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Molecular Endocrinology 18 (1): 105-116
Copyright © 2004 by The Endocrine Society

Position of Pro and Ser near Glu7.32 in the Extracellular Loop 3 of Mammalian and Nonmammalian Gonadotropin-Releasing Hormone (GnRH) Receptors Is a Critical Determinant for Differential Ligand Selectivity for Mammalian GnRH and Chicken GnRH-II

Chengbing Wang, Oim Yun, Kaushik Maiti, Da Young Oh, Kyeong Kyu Kim, Chong Hak Chae, Chang Jun Lee, Jae Young Seong and Hyuk Bang Kwon

Hormone Research Center (C.W., O.Y., K.M., D.Y.O., J.Y.S., H.B.K.), Chonnam National University, Gwangju 500-757, Republic of Korea; Department of Molecular Cell Biology (K.K.K.), Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Republic of Korea; Korea Chemical Bank (C.H.C.), Korea Research Institute of Chemical Technology, Daejeon 305-606, Republic of Korea; and Department of Chemistry (C.J.L.), Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea

Address all correspondence and requests for reprints to: Jae Young Seong, Ph.D., Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea. E-mail: jyseong{at}chonnam.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A Glu/Asp7.32 residue in the extracellular loop 3 of the mammalian GnRH receptor (GnRHR) is known to interact with Arg8 of mammalian GnRH (mGnRH), which may confer preferential ligand selectivity for mGnRH than for chicken GnRH-II (cGnRH-II). However, some nonmammalian GnRHRs also have the Glu/Asp residue at the same position, yet respond better to cGnRH-II than mGnRH. Amino acids flanking Glu/Asp7.32 are differentially arranged such that mammalian and nonmammalian GnRHRs have an S-E/D-P motif and P-X-S/Y motif, respectively. We presumed the position of Ser7.31 or Pro7.33 of rat GnRHR as a potential determinant for ligand selectivity. Either placing Pro before Glu7.32 or placing Ser after Glu7.32 significantly decreased the sensitivity and/or efficacy for mGnRH, but slightly increased that for cGnRH-II in several mutant receptors. Among them, those with a PEV, PES, or SES motif exhibited a marked decrease in sensitivity for mGnRH such that cGnRH-II had a higher potency than mGnRH, showing a reversed preferential ligand selectivity. Chimeric mGnRHs in which positions 5, 7, and/or 8 were replaced by those of cGnRH-II revealed a greater ability to activate these mutant receptors than mGnRH, whereas they were less potent to activate wild-type rat GnRHR than mGnRH. Interestingly, a mutant bullfrog type I receptor with the SEP motif exhibited an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II. These results indicate that the position of Pro and Ser near Glu7.32 in the extracellular loop 3 is critical for the differential ligand selectivity between mammalian and nonmammalian GnRHRs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GNRH IS a pivotal neuropeptide controlling reproduction and sexual development via interaction with the heptahelical GnRH receptor (GnRHR) (1). To date, 16 GnRH variants have been identified in a variety of vertebrates and invertebrates, and at least two or more forms of GnRH exist in the brain of a single species (2, 3, 4, 5, 6). Mammalian GnRH (mGnRH, pyro-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), a representative GnRH-1 is predominant in the hypothalamus and regulates the release of gonadotropins from the pituitary (7). Chicken GnRH-II (cGnRH-II, [His5, Trp7, Tyr8]GnRH, or GnRH-2) that differs from mGnRH by three amino acids at positions 5, 7, and 8 is evolutionally conserved throughout vertebrates (8, 9). In contrast, cGnRH-II has a widespread distribution in the brain but is most prominent in the hindbrain and spinal cord (4, 9). Although the exact function of cGnRH-II is unclear, it was proposed that cGnRH-II contributes to neuromodulation and sexual behavior (10, 11, 12).

The presence of two or more forms of GnRH in a single species suggested the existence of cognate receptors (13). Recently, we and others demonstrated the presence of two types of GnRHR in fish (14, 15, 16) and three types in frogs (17). More recently, a type II GnRHR that is structurally and functionally similar to nonmammalian GnRHRs was identified in the monkey (18, 19). That two or more forms of GnRHR exist in a single species suggests diverse roles for GnRH in vertebrate reproduction through differential affinities for different GnRHRs.

The type I mammalian GnRHR, the most predominant form in the pituitary, is more sensitive to mGnRH than cGnRH-II (20), whereas nonmammalian and type II mammalian GnRHRs respond better to cGnRH-II than mGnRH (14, 15, 16, 17, 18, 19). Thus, it is quite interesting to elucidate factors that determine differential ligand selectivity. The amino- and carboxyl-terminal residues, and His2 of GnRH, and their cognate binding sites of GnRHR (Asp2.61, Asn2.65, Lys3.32, Asn5.39, Trp6.48, Tyr6.51, Tyr6.59, Phe7.37, and Phe7.41) are all conserved in fish, amphibians, birds, and mammals (14, 20, 21, 22, 23, 24, 25). It was thought that the specificity of type I mammalian GnRHR for mGnRH is conferred by an electrostatic interaction of Arg8 of mGnRH with negatively charged amino acids, i.e. Glu7.32(301) (in rat and mouse GnRHR) or Asp7.32(302) (in human GnRHR) in the extracellular loop 3 (ECL3) (26, 27). The amino acid at position 8 of GnRH is the most variable (2, 3, 4, 5, 6). Except for mGnRH, others contain an uncharged residue, such as Tyr (for cGnRH-II) or Leu (for salmon GnRH) at this position. Mutations of Glu7.32 to other uncharged amino acids in mouse GnRHR greatly decreased the affinity for mGnRH, and mutations of Arg8 in mGnRH to other negatively or uncharged amino acids also decreased the affinity for wild-type mouse GnRHR (24). Thus, it appears that the interaction between Arg8 of mGnRH and Glu/Asp7.32 of mammalian type I GnRHR is responsible for the preferential ligand selectivity for mGnRH.

However, such an electrostatic interaction cannot fully account for differential ligand selectivity between mammalian and nonmammalian GnRHRs, as some nonmammalian GnRHRs have an acidic amino acid, e.g. Glu7.32(332) [for bullfrog type II receptor (bfGnRHR-2)] and Asp7.32(304) (catfish GnRHR), at the same position in ECL3, yet these receptors respond better to cGnRH-II than mGnRH (14, 15, 16, 17). It is of interest to note that in type I mammalian GnRHRs, Glu/Asp7.32 is always preceded by Ser7.31 and followed by Pro7.33, respectively, generating an S-E/D-P motif. At the same position, however, in nonmammalian and type II mammalian GnRHRs, Pro usually comes first, followed by an amino acid (X) including Glu or Asp, and then a Ser or Tyr residue, producing a P-X-S/Y motif (Fig. 1Go). Only bullfrog type I receptor (bfGnRHR-1) contains an SQS motif (17). It is well known that Pro leads to a local constraint on the peptide chain conformation due to its pyrrolidine ring structure. As the motif is located at the junction between ECL3 and transmembrane helix 7 (TMH7) (28), it is possible that Pro can affect an extension between TMH7 to ECL3 (29), and/or induce a different conformation of ECL3. Tyr may contribute to an aromatic interaction with aromatic residues of ligand (30). These facts raise the possibility that the position of Pro, Ser, or Tyr near Glu/Asp7.32 is another factor responsible for differential ligand selectivity between mammalian and nonmammalian GnRHRs. The present study addresses this possibility. The Pro and Ser residues near Glu7.32 of rat GnRHR were reciprocally changed or replaced by other amino acids. Moreover, the PEY motif of bfGnRHR-2 and the SQS motif of bfGnRHR-1 were modified to those present in mammalian GnRHRs. Ligand selectivity of these mutant receptors was determined using natural and chimeric GnRHs.



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  		Fig. 1. Schematic Diagram of the GnRHR and Species Comparison of the S-E/D-P Motif Sequence

The GnRHR structure with seven cylinders representing transmembrane domains, is shown. The amino acids indicated in the transmembrane domains are important for ligand binding and highly conserved throughout mammalian and nonmammalian GnRHRs. Sequence alignment of amino acids in the ECL3 shows that mammalian type I receptor has a conserved S-E/D-P motif, whereas nonmammalian and mammalian type II receptors have a P-X-S/Y motif. Various mutations used in this study are shown to the right of the SEP motif of rat GnRHR, the SQS motif of bfGnRHR-1, and the PEY motif of bfGnRHR-2, respectively.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Differential Ligand Selectivity of Mammalian and Nonmammalian GnRHRs
Differential ligand selectivity of rat GnRHR (a representative mammalian type I receptor) and bullfrog type II GnRHR (bfGnRHR-2, a representative nonmammalian receptor) was examined using two different assay systems, inositol phosphate (IP) production and c-fos promoter-driven luciferase assays. The plasmid containing wild-type rat GnRHR or bfGnRHR-2 cDNA was transfected into CV-1 cells, and either IP production or c-fos promoter-driven luciferase activity was examined after stimulation with cGnRH-II or mGnRH (Fig. 2Go). As shown in Fig. 2Go, A and C, rat GnRHR was more sensitive to mGnRH than cGnRH-II. mGnRH was approximately 5–10 times more potent in activating rat GnRHR than cGnRH-II as revealed by the EC50 value (Table 1Go). In addition, maximal fold increases (Emax) in IP production and c-fos-luc activity by mGnRH were approximately 1.7-fold higher than those by cGnRH-II. In contrast to rat GnRHR, bfGnRHR-2 exhibited a higher sensitivity for cGnRH-II than mGnRH. cGnRH-II is about 4000 times more potent in activating bfGnRHR-2 than mGnRH. The maximal fold induction by cGnRH-II was similar to that by mGnRH. It should be noted that EC50 values obtained by IP production and c-fos-luc assays were very similar to each other, but the maximal fold induction obtained by c-fos-luc assay was much higher than that in the IP production assay. As the c-fos-luc assay system exhibited a higher sensitivity than the IP production assay system (31), we examined GnRH-induced activity of mutant GnRHRs using the c-fos-luc assay system in the ensuing experiments.



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  		Fig. 2. Ligand Selectivity of Rat GnRHR and bfGnRHR-2

Ligand selectivity of rat GnRHR and bfGnRHR-2 was examined using two different assay systems. Panels A and B show GnRH-stimulated IP accumulation in cells transfected with rat GnRHR (A) and bfGnRHR-2 (B). IP accumulation assay was performed as described in Materials and Methods. Panels C and D show c-fos promoter-driven luciferase activity by GnRH in cells expressing rat GnRHR (C) and bfGnRHR-2 (D). Each GnRHR expression vector was cotransfected with the c-fos-luc reporter into CV-1 cells. To determine the c-fos-luc activity, cells were maintained in serum-free medium for 24 h before GnRH treatment. Six hours after treatment with GnRH, luciferase activities were determined.

 

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  		Table 1. Log EC50 and Emax, Percentage of Binding of Mutant Rat GnRHR for mGnRH and cGnRH-II

 
Mutations of Ser7.31 and Pro7.33 Residues Neighboring Glu7.32 of Rat GnRHR Caused Alterations in Ligand Sensitivity
To elucidate the effect of the position of Ser and Pro near Glu7.32 on ligand selectivity of rat GnRHR, we constructed several rat GnRHR mutants in which the Ser7.31 and Pro7.33 residues were reciprocally exchanged or replaced by some other amino acids. In the first series of mutations, we mutated Ser7.31 to Ala, Val, or Pro, respectively. Mutation of Ser7.31 to Ala (AEP mutant) did not significantly alter ligand sensitivity for either mGnRH or cGnRH-II but showed a 1.6-fold increased efficacy for cGnRH-II. Mutation of Ser7.31 to Val (VEP) reduced ligand sensitivity for both mGnRH and cGnRH-II. Interestingly, change of Ser7.31 to Pro (PEP) decreased ligand sensitivity for mGnRH but slightly increased sensitivity/efficacy for cGnRH-II (Table 1Go).

In a second series of experiments, Pro7.33 was mutated to Tyr, Ser, Ala, or Val, respectively. The SES, SEY, and SEA mutants exhibited decreased ligand sensitivity for mGnRH but no significant changes in ligand sensitivity for cGnRH-II. For the SEV mutant, we observed decreases in sensitivity and efficacy for both mGnRH and cGnRH-II. It is interesting that the SES mutant showed a decrease in efficacy for mGnRH but 1.9-fold increase in efficacy for cGnRH-II (Fig. 3AGo). Thus, the potency of cGnRH-II for the SES mutant is higher than that of mGnRH, showing a reversed ligand sensitivity for mGnRH and cGnRH-II compared with wild-type rat GnRHR.



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  		Fig. 3. Ligand Selectivity of Rat GnRHRs with Various Mutations at Position 7.31

The Ser-Glu-Pro (SEP) motif of rat GnRHR was changed to SES (A), PEV (B), PES (C), and PQS (D), respectively. The mutant receptors were cotransfected with the c-fos-luc reporter into CV-1 cells. Six hours after treatment with GnRH, luciferase activities were determined. Triangles and circles indicate mGnRH- and cGnRH-II-treated group, respectively. Dashed lines are data obtained from rat GnRHR wild type (WT), and solid lines are data observed from each mutant.

 
In the ensuing experiment, we made four double mutants in which Ser7.31 was replaced by Pro, and Pro7.33 was replaced by Ser, Tyr, Ala, or Val, generating PES, PEY, PEA, and PEV mutants, respectively. Placing Pro before Glu7.32 greatly affected ligand sensitivity. Among the mutants, PES and PEV mutants exhibited reversed ligand sensitivity for mGnRH and cGnRH-II (Fig. 3Go, B and C).

To examine the role of Glu7.32, this amino acid of the PEX mutants was changed to Gln or Leu, generating PQS, PQY, PLS, and PQV, respectively. All of these mutants showed great decreases in sensitivity/efficacy for mGnRH. Compared with PES, PEV, and PEY mutants, PQS, PQY, and PLS mutants maintained a similar sensitivity/efficacy for cGnRH-II, whereas the PQV mutant showed a decrease in sensitivity for cGnRH-II (Fig. 3DGo and Table 1Go). This result indicates that the Glu7.32 residue in the mutant receptors is still important for sensitivity toward mGnRH but not cGnRH-II.

The whole-cell binding assay with a high concentration of radiolabeled GnRH analogs revealed that the total binding of most mutant receptors varied by approximately 15% over wild-type receptor. The total binding of SEV, PLS, and PQV mutants was about 40–67% of that of the wild-type receptor (Table 1Go).

Ligand Sensitivity of the PEV/PES Mutant for GnRH Analogs
To examine which amino acids of GnRH are responsible for the interaction with the receptor, we employed a variety of natural GnRHs and chimeric GnRHs in which amino acids at position 5, 7, and/or 8 were substituted. mGnRH variants with Gln, Trp or Leu at position 8 showed a lower potency in the activation of wild-type rat GnRHR compared with mGnRH. Interestingly, these GnRHs exhibited a higher potency in activation of bfGnRHR-2 than mGnRH. Thus, Arg8 hampers the interaction of mGnRH with the nonmammalian GnRHR. The potency of [Gln8]mGnRH, [Trp8]mGnRH, and [Trp7,Leu8]mGnRH in activation of the PEV and PES mutants was similar to that of mGnRH (Fig. 4Go and Table 2Go). The potency of [Trp7]mGnRH and [His5]mGnRH in activation of rat GnRHR was similar to that of mGnRH, while the potency of [Trp7, Tyr8]mGnRH in activation of rat GnRHR was lower than mGnRH. Together with the natural GnRH study, these results imply that the amino acids at positions 5 and 7 of GnRH do not contribute much to the ligand selectivity of rat GnRHR. Regarding bfGnRHR-2, all chimeric GnRHs were much more active than mGnRH. In particular, [Trp7, Tyr8]mGnRH exhibited a greatly improved activity for bfGnRHR-2, indicating that positions 7 and 8 of GnRH have considerable involvement in ligand selectivity of bfGnRHR-2. Interestingly, all of these chimeric GnRHs revealed an approximately 2- to 3-fold higher potency in activating PEV and PES mutants than mGnRH itself (Fig. 4Go and Table 2Go).



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  		Fig. 4. Ligand Sensitivity of bfGnRHR-2, Rat GnRHR, and Rat GnRHR with PEV and PES Mutations for Various GnRH Analogs

Rat GnRHR wild type (A), bfGnRHR-2 (B), and rat GnRHR with PEV (C) and PES (D) mutations were cotransfected with the c-fos-luc reporter into CV-1 cells. Cells were treated 48 h after transfection with various GnRH analogs; mGnRH ({triangleup}), cGnRH-II, ({circ}), [Trp7, Tyr8]cGnRH-II ({blacksquare}), [Trp7, Leu8]mGnRH ({diamondsuit}), [Trp8]mGnRH ({diamond}), [His5]mGnRH ({square}), [Trp7]mGnRH (X), and [Glu8]mGnRH ({blacktriangledown}). Luciferase activities were determined were determined 6 h after treatment with GnRH.

 

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  		Table 2. Log EC50 and Emax Values of bfGnRHR and Rat GnRHR with PEV or PES Mutation for Various mGnRH and cGnRH-II Analogs

 
In a third series of experiments, we used GnRH analogs with D-Ala at position 6. These analogs have a constrained and stable ß-II-turn structure (20, 32). They were applied to cells expressing rat GnRHR, bfGnRHR-2, PEV, and PES mutant GnRHRs. For rat GnRHR, [D-Ala6]mGnRH had the highest power to activate the receptor among the analogs. [D-Ala6]cGnRH-II activated bfGnRHR-2 and rat GnRHR PES mutant more than [D-Ala6]mGnRH (Table 2Go).

Mutations of the PEY Motif of bfGnRHR-2 and SQS Motif of bfGnRHR-1 to Those Found in Mammalian GnRHRs
To examine the significance of the Pro and Ser positions in nonmammalian GnRHRs, the PEY motif of bfGnRHR-2 and the SQS motif of bfGnRHR-1 were mutated to those found in mammalian GnRHRs. bfGnRHR-2 with an SEP mutation slightly decreased the sensitivity for both mGnRH and cGnRH-II, whereas bfGnRHR-2 with the SDY mutation showed a decrease in sensitivity for mGnRH but no change in that for cGnRH-II. The mutation to SEY in bfGnRHR-2 greatly reduced ligand sensitivity for both mGnRH and cGnRH-II. We failed to observe a reversed ligand sensitivity in bfGnRHR-2 mutants. Interestingly, bfGnRHR-1 with an SES mutation showed an increased ligand sensitivity for mGnRH but no changes for cGnRH-II. Further, bfGnRHR-1 with the SEP mutation had an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II (Fig. 5Go and Table 3Go), showing a reversed ligand selectivity.



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  		Fig. 5. Ligand Selectivity of bfGnRHR with Mutations of the PEY/SQS Motif

The PEY motif of the bfGnRHR-2 was mutated to SEP (A), SDY (B), and SEY (C), respectively. The SQS motif of the bfGnRHR-1 was mutated to SES (D) and SEP (E), respectively. Mutant receptors were cotransfected with the c-fos-luc reporter into CV-1 cells. Luciferase activities were determined 6 h after treatment with GnRH. Triangles and circles indicate mGnRH- and cGnRH-II-treated groups, respectively. Dashed lines are data obtained from rat GnRHR wild type (WT), and solid lines are data observed from each mutant.

 

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  		Table 3. Log EC50 and Emax Values of Mutant bfGnRHR-1 and bfGnRHR-2 for mGnRH and cGnRH-II

 
Molecular Modeling
To support the biochemical data, we made the models for the interaction of GnRHs with the wild-type and PES mutant GnRHRs (Fig. 6Go). Intermolecular interactions between GnRH and receptor observed in current models appear to be in good agreement with previous observations (25). For instance, Arg8 of mGnRH is in close contact with Glu301 of rat GnRHR (Fig. 6AGo). Two cysteine residues (Cys14 and Cys199) also moved together to form a disulfide bond (Fig. 6AGo). Applying cGnRH-II to rat GnRHR altered intermolecular interactions between the receptor and ligand. A salt bridge between Arg8 of mGnRH and Glu301(7.32) of rat GnRHR was replaced by a hydrogen bond between Tyr8 and Glu301 (Fig. 6BGo). In addition, the possible hydrophobic interactions between Tyr5 of mGnRH and hydrophobic pocket formed by residues (Cys14, Leu183, Cys199, Trp288, Tyr289) in rat GnRHR were abolished as Tyr5 was changed to His5 in cGnRH-II (Fig. 6BGo). A double mutation [Ser300(7.31) to Pro and Pro302(7.33) to Ser, PES mutant] induced a local conformational change in the loop of rat GnRHR near residues 7 and 8 of mGnRH led to an alteration in the intermolecular interaction (Fig. 6CGo). Interestingly, the hydrogen bond interaction between Tyr8 in cGnRH-II and Glu301 in the PES mutant was not observed. Rather, Tyr8 moved to a hydrophobic pocket formed by Pro189, Ala190, and Val191 (Fig. 6DGo).



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  		Fig. 6. Molecular Models for the Interaction of mGnRH with Wild-Type Rat GnRHR (A), cGnRH-II with Wild-Type Rat GnRHR (B), mGnRH with the PES Mutant GnRHR (C), and cGnRH-II with the PES Mutant GnRHR (D)

The secondary structures of the GnRHR were drawn as ribbon diagrams in cyan. GnRHs were shown in white tubes. GnRHR residues that have a tight contact with GnRH are drawn in a yellow stick model. The residues 300(7.31), 301(7.32), and 302(7.33) were labeled. GnRH residues were drawn as a ball-and-stick model with carbon atoms in black, oxygen atoms in red, and nitrogen atoms in blue. Each residue in GnRH was labeled in green.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
As electrostatic interaction of Arg8 with Glu7.32 cannot fully account for the differential ligand selectivity between mammalian and nonmammalian GnRHRs, we noticed the S-E/D-P motif and P-X-S/Y motif in the mammalian type I and nonmammalian GnRHRs, respectively. We focused on the position of Pro as it may affect the structure and conformation of ECL3 in GnRHRs. Most Ser7.31 -> Pro mutant rat GnRHRs revealed a decreased ligand sensitivity and/or efficacy for mGnRH but an increased or similar ligand sensitivity and/or efficacy for cGnRH-II, compared with the wild-type receptor. This observation indicates that change of Pro position may alter the ECL3 local structure, probably modifying the side chain orientation of Glu7.32, consequently weakening the interaction of Arg8 of GnRH with Glu7.32 of the mutant receptors. However, it seems that substituting Pro for Ser7.31 did not completely abolish the electrostatic interaction of Arg8 with Glu7.32, as additional mutations of Glu7.32 to Leu or Gln further decreased sensitivity for mGnRH. Indeed, our computer modeling data indicated that Arg8 still has an interaction with Glu7.32 in the PES mutant receptor (Fig. 6Go, A and C). It is likely that the substitution of Pro for Ser7.31 led to a local conformational change that may affect the electrostatic interaction between Glu7.32 and Arg8 of mGnRH. Interestingly, a Pro substitution for Ser7.31 in rat GnRHR led to an increase in ligand sensitivity and/or receptor efficacy for cGnRH-II. Based on the fact that mutant rat GnRHRs had a higher affinity for GnRH with Tyr8 rather than GnRH with Trp8, Leu8, or Gln8, we postulated that Tyr8 plays an important role in ligand selectivity. It was suggested that Tyr8 of cGnRH-II is able to form a hydrogen bond with Asp7.32 in catfish GnRHR (24, 27). Modeling data also showed that Tyr8 and Glu7.32 of the wild-type rat GnRHR are close enough to form a hydrogen bond (Fig. 6BGo). Interestingly, mutation of the SEP motif to PES increased distance between Tyr8 and Glu7.32 to abolish such a hydrogen bond (Fig. 6DGo). This finding indicates that the interaction of Tyr8 of cGnRH-II with Glu7.32 of the wild-type or mutant rat GnRHRs is not critically involved in the activation of those receptors. This notion can be further supported by the observation that additional mutations of Glu7.32 to Leu or Gln (PQS, PQY, and PLS mutants) did not significantly decrease the sensitivity for cGnRH-II. Interestingly, Tyr8 moved to Pro189, Ala190, Val191 in ECL2 in the PES mutant receptor instead of with Glu7.32 when it binds to mutant GnRHR. Thus, it is possible that Tyr8 appears to interact with other amino acid residues or hydrophobic pockets. The substitution of Trp for Leu7 of mGnRH had no significant effect on sensitivity for either wild-type or mutant rat GnRHR, yet this substitution led to an increase in the ability to activate bfGnRHR-2. Thus, it is likely that an amino acid at position 7 in GnRHs does not greatly contribute to a reversed ligand sensitivity of mutant rat GnRHR, whereas this position is important for sensitivity of bfGnRHR-2. The amino acid at position 5 of GnRH seems to play a role in ligand selectivity; [His5]mGnRH exhibited a similar ligand sensitivity but a decreased efficacy of wild-type rat GnRHR. This substitution increased the power to activate the mutant GnRHRs. Tyr5 of mGnRH is known to interact with the Tyr6.58 of the receptor (27). Because Tyr6.58 was not mutated, the increase in sensitivity of mutant rat GnRHR for [His5]mGnRH indicates that such a substitution may affect ligand conformation and/or alteration of a binding pocket for His5 by the receptor mutation. In support of this possibility, our computer modeling data suggest that His5 may interact with the backbone of Asn12, Cys14, Asp185 in the PES mutant.

Increases in activation of the PEV and PES mutant receptors by substitutions of positions 5, 7, and/or 8 of mGnRH suggest that the secondary structure of GnRH as well as composition of an individual amino acid is important for increased sensitivity (33). Arg8 is known to be important not only for interaction with the acidic side chain of ECL3, but also for secondary structure stabilization of mGnRH. Arg8 appears to have a hydrogen bond interaction with Ser4; Tyr5 also contributes to the conformation of mGnRH through an interaction with Arg8 (20). Trp7 of cGnRH-II has a bulkier side chain than that of Leu7 in mGnRH. Therefore, the absence of Arg8 and substitutions of His5 and Trp7 in cGnRH-II critically affect the secondary structure.

Our study demonstrates that the S-E/D-P motif in ECL3 of type I mammalian GnRHR confers specificity for Arg8 of mGnRH. It can be postulated that the evolutionary development of the S-E/D-P motif in mammalian type I GnRHR is one of the prerequisites to achieve high-affinity binding to mGnRH. One interesting finding of this study is that the mutation of the SQS motif to SEP in the bfGnRHR-1 greatly increased the ligand sensitivity for mGnRH whereas it decreased ligand sensitivity for cGnRH-II, behaving like wild-type rat GnRHR. These results demonstrates that placing the Pro after Glu is critical for decreasing ligand sensitivity for cGnRH-II, suggesting that the position of Pro and Ser in ECL3 is important for differential ligand selectivity in some nonmammalian GnRHRs as well as type I mammalian GnRHRs. It should be noted that a nonmammalian GnRHR with Ser7.31, like bfGnRHR-1, is a small subfamily compared with nonmammalian GnRHRs with Pro7.31 (16). BfGnRHR-1 appears to be a functional mammalian type-I GnRHR analog. bfGnRHR-1 is expressed mainly in the pituitary and has a relatively high sensitivity for mGnRH (17, 31). Furthermore, bfGnRHR-1 preferred a Gq-coupled signaling pathway like mammalian type I receptors, whereas bfGnRHR-2 and bfGnRHR-3 revealed Gs-preferred and Gs/Gq equivalent signaling pathways, respectively (34). Thus, it appears that bfGnRHR-1 is an evolutionary intermediate from nonmammalian to mammalian type I GnRHRs.

However, a change in the Pro position does not fully account for the evolutionary change in ligand selectivity, as none of the mutations in which a PEY motif of bfGnRHR-2 was changed to SEP, SDY, or SEY exhibited reversed ligand selectivity. We suggest that, in addition to the changes in Pro position, other sequence or structural variability between the mammalian type I and nonmammalian GnRHRs may account for differential ligand selectivity. Our unpublished data showed that a chimeric GnRHR, in which whole sequences for ECL3 and a part of TMH7 of monkey type II GnRHR were replaced with those of rat GnRHR, significantly increased the ligand sensitivity for mGnRH but decreased the sensitivity for cGnRH-II. This result indicates that structure of the amino acid chain from ECL3 to TMH7 is critical for the SEP motif conformation (35), thereby affecting ligand selectivity.

In summary, this study suggests that the position of Pro and Ser near Glu/Asp7.32, in addition to Glu/Asp7.32 itself, is important for ligand selectivity for cGnRH-II and mGnRH. Understanding structure-function relationships of mammalian and nonmammalian GnRHRs may provide clues for evolutionary changes of these receptors and promote the development of selective agonists and antagonists with therapeutic value.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Peptides
mGnRH, cGnRH-I ([Glu8]mGnRH) and [D-Ala6]mGnRH were purchased from Sigma Chemical Co. (St. Louis, MO). Chimeric GnRHs, ([Trp8]mGnRH, [His5]mGnRH, [Trp7]mGnRH, [Trp7, Tyr8]mGnRH), salmon GnRH, cGnRH-II, and [D-Ala6] cGnRH-II were synthesized by AnyGen (Gwangju, Korea).

Mutagenesis
Rat GnRHR, bfGnRHR-1, and bfGnRHR-2 mutants were constructed by PCR-based site-directed mutagenesis. Primers for mutations were as follows:

Rat-R, 5'-CACCCTGTTTAACATTTCCGG-3'; rat-AEP, 5'-CCGGAAATGTTAAACAGGGTGGCAGAGCCA-3'; rat-VEP, 5'-CCGGAAATGTTAAACAGGGTGGTAGAGCCAG-3'; rat-PEP,5'-CCGGAAATGTTAAACAGGGTGCCAAGAGCCAG-3'; rat-SEY, 5'-CCGGAAATGTTAAACAGGGTGTCAGAGTATGTCAATCA-3'; rat-SES, 5'-CCGGAAATGTTAAACAGGGTGTCAGAGTCAGTCAATCA-3'; rat-SEA, 5'-CCGGAAATGTTAAACAGGGTGTCAGAGGCAGTCAATCA-3'; rat-SEV, 5'-CCGGAAATGTTAAACAGGGTGTCAGAGGTAGTCAAT-3'; rat-PEY, 5'-CCGGAAATGTTAAACAGGGT-GCCAGAGTATGTCAATCACTTC-3'; rat-PES, 5'-CCGGAAATGTTAAACAGGGTGCCAGAGTCAGTCAAT-3'; rat-PEV, 5'-CCGGAAATGTTAAACAGGGTGCCAGAGGTAGTCAAT-3'; rat-PEA, 5'-CCGGAAATGTTAAACAGGGTGCCAGAGGCAGTCAAT-3'; rat-PQY, 5'-CCGGAAATGTTAAACAGGGTGCCACAGTATGTCAAT-3'; rat-PQV, 5'-CCGGAAATGTTAAACAGGGTGCCACAGGTAGTC-3'; rat-PQS, 5'-CCGGAAA-TGTTAAACAGGGTGCCACAGTCAGTC-3'; rat-PLS, 5'-CCGGAAATGTTAAAC-AGGGTGCCACTGTCAGTC-3'; Bf1-R, 5'-CACCTTCTCCTCCATTATCT-3'; Bf1-SES, 5'-AGATAATGGAGGAGAAGGTGTCAGAGTCCACC-3'; Bf1-SEP, 5'-AGATAATGGAGGAGAAGGTGTCAGAGCCCACCACA-3'; Bf2-R, 5'-AGTCAGGTAGATCATCTCTGG-3'; Bf2-SEP, 5'-CCAGAGATGATCTACCTGACTTCCGAGCCTGTCCACCACAGC-3'; Bf2-SDY, 5'-CCAGAGATGATCTACCTGACTTCCGACTATGTC-3'; Bf2-SEY, 5'-CCAGAGATGATCTACCTGACTTCCGAG-3'. The mutated PCR products were cloned into pcDNA3 (Invitrogen, San Diego, CA) at the EcoRI and XbaI restriction enzyme sites. Mutated sequences were confirmed using a Sequenase version 2.0 DNA Sequencing Kit (United States Biochemical Corp., Cleveland, OH) according to the manufacturer’s instructions.

Cell Culture, Transfection, and Luciferase Assay
CV-1 cells were maintained at 37 C in DMEM with 10% heat-inactivated fetal bovine serum, 1 mM glutamate, 100 U of penicillin, and 100 µg/ml streptomycin. Cells were cultured in 24-well plates and transfection was performed using the SuperFect transfection kit (QIAGEN, Chatsworth, CA) following the manufacturer’s protocol. For each transfection, 100 ng of each receptor cDNA, and 200 ng of c-fos-luc (containing ~ -711 to +45 sequence of the human c-fos promoter constructed in the pFLASH vector (a gift from Dr. R. Prywes, Columbia University, New York, NY), along with 200 ng of the internal control plasmid pCMVß Gal, were used. The pcDNA3 was used to adjust the total amount of DNA transfected to 0.7 µg. Transfected cells were incubated in serum-free DMEM for 18 h before treatment with GnRH. Six hours after adding GnRH, cells were harvested, and the luciferase activity in the cell extract was determined as previously described (31, 36). The luciferase activities were normalized using the ß-galactosidase values. Transfection experiments were performed in duplicate and repeated three to five times.

IP Production Assay
The IP production assay was performed as previously described (17). CV-1 cells (1 x 105 per well) were seeded in 12-well plates, and the following day cells were transfected with SuperFect (QIAGEN). Twenty-four hours after transfection, the cells were incubated in inositol-free DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 2% dialyzed fetal calf serum and labeled with 1 µCi myo-[3H]inositol/well (Amersham Pharmacia Biotech, Buckinghamshire, UK) for 18 h. Medium was then removed and cells were washed with 0.5 ml buffer A (140 mM NaCl, 20 mM HEPES, 4 mM KCl, 8 mM D-glucose, 1 mM MgCl2, 1 mM CaCl2, 1 mg/ml fatty acid-free BSA). Then cells were preincubated with buffer A containing 10 mM LiCl for 15 min, followed by addition of graded concentration (0.01 nM to 10 µM) of GnRHs at 37 C for 45 min. The reaction was stopped by removing the incubation medium and adding 0.5 ml of ice-cold 10 mM formic acid. After 30 min at 4 C, the formic acid extracts were transferred into columns containing Dowex anion exchange resin. Total IPs were then eluted with 1 ml of 1 M ammonium formate/0.1 M formic acid, and their radioactivity was determined.

Ligand Binding Assay
[His5, D-Tyr6]GnRH was radioiodinated using the chloramine-T method and purified by chromatography on a Sephadex G-25 (Sigma) column in 0.01 M acetic acid, 0.1% BSA. The ligand binding of the GnRHR was quantified using a whole-cell binding assay (37). Cells (1.3 x 105) were grown in 12-well plates 24 h before assay. Cells were washed with PBS and then incubated at 4 C for 3–4 h in the presence of [125I]cGnRH-II (200,000 cpm/well). Cells were then washed twice with PBS, and radioactivity was determined by resolving cells in 1% sodium dodecyl sulfate and 0.2 M NaOH. Nonspecific binding for each time point was determined in the presence of 1 µM unlabeled [His5, D-Tyr6]GnRH.

Molecular Modeling
Seven helices of rat GnRHR were built by a G protein-coupled receptor mode in Swiss Model (38) with bovine rhodopsin as a template (Protein Data Bank identification no. 1F88). The extra and intracellular domains of the GnRHR were built by a protein-modeling routine in Molecular Operating Environment Chemical Software (MOE, Chemical Computing Inc., Quebec, Canada). The GnRHR model was minimized with AMBER94 force filed using MOE. Cys114 and Cys195 were located close enough for making a disulfide bond, validating the current GnRHR model. Cys14 and Cys199 were also within a distance to form a disulfide bond. mGnRH was docked into the putative binding site of the rat GnRHR manually and the complex model was minimized in MOE. The models for wild-type GnRHR/cGnRH-II, PES mutant GnRHR/mGnRH, and PES mutant GnRHR/cGnRH-II were also built by mutating corresponding residues in wild-type rat GnRHR and mGnRH. Molecular dynamics simulation of four complexes were performed for 700 psec [time step = 1 fsec, T = 300K, {epsilon} = 1] with CHARMM (39). Equilibrated structures of the complexes were obtained from the energy-minimized average over the final period of the simulation covering the last 200 psec. The final models showed the good geometry confirmed by PROCHECK (40). Ligand binding energies of four models were calculated with CHARMM.

Data Analysis
All assays were performed in triplicate and repeated three times. Data analysis was performed using nonlinear regression, with sigmoid dose response. The GnRH concentrations inducing half-maximal stimulation (EC50) and maximal fold increase (Emax) were calculated using the GraphPad PRISM2 software (GraphPad, San Diego, CA). All data are presented as mean ± SEM of at least three independent experiments. The data were analyzed by one-way ANOVA followed by Newman-Keuls posttest. P < 0.05 was considered statistically significant.


   FOOTNOTES
 
This work was supported by the Korea Research Foundation (Grant 2001-DP0493). D.Y.O. was supported by a Brain Korea 21 Research Fellowship from the Korea Ministry of Education.

Abbreviations: bfGnRHR, Bullfrog GnRHR; cGnRH, chicken GnRH; ECL3, extracellular loop 3; GnRHR, GnRH receptor; IP, inositol phosphate; mGnRH, mammalian GnRH; TMH7, transmembrane helix 7.

Received for publication March 25, 2003. Accepted for publication September 26, 2003.


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