1 shown on the left and those of CRFR2 shown on the right. The conserved aa are shown in solid circles and divergent aa are shown in open symbols. The regions that are important for binding all three peptide ligands r/hCRF, urocortin, and sauvagine, are shown in squares; the region that is important for binding sauvagine only is shown as a diamond; the two aa that are important for NBI 27914 binding are shown in triangles. The numbers indicate the aa positions of those residues flanking the postulated TMs. All numberings are based on the sequence of CRFR1.

 
The two CRF receptors (CRFR1 and CRFR2) have more than 2 orders of magnitude difference in binding affinity for r/hCRF. Each of the three regions described above, when mutated from CRFR1 to the corresponding CRFR2 sequences, shifted the apparent EC50 value in stimulating intracellular cAMP by ~7- to 10-fold (18). As shown in Table 1Go, each of the three regions caused a ~2- to 3-fold shift in the apparent EC50 value for urocortin when changed from the CRFR1 to the corresponding CRFR2 sequences. These results suggest that either these three regions play a less significant role relative to the rest of the molecule in binding urocortin than binding r/hCRF or that these specific CRFR1 to CRFR2 sequence changes in the three regions are more compatible with urocortin binding than r/hCRF binding. Overall, the relatively minimal change in EC50 value for urocortin caused by chimeric mutation in each of these three regions is consistent with the fact that the two CRF receptors have a more similar affinity for urocortin (~7-fold difference) than for r/hCRF (~360-fold difference).


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  		Table 1. EC50 of r/hCRF, Urocortin and Sauvagine in Stimulation of Intracellular cAMP for CRFR1, CRFR2, and the Three Chimeric Receptors that have been shown to have decreased affinity for r/hCRF

 
For sauvagine, the EC50 value of CRFR2 was ~6-fold higher than that of CRFR1 (Table 1Go). Unexpectedly, the D266L267V268 triple mutation alone caused an almost 50-fold shift in EC50 value for sauvagine (Table 1Go). This same triple mutation increased the EC50 value for r/hCRF by a much smaller magnitude (~10-fold), suggesting that either aa 266–268 plays a more significant role in sauvagine binding than r/hCRF binding, or that this particular triple mutation (CRFR1 to CRFR2 sequence) decreased the binding affinity for sauvagine more so than for r/hCRF.

The Arg to His mutation at aa 189, in conjunction with the D266L267V268 mutations (H189DLV), increased the EC50 value for sauvagine by another 10-fold (Table 1Go), a magnitude similar to that for r/hCRF (~8-fold); the EC50 value of the chimeric receptor R1174R2178DLVR1 for sauvagine was only slightly higher (~1.5-fold) than that of D266L267V268 mutant, a relatively small change compared with that for r/hCRF (~7-fold). Previously, we have shown that while aa 266–268 plays a primary role in securing the binding of r/hCRF, the roles of His189 and aa 175–178 appear to be secondary and become significant only in the presence of the D266L267V268 mutations (18). This also seems to be the case for sauvagine and urocortin binding since both H189 and R1174R2178 R1 mutants have EC50 values for sauvagine and urocortin comparable to those of CRFR1 (Table 1Go).

The fact that the D266L267V268 triple mutation alone had significantly higher EC50 value (lower binding affinity) for sauvagine than CRFR2 (Table 1Go), suggests that some other CRFR1 to CRFR2 sequence change(s) is (are) capable of rescuing or reverting part of the affinity decrease for sauvagine caused by the D266L267V268 mutations. To localize such a rescuer region, the EC50 value for sauvagine of the chimeric receptor R1228R2268R1, in which aa 229–265 of CRFR1 was replaced by the corresponding CRFR2 sequence, in conjunction with the D266L267V268 triple mutation, was determined and found to be 12-fold lower than that of D266L267V268 mutant (Table 2Go), indicating that the rescuer is located between aa 229 and aa 265.


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  		Table 2. EC50 of Sauvagine and r/hCRF in Stimulation of Intracellular cAMP for the Chimeric Receptors Containing D266L267V268 Mutations

 
To further map the rescuer region, two chimeric receptors, R1228R2238DLVR1 and R1238R2268R1, were constructed and assayed. The chimeric receptor R1228R2238DLVR1, with aa 229–238 of CRFR1 replaced by the corresponding CRFR2 sequences in conjunction with the D266L267V268 mutations, had very similar EC50 value to the D266L267V268 mutant (Table 2Go); on the other hand, the R1238R2268R1 chimeric receptor, with aa 239–265 replaced by the corresponding CRFR2 sequence in conjunction with the D266L267V268 mutations, had an EC50 value of 0.3 nM (Table 2Go), significantly lower than that of the D266L267V268 mutant and similar to that of R1228R2268R1. These results suggest that the rescuer is located between aa 239 and aa 265, where there are three aa differences between CRFR1 and CRFR2, i.e. aa 254, aa 257, and aa 263 (Fig. 1Go). Each of these three aa was then individually mutated from CRFR1 to the corresponding CRFR2 aa in conjunction with the D266L267V268 mutations. As shown in Table 2Go, while Q257DLV and E263DLV still have significantly higher EC50 values than R1228R2268R1, E254DLV had an EC50 value of 0.7 nM, which is 10-fold lower than that of the D266L267V268 mutant and is comparable to the EC50 values of R1228R2268R1 (0.6 nM) and R1238R2268R1 (0.3 nM).

For r/hCRF and urocortin, we did not detect the rescuing effect described above, i.e. the EC50 values of the D266L267V268 mutant were lower than those of CRFR2 (Table 1Go) and comparable to those of the chimeric receptor R1228R2268R1 (Table 2Go). Thus, it is not surprising that the same Asp254 to Glu254 mutation had relatively little effect on the EC50 values of E254DLV for r/hCRF and urocortin (compare the EC50 values of E254DLV and D266L267V268 in Table 2Go).

The Asp254 to Glu254 mutation increased the binding affinity of the D266L267V268 mutant for sauvagine by 10-fold, implicating the importance of aa 254 in sauvagine binding. This was further confirmed by analyzing a mutant receptor with a CRFR2 backbone D254R2, in which the Glu residue of CRFR2 (corresponding to CRFR1 aa 254) was mutated to Asp (for consistency in nomenclature, the numbering of aa of CRFR2 is based on the CRFR1 sequences). With a single conservative aa substitution by eliminating one carbon from the side chain of aa 254, D254R2 had an EC50 value of 5.8 ± 0.9 nM (n = 3), 6.5-fold higher than that of CRFR2. It is possible that the negatively charged carboxyl group of Glu interacts with a basic functional group on sauvagine such that a change in the length of the side chain (Glu to Asp mutation) changes the effective distance of interaction and thus lowers the binding affinity.

We have recently described the synthesis of a series of CRFR1-specific CRF receptor antagonists (20). One of these antagonists NBI 27914 (see compound 3b in Ref.20) has a Ki value in the low nanomolar range for CRFR1 and no affinity for the CRFR2 subtype (20). To initially identify some of the aa residues involved in NBI 27914 binding, we transiently transfected VIP2.0Zc cells with CRFR1 and two chimeric receptors R2188R1 (where the N-terminal 188 aa of CRFR1 was replaced by corresponding CRFR2 sequences) and R1334R2 (where the C-terminal sequence of CRFR1 after aa 334 was replaced by the corresponding CRFR2 sequences), all of which have about the same affinity for r/hCRF (EC50 values are 0.16, 0.26, and 0.10 nM, respectively). We then measured the inhibition of r/hCRF-stimulated cAMP production by NBI 27914 for the three transfectants and determined that both R2188R1 and R1334R2 appeared to have approximately the same affinity for NBI 27914 as CRFR1 (data not shown), suggesting that some aa differences between CRFR1 and CRFR2 within the aa 188 (beginning of TM3) to aa 334 (end of TM6) region are responsible for the CRFR1 selectivity of NBI 27914.

To determine more precisely which aa residues are important for the binding of NBI 27914, some CRFR1 to CRFR2 point mutations within TM3, TM4, and TM5 (CRFR1 and CRFR2 have identical aa sequence for TM6) were introduced into the CRFR1 receptor. All these mutants have approximately the same EC50 values for sauvagine in stimulating intracellular cAMP (0.09–0.18 nM) as CRFR1. The apparent affinity of NBI 27914 for these mutants was determined by direct measurement of [125I]sauvagine binding. None of the six mutants with a single aa substitution within TM4, i.e. C229, L230, L232, F233, C237, and I238 significantly altered the affinity for NBI 27914 (data not shown). However, two point mutations, one involving a His to Val mutation at aa 199 in TM3 (mutant V199), and the other a Met to Ile mutation at aa 276 in TM5 (mutant I276), reduced the affinity of NBI 27914, while having no effect on the ability of r/hCRF to inhibit [125I]sauvagine binding. Figure 2Go demonstrates that the apparent affinity of NBI 27914 was shifted by approximately 40- and 200-fold, in the two mutants V199 and I276, respectively. In the stable cell line expressing the native CRFR1 receptor subtype, the apparent affinity of NBI 27914 in inhibiting [125I]sauvagine binding was 17 ± 1.5 nM, and this was decreased to 750 ± 82 nM and 4162 ± 140 nM in the V199 and I276 mutants, respectively (see Fig. 2Go). Thus, these two residues appear to be either directly interacting with a critical binding site on NBI 27914 or are required to maintain the local conformation of the binding pocket for NBI 27914. These results also suggest that the binding domain of NBI 27914 is likely to be at least partially incorporated within the transmembrane regions. The lack of a molecular model for CRF receptor makes it difficult to predict whether NBI 27914 is large enough to also interact with some aa residues in the extracellular domains. Unfortunately, all mutations in the extracellular domains introduced thus far also affect the binding affinity for the peptide ligands, making measurement of the relative binding affinity of NBI 27914 for these mutants difficult. Future studies using radiolabeled non-peptide CRFR1 antagonists may help elucidate the absolute binding requirements. It is noteworthy however, that for all non-peptide ligands of G protein-coupled receptors whose binding domains have been characterized so far, whether the natural ligands are small molecules such as biogenic amines or peptides such as neurokinins, it is the TMs of the receptor that form the major binding sites (for review see Ref.21).



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  		Figure 2. Competition of r/hCRF and NBI 27914 for Native CRFR1 and I276 and V199 Mutant Receptors

Competition of r/hCRF (top panel) or NBI 27914 (bottom panel) for [125I]sauvagine binding was performed using radioligand binding studies described. The apparent affinity of r/hCRF (top panel) for sauvagine binding remained unaltered with apparent inhibition constant (Ki) values of 3.5 ± 0.6 nM, 4.0 ± 0.6 nM, and 5.6 ± 0.3 nM for the native CRFR1 receptor subtype (squares) and the two mutant receptors expressed transiently, the V199 mutant (triangles) and the I276 mutant (circles), respectively. In contrast, the apparent affinity of NBI 27914 (bottom panel) was drastically reduced in the mutants from 17 ± 1.5 nM in the native receptor (squares) to 750 ± 82 nM and 4162 ± 140 nM in the V199 (triangles) and I276 (circles) mutants, respectively. The graphs are representative of triplicate values from three independent determinations. In all cases, analysis of the competition data using nonlinear least squares curve fitting indicated binding to a single homogeneous class of binding sites.

 
Since NBI 27914 is much smaller in size than r/hCRF, it is likely that the non-peptide antagonist only interacts with a subset of those domains that are involved in peptide ligand binding. The fact that His199 and Met 276 are important for NBI 27914, but not r/hCRF binding, and the competitive nature of NBI 27914 antagonism (our unpublished results) raise the intriguing possibility that there is some overlap in the binding pockets for NBI 27914 and r/hCRF such that binding of the two ligands becomes mutually exclusive. A similar phenomenon has been described for the neurokinin receptor NK1, where mutations of His197 in TM5 and His265 in TM6 reduced the affinities for non-peptide competitive antagonists while those same mutations had no effect on the binding of peptide ligand substance P (22, 23).

In summary, the present study demonstrates that the three regions of CRF receptor previously identified to be important for r/hCRF binding are also important for urocortin and sauvagine binding. However, the contributions of these three regions appear to be different among the three peptide ligands. This can at least partially account for the different affinities of CRFR2 for the three ligands. In addition, a fourth region was identified to be important for sauvagine, but not r/hCRF or urocortin binding, suggesting that different peptide ligands, despite their sequence similarity, may not interact with the same set of molecules on the receptor. We have also identified two aa residues, His 199 and Met276, that are important for binding the non-peptide antagonist NB I27914. Both of these aa residues are located within TMs, suggesting that TMs are important in forming the binding pocket for the non-peptide antagonists.

Finally, although the chimeric receptor approach has been used extensively to localize regions that are important for ligand binding, it is important to understand the limitations of such an approach. First, it does not address the significance of conserved aa residues. Second, with any specific chimeric receptor, although a lack of observed effects on ligand binding might suggest a noncritical role for the nonconserved region(s) that has gone through the chimeric substitution, it is also possible that the particular chimeric sequence change is as compatible with binding the ligand as is the native receptor. Thus, the chimeric receptor approach used in conjunction with specific point mutations within critical regions of the protein are required to elucidate the importance of certain regions of the receptor in binding the ligands.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
All chimeric and mutant receptors were constructed and transiently expressed in LVIP2.0Zc cells (24), a cell line containing a cAMP-responsive ß-galactosidase reporter gene as described (18). The EC50 values of peptide ligands in increasing the levels of intracellular cAMP for various receptors were then determined by assaying the ß-galactosidase activity as described (18, 25). To measure inhibition of r/hCRF-stimulated intracellular cAMP by NBI 27914, VIP2.0Zc cells were transiently transfected with various receptors and incubated with 0.6 nM r/hCRF, which caused ~ 70–80% of maximal cAMP induction in the absence or presence of 0.1–10 µM NBI 27914.

The binding affinities of NBI 27914 and r/hCRF for the expressed CRFR1 and various transmembrane mutant receptors were determined using [125I]-Tyr0-sauvagine binding in the presence of varying concentrations of unlabeled ligands as described previously (26). All drugs and reagents were made up in assay buffer (PBS containing 10 mM MgCl2, 2 mM EGTA, and 0.15 mM bacitracin, pH 7.0, at 22 C). Eppendorf tubes received in order, 100 µl buffer (with or without competing r/hCRF or NBI 27914), 50 µl of [125I]-Tyr0-sauvagine (final concentration 100–200 pM), and 150 µl membrane suspension for a total assay volume of 300 µl. The assay was incubated at equilibrium for 2 h at 22 C. Reactions were terminated by centrifugation in a Beckman microfuge for 10 min at 12,000 x g. The resulting pellets were washed gently with 1 ml of ice-cold PBS containing 0.01% Triton X-100 and centrifuged again for 10 min at 12,000 x g. The supernatants were aspirated and the tubes cut just above the pellet and placed into 12 x 75-mm polystyrene tubes and monitored for radioactivity in a Packard Cobra II {gamma}-counter at approximately 80% efficiency. Data were analyzed using the iterative nonlinear least-squares curve-fitting program Prism (GraphPad Inc., San Diego, CA). Competition curves were routinely fit to single- and multiple-site models, and the fits were compared statistically to determine whether a more complex data model was justified.


   ACKNOWLEDGMENTS
 
We thank Drs. Michael Brownstein and Monika König for providing the LVIP2.0Zc cell line, and Dr. Wylie Vale for the human CRFR1 cDNA clone. We also thank Dr. Nick Ling for synthesizing the peptide ligands, Dr. Chen Chen for synthesizing NBI 27914, and Guy Barry for sequencing the mutant and chimeric receptor clones.


   FOOTNOTES
 
Address requests for reprints to: Dr. Chen W. Liaw, Departments of Molecular Neurobiology and Neuroscience, Neurocrine Biosciences, Inc., 3050 Science Park Road, San Diego, California 92121.

This work was supported in part by SBIR Grants R43 NS34203 and R44 NS33489–02.

Received for publication August 27, 1997. Accepted for publication September 17, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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