Membrane-Proximal Region of the Carboxyl Terminus of the Gonadotropin-Releasing Hormone Receptor (GnRHR) Confers Differential Signal Transduction between Mammalian and Nonmammalian GnRHRs

Da Young Oh, Jung Ah Song, Jung Sun Moon, Mi Jin Moon, Jae Il Kim, Kyungjin Kim, Hyuk Bang Kwon and Jae Young Seong

Hormone Research Center (D.Y.O., J.A.S., J.S.M., M.J.M., H.B.K., J.Y.S.), School of Biological Sciences and Technology, Chonnam National University, Gwangju 500-757; School of Biological Sciences (K.K.), Seoul National University, Seoul 151-742; and Department of Life Science (J.I.K.), Kwangju Institute of Science and Technology, Gwangju 500-712, Korea

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recently, we demonstrated that the mammalian type-I GnRH receptor (GnRHR) has a high preference for the phospholipase C/protein kinase C (PLC/PKC)-linked signaling pathway, whereas non-mammalian bullfrog (bf) GnRHRs couple to both adenylate cyclase/protein kinase A (AC/PKA)- and PLC/PKC-linked signaling pathways. In the pre-sent study, using AC/PKA-specific reporter (cAMP-responsive element-luciferase) and PLC/PKC-specific reporter (serum-responsive element-luciferase) systems, we attempted to identify the motif responsible for this difference. A deletion of the intracellular carboxyl-terminal tail (C tail) of bfGnRHR-1 remarkably decreased its ability to induce the AC/PKA-linked signaling pathway. Further dissection of the C tail indicated that an HFRK motif in the membrane-proximal sequence of bfGnRHR-1 C tail is a minimal requirement for the AC/PKA-linked signaling pathway as the addition of this motif to rat GnRHR or deletion of it from bfGnRHR-1 significantly affected the ability to induce the AC/PKA-linked signaling pathway. Deletion or addition of the HFRK motif, however, did not critically influence the PLC/PKC-linked signaling pathway. These results indicate that the HFRK motif in the membrane-proximal region confers the differential signal transduction pathways between mammalian and nonmammalian GnRHRs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GNRH IS A CENTRAL regulator in the mammalian reproductive system. GnRH controls the synthesis and secretion of LH and FSH in the anterior pituitary via its receptors (GnRHRs) (1). Functional GnRHR cDNA was first cloned from mouse pituitary cells (2, 3). Subsequently, GnRHR cDNAs have been cloned from other mammalian and nonmammalian species (4, 5, 6, 7). Recently, we identified cDNAs encoding three distinct types of GnRHR in the bullfrog, designated as bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 (8, 9). Further, the cDNA for a second GnRHR was isolated in the monkey (10, 11) and was named mammalian type-II GnRHR to distinguish it from mammalian type-I GnRHR. The presence of two or more types of GnRH (12, 13) and the existence of their cognate receptors suggests the complex nature of this GnRH-GnRHR system that controls reproduction and sexual behavior.

GnRHRs belong to the rhodopsin family of G protein-coupled receptors (GPCRs) that transmits their signals through heterotrimeric G proteins. The mammalian type-I GnRHR is unique because it lacks the common cytoplasmic C-terminal tail (C tail) (2, 3). This C tail is known to be important for the internalization and desensitization of GnRHR (14, 15) as well as many other GPCRs (16, 17). Nonmammalian and mammalian type-II GnRHRs, however, contain the C tail as other GPCRs (5, 6, 7, 8, 9, 10, 11). Another important structural difference is an amino acid variability (Asn for mammalian type-I GnRHR vs. Asp for nonmammalian GnRHR) at position 7.49 in transmembrane domain VII, which is known to interact with Asp at position 2.50 in transmembrane domain II, contributing to receptor conformation and stabilization (18). Moreover, the conserved DRY motif of the proximal second intracellular loop in other rhodopsin family GPCRs is changed to DRS in most mammalian type-I GnRHRs (2, 3, 4), and this motif is modified to DRH or DRQ in nonmammalian GnRHRs (5, 6, 7, 8).

A number of studies regarding GnRH-mediated signal transduction have proposed that GnRHR exclusively activates phospholipase C (PLC) via Gq/11 coupling (19). PLC, in turn, hydrolyzes phosphatidylinositol 4, 5-bisphosphate to generate inositol 1,4,5-triphosphate and diacylglycerol (20). However, other studies indicate that activation of GnRHRs produces cAMP via Gs/adenylate cyclase (AC) (21, 22). Thus, the signaling cascade of the mammalian type-I GnRHR is still controversial and under debate. Moreover, the signaling pathways of nonmammalian GnRHRs are poorly understood. Recently, we demonstrated that the mammalian type-I GnRHRs preferred a PLC/protein kinase C (PKC)-linked signaling pathway, whereas bfGnRHRs, representative nonmammalian GnRHRs, may couple to both the PLC/PKC- and AC/PKA-linked signaling pathways with either similar strength or with a higher preference for the AC/PKA- than PLC/PKC-linked pathway (23).

In this study we attempted to elucidate the motif responsible for such a differential signaling pathway between mammalian type-I and nonmammalian GnRHRs. As the most striking structural difference between these receptors is the presence or absence of a C tail, we wondered whether this difference may influence the signal transduction pathway. To address this issue, we constructed a series of mutant mammalian type-I (rat) GnRHRs containing the C tail of bfGnRHRs and mutant bfGnRHRs the C tails of which were deleted. Further, using a serial deletion of the C tail and site-directed mutagenesis we demonstrated that an HFRK motif in the membrane-proximal region of the C tail is critical to the difference in signal transduction between nonmammalian and mammalian type-I GnRHRs.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Different Signaling Pathways of Rat GnRHR and bfGnRHRs
To validate the specificity of the cAMP-responsive element (CRE)-luc and serum-responsive element (SRE)-luc reporter systems, specific chemical activators or inhibitors for AC/PKA and PLC/PKC signaling pathways were used in control experiments. The CRE-luc reporter system was significantly activated by forskolin (FKN), an AC activator but not by 12-O-tetradecanoylphenol-13-acetate (TPA), a PKC activator (Fig. 1AGo), whereas the SRE-luc reporter system was activated by TPA but not by FKN (Fig. 1BGo). FKN-induced CRE-luc activity was completely inhibited by H-89 (10 µM), a specific PKA inhibitor, but only partially inhibited by GF109203X (5 µM), a specific PKC inhibitor. Similarly, TPA-induced SRE-luc activity was completely blocked by GF109203X, but to a lesser extent by H-89. To further confirm the specificity of the reporter system, we examined the reporter activity in cells expressing human ß2-adrenergic receptor (hß2AR) or human oxytocin receptor (hOTR). hß2AR induced CRE-luc activity, but only marginally induced that of SRE-luc. hOTR evoked SRE-luc activity more than CRE-luc (Fig. 1CGo). This result agrees well with previous findings that hß2AR specifically activates the AC/PKA signaling pathway, whereas hOTR induces that of PLC/PKC (24, 25). Together, these results indicate that the CRE-luc and SRE-luc reporter systems are useful tools to discriminate the AC/PKA and PLC/PKC signaling pathway, respectively. The different signaling pathways of rat GnRHR and bfGnRHRs were examined using these reporters. Consistent with the previous results (23), rat GnRHR strongly induces SRE-luc activity but only marginally induces CRE-luc activity. BfGnRHRs trigger both SRE- and CRE-luc activities with a similar (bfGnRHR-1 and -3) or higher preference for CRE-luc (bfGnRHR-2) (Fig. 1DGo).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1. GnRH-Induced CRE- and SRE-Derived Transcriptional Activity

For the validation of the CRE-luc and SRE-luc reporter systems (panel A), after transfection of HeLa cells with CRE-luc (panel A) or SRE-luc (panel B), cells were treated with TPA (200 nM) or FKN (10 µM) for 6 h. GF109203X (5 µM) or H-89 (10 µM) was added 15 min before the treatment with TPA or FKN. To examine the signaling pathways of hß2AR and hOTR (panel C), and GnRHRs (panel D), HeLa cells were transfected with each receptor plasmid in combination with CRE-luc or SRE-luc. Cells were treated 48 h after transfection with 1 µM of the appropriate ligand for 6 h. Luciferase activity was measured and normalized against ß-galactosidase activity. The results were plotted as fold activity over basal luciferase activity. Each bar represents means ± SEM of three independent experiments performed in duplicate. bf1, bf2, bf3, and rat indicate bfGnRHR-1, bfGnRHR-2, bfGnRHR-3, and rat GnRHR, respectively. *, P < 0.05

 
C Tail of GnRHR Confers Differential Signal Transduction
To identify the motif responsible for such a differential signal transduction, we constructed mutant rat GnRHRs containing the C tail of bfGnRHRs and mutant bfGnRHRs the C tails of which were deleted. All rat GnRHR mutants containing the bfGnRHR C tail exhibited a decreased SRE-luc activity. The rat GnRHR mutants with the C tail of bfGnRHR-2 and bfGnRHR-3 had an increased CRE-luc activity (Fig. 2AGo). Consistently, the bfGnRHR mutants without a C tail showed a reduced CRE-luc activity (90% compared with wild-type bfGnRHR-1 and -2 and by 50% of wild-type bfGnRHR-3, respectively) (Fig. 2BGo). No significant changes in SRE-luc activity were observed in C tail-less bfGnRHR-1 and bfGnRHR-3. C tail-deleted bfGnRHR-2 showed a decreased SRE-luc activity (Fig. 2CGo). To further clarify the signaling preferences of these mutant receptors, the ratio of CRE-luc to SRE-luc activity was examined (Table 1Go). Mutant rat GnRHRs with the C tail of bfGnRHRs showed an increased ratio whereas the C tail-less bfGnRHRs exhibited a decreased ratio, indicating that the C tail of bfGnRHR does play a key role in signaling preference. Whole-cell and saturation binding assays were performed to examine mutant receptor expression. All mutant GnRHRs, except for the C tail-less bfGnRHR-2, revealed slight changes in expression level compared with wild-type receptors (Table 1Go). A remarkable decrease in receptor expression was observed in bfGnRHR-2 without the C tail. Considerable decreases in both CRE- and SRE-luc activities of C tail-less bfGnRHR-2 are, therefore, most likely due to decreased receptor expression.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2. Effect of C Tail on CRE- and SRE-luc Activities

CRE- and SRE-luc activities of wild-type and mutant rat GnRHRs with the C tail of each bfGnRHR (+bf1, +bf2, and +bf3) were examined (panel A). Effects of C tail deletion from the bfGnRHRs on CRE-luc (panel B) and SRE-luc (panel C) activities were tested. Cells were transfected with wild-type or mutant receptors in combination with CRE- or SRE-luc reporters and treated with 1 µM cGnRH-II for 6 h.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Ratio of CRE-luc to SRE-luc Activity and Receptor Expression of Wild-Type (WT) and Mutant GnRHRs

 
Membrane-Proximal Region of bfGnRHR-1 C Tail Is Involved in Differential Signaling Preference
As the deletion of bfGnRHR-1 C tail remarkably decreased CRE-luc activity, we further investigated the C tail of bfGnRHR-1. Serial deletions from the distal part of the C tail (designated 2/3, 1/3, and 0/3) were made. For rat GnRHR, the same deleted C tail fragments from the bfGnRHR-1 were added (designated +1/3, +2/3, and +3/3). All rat GnRHR mutants with these C tail fragments had a decreased SRE-luc activity, whereas the addition of 2/3 and 3/3 C tail fragments to rat GnRHR slightly increased CRE-luc activity. Interestingly, the addition of membrane-proximal 1/3 fragment to rat GnRHR considerably augmented the CRE-luc activity (Fig. 3AGo), indicating that this fragment of bfGnRHR-1 C tail is critical for the AC/PKA-linked signaling pathway. The serial deletion of the C tail from bfGnRHR-1 further supports this idea. Mutant bfGnRHR-1 without the C tail showed a drastic decrease in CRE-luc activity, whereas the mutant bfGnRHR-1 with 1/3 or 2/3 fragments showed an increased CRE-luc activity compared with that of the wild-type receptor (Fig. 3BGo).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3. Effect of Serial Deletion or Addition of bfGnRHR-1 C Tail on CRE- and SRE-luc Activities

Rat GnRHR mutants with different lengths of bfGnRHR-1 C tail (panel A) and the mutant bfGnRHR-1 with serial deletions of C tail (panel B) were cotransfected with either the CRE- or SRE-luc reporter. The diagram in panel B represents a serial deletion of C tail from the distal part of bfGnRHR-1. The +1/3, +2/3, and +3/3 in panel A indicate the addition of 1/3, 2/3, and 3/3 of bfGnRHR-1 C tail to rat GnRHR. *, P < 0.05.

 
The HFRK Motif Is Critical for the AC/PKA-Linked Signaling Pathway
The 1/3 part of the bfGnRHR-1 C tail contains 25 amino acids. To identify the specific motif responsible for the AC/PKA-linked pathway, we made serial deletions of 1/3 fragment from bfGnRHR-1 and added these fragments to rat GnRHR. Deletion of the distal TSSSVT sequence slightly decreased the CRE-luc activity compared with that of the 1/3 bfGnRHR-1 mutant. CRE-luc activity similar to that of the wild type was maintained in the mutant harboring a membrane-proximal HFRK sequence (Fig. 4AGo). Interestingly, a deletion of Lys from the HFRK sequence remarkably diminished CRE-luc activity. The deletion of two amino acids, Arg and Lys, from the HFRK motif further lessened the CRE-luc activity such that it was similar to that of the tailless bfGnRHR-1 mutant. The serial deletion of the 1/3 fragment did not critically affect SRE-luc activity. This observation strongly suggests that the membrane-proximal HFRK sequence is critical for the AC/PKA-linked signaling pathway of bfGnRHR-1.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 4. Requirement of the Membrane-Proximal HFRK Motif for CRE-luc Activity

Amino acid segments of the 1/3 part of bfGnRHR-1 C tail were sequentially deleted (panel A), and the same segments were added to rat GnRHR (panel B) as shown in a schematic diagram. These mutant rat and bfGnRHRs were cotransfected with CRE- or SRE-luc reporter in HeLa cells and treated with 1 µM cGnRH-II for 6 h. Luciferase activities were measured and normalized against ß-galactosidase activity. *, P < 0.05.

 
The same C tail fragments were added to rat GnRHR. A significant rise in CRE-luc activity of the mutant rat GnRHR with the HFRK motif was observed, and the addition of other sequences gradually increased CRE-luc activity (Fig. 4BGo). However, the increase in CRE-luc activity by addition of the HFRK motif or the 1/3 fragment was not as drastic as that of bfGnRHR-1 mutants. It should be noted that SRE-luc activity gradually decreased after the serial addition of amino acids. The decrease in SRE-luc activity cannot be attributable to a decreased receptor expression, as the addition of C tails to rat GnRHR did not critically affect their expression (Table 1Go). Thus, it is likely that the total activity of mutant rat GnRHR gradually decreases as a function of an increasing number of amino acids.

To quantify signaling preferences, the ratios of CRE-luc to SRE-luc activity were determined (Table 2Go). Consistent with Fig. 4Go, the bfGnRHR-1 C tail mutants that contained only 1/3 part of the C tail exhibited a 30% higher ratio of CRE-luc to SRE-luc activity compared with that of wild-type bfGnRHR-1. The further deletion of the 1/3 tail gradually decreased this ratio, and a drastic decrease in the ratio was observed in the mutant with the HFR or HF sequence. Interestingly, rat GnRHR with the HFRK motif exhibited a 3-fold higher ratio of CRE-luc to SRE-luc activity compared with the wild-type receptor. The addition of other downstream sequences gradually increased the ratio of CRE-luc to SRE-luc activity (Table 2Go).


View this table:
[in this window]
[in a new window]
 
Table 2. Ratio of CRE-luc to SRE-luc Activity of Wild-Type (WT) and Mutant GnRHRs

 
The effect of the HFRK motif on AC/PKA-linked signaling was further determined by measuring cAMP and inositol phosphate (IP) production in cells expressing wild-type and C tail mutant GnRHRs (Fig. 5Go). Consistent with the data obtained from the CRE- and SRE-luc assay system, addition of the HFRK sequence and the 1/3 fragment of bfGnRHR-1 C tail to rat GnRHR significantly increased cAMP production, whereas IP production decreased somewhat (Fig. 5AGo). Deletion of the C tail from bfGnRHR-1 led to a decrease in cAMP production and did not affect IP production (Fig. 5BGo). These results provide further evidence that the HFRK motif of bfGnRHR-1 C tail are primarily involved in the AC/PKA-linked signaling pathway.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5. GnRH-Induced cAMP Production and IP production in Cells Expressing Rat and bfGnRHR-1 Wild Types and C Tail Mutants

A, HeLa cells were transfected with plasmids containing rat GnRHR wild type (wt), and the mutant receptors with the HFRK motif (+HFRK), 1/3 fragment (+1/3), and 3/3 fragment (+3/3) of bfGnRHR-1 C tail. B, HeLa cells were transfected with plasmids containing wild-type bfGnRHR-1 (wt) or the mutant receptors having the 1/3 fragment (1/3), HFRK motif (HFRK), and no C tail fragment (0/3), respectively. The cells were treated with 1 µM cGnRH-II for 30 min at 37 C. cAMP and IP assays were performed as described in Materials and Methods. Data were expressed as fold-induction over basal. a, P < 0.05 vs. basal; b, P < 0.05 vs. wild type.

 
To examine sequence specificity of the HFRK motif in the AC/PKA-linked signaling pathway, each of the amino acids was replaced by Ala within the HFRK motif or the full-length C tail sequence of bfGnRHR-1 (Fig. 6Go). Substitution of Ala for His in the HFRK motif decreased CRE-luc activity by 30%. Other substitutions (Ala for Phe, Arg, or Lys) greatly reduced CRE-luc activity such that the activity was similar to that of tailless bfGnRHR-1 (Fig. 6AGo). The substitution of HAAA or AAAA for the HFRK motif in the context of a full-length C tail significantly lowered CRE-luc activity by 40–50% (Fig. 6BGo). However, all these substitutions did not alter the SRE-luc activity. Taken together, these results strongly indicate that the HFRK motif is essential for triggering AC/PKA-linked signaling.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 6. Effect of Ala Substitution in the HFRK Motif

Each or all amino acids in the HFRK motif were substituted with Ala in the context of the HFRK motif (panel A) and in the context of full-length bfGnRHR-1 (panel B). The mutant receptors with the HFRK motif or the Ala-substituted sequence were cotransfected with CRE- or SRE-luc. After a 48-h incubation, cells were treated with 1 µM cGnRH-II for 6 h, and luciferase activities were measured. a, P < 0.05 vs. wild type; b, P < 0.05 vs. bfGnRHR-1 mutant with HFRK.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mammalian type-I GnRHR is the only GPCR that lacks an intracellular C tail. The absence of the C tail was regarded as the main reason for delayed desensitization and internalization (14, 15). Indeed, the addition of the C tail from the catfish GnRHR or TRH receptor to rat GnRHR facilitates receptor desensitization (14). In the present study, we demonstrate that the C tail of GnRHR can control the AC/PKA-linked signaling pathway, probably through G{alpha} protein. We also show that a deletion of the C tail from bfGnRHR-1 remarkably decreases the ability to induce the AC/PKA-linked signaling pathway. In particular, the HFRK motif in the membrane-proximal sequence of bfGnRHR-1 C tail appears to be critical for the AC/PKA-linked signaling pathway, as the addition of this motif to rat GnRHR or the deletion of it from bfGnRHR-1 significantly affects the ability of the receptors to induce AC/PKA-linked signaling.

Attempts to identify a specific motif responsible for G protein coupling to, or preference for, a certain signal transduction pathway were made by producing chimeric or site-directed mutant receptors (26, 27, 28, 29). Now it is well established that multiple intracellular regions, particularly the second and third intracellular segments of GPCRs, are involved in differential signaling preference (30). A considerable body of evidence suggests that these regions act in a cooperative fashion to dictate proper G protein recognition and efficient G protein activation (31, 32). However, to date, no consensus sequences responsible for differential G protein coupling have been identified; thus it is postulated that the receptor coupling is dependent on three-dimensional structures or receptor conformation and not on primary sequence (33).

In the present study, we found that an HFRK motif in the membrane-proximal sequence of the bfGnRHR-1 C tail is an essential motif for the AC/PKA-linked signaling pathway. Generally, the C tails of GPCRs are known to be responsible for receptor desensitization by serving as substrates for various receptor kinases or binding sites for endocytosis machinery proteins (34, 35, 36, 37). Indeed, we demonstrated that the deletion or addition of a C tail considerably affects receptor desensitization (data not shown). C tail involvement in signaling cascades, however, has rarely been discussed. Some deletion or mutagenesis studies have shown that most of the C tail is not required for efficient G protein coupling (38, 39). However, other studies demonstrated that C-terminal-truncated receptors can couple to G proteins with improved efficacy (40, 41, 42) or display an increased basal activity (43). It is known that the C tail of metabotropic glutamate receptor 7 binds to {gamma}-subunits, which mediate Ca2+ channel closure and an inhibition of neurotransmitter release (44, 45, 46). Moreover, Ca2+/CaM binding to the C tail promotes G protein-mediated signaling by displacing Gß{gamma}-subunits from the C tail (47). In this study, we demonstrate that the C tail, especially the HFRK motif, markedly affects the AC/PKA-linked signaling for either bfGnRHRs or rat GnRHR without influencing the PLC/PKC-linked signaling pathway.

It should be noted that Ala substitution in the HFRK motif greatly decreases the ability to induce AC/PKA-linked signaling, indicating sequence specificity for a particular pathway. Further, it is of interest to note that bfGnRHR-2 and bfGnRHR-3 possess an HFRK-like motif (SFK for bfGnRHR-2 and HFRR for bfGnRHR3) in the membrane-proximal sequence. Because deleting the C tail of bfGnRHR-2 and bfGnRHR-3 caused a significant decrease in the ratio of CRE-luc to SRE-luc activity, it is possible that such HFRK-like motifs are involved in the signaling pathway regulation of bfGnRHRs. Together with the sequence analysis of bfGnRHRs, at least Phe, followed by a basic amino acid (Lys or Arg), is conserved in these receptors. Whether such a consensus sequence is recognized by a particular G protein remains to be elucidated.

In addition to the HFRK motif, a Ser/Thr cluster followed by Val and Thr (TSSSVT) or a neighboring sequence within the 1/3 fragment appears to aid the association of the receptor with AC/PKA-linked signaling, as the deletion of the TSSSVT sequence significantly reduces CRE-luc activity (Fig. 4AGo). The still considerable CRE-luc activity of the receptor with the HFRK mutation in the context of the full-length C tail, despite a significantly decreased CRE-luc activity compared with that of wild type receptor, further supports the above notion. Moreover, it is of interest to note that GnRHR with the 1/3 C tail showed a higher CRE-luc activity than the receptors with the longer C tail, suggesting the presence of an inhibitory cis-element in the distal part of the C tail. It is possible that the 2/3 fragment of the C tail may induce a steric hindrance by random protein folding, which could partly constrain G protein coupling of the receptor (33). Alternatively, protein binding to a specific sequence in the distal part of the C tail may influence coupling to the AC/PKC-linked pathway. The C tail fragment contains many potential phosphorylation and protein binding sites. DTSSS and SDAD motifs in the 1/3 fragment are potential candidates for substrates of GPCR kinase and casein kinase II, respectively (37, 48). The SFR motif and SMSS in the 2/3 fragment might serve as binding sites for PKC and ß-arrestin, respectively (37, 49). However, it remains to be elucidated how these motifs, or others yet unknown, participate positively or negatively in the AC/PKC-linked pathway.

It is possible that a nonmammalian GnRHR may have evolved into the mammalian type-I GnRHR and, in doing so, lost the C tail. This loss then altered the ability of the receptor to activate the AC/PKA-linked signaling pathway. However, by losing the C tail, the mammalian type-I GnRHR acquired an ability to maintain its activity for a longer period. Although the physiological relevance of a C tail is uncertain, our study suggests that the regulatory mechanisms underlying secretion and synthesis of LH/FSH through the GnRHRs of mammalian and nonmammalian species are definitely different (50).

In conclusion, it is still not clear whether G protein coupling is regulated by consensus sequence motifs or mediated through conformational recognition. However, the HFRK-like motif in the membrane-proximal portion of the C tail could prove to be important for the AC/PKA-linked signaling pathway; direct evidence, including studies of the physical interaction between this HFRK-like motif and Gs protein by cross-linking, immunoprecipitation, or GTP-loading, needs to be provided. Elucidating precise G protein-coupling mechanisms may eventually lead to new insights into the complex processes governing receptor-G protein coupling. Moreover, specific motifs and conformational structures responsible for differential G protein coupling may lead to an understanding of the evolutionary process of GnRHRs as well as how they control the neuroendocrine and reproductive systems.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Reagents
Chicken GnRH-II and mammalian GnRH were synthesized by AnyGen (Gwangju, Korea). TPA, FKN, and alumina (Neutral WN-3) were purchased from Sigma Chemical Co. (St. Louis, MO), and GF109203X and H-89 were obtained from Calbiochem (San Diego, CA).

Plasmids
The cDNAs for bfGnRHRs were constructed at the EcoRI and XbaI sites of pcDNA3 (Invitrogen, San Diego, CA) as previously described (8). Tail mutants of rat GnRHR and bfGnRHRs were generated by PCR. All PCR-derived sequences were verified as correct by automatic sequencing. hß2AR and hOTR were kind gifts from Dr. K. M. Kim, Chonnam National University, and Dr. Thierry Durroux, INSERM U469. The pCMV ß-Gal was purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The CRE-luc vector containing four copies of the CRE (TGACGTCA) was from Stratagene (La Jolla, CA). SRE-luc, containing a single copy of the SRE (CCATATTAGG) followed by c-fos basic promoter, was constructed in our laboratory.

Luciferase Assays
HeLa cells and HEK 293T cells were maintained in DMEM in the presence of 10% FBS (Biowhittaker, Walkersville, MD). For luciferase assays, the cells were plated into 24-well plates and transfected 24 h later with SuperFect reagent (QIAGEN, Valencia, CA). The total amount of DNA used in each transfection was adjusted to 1 µg by adding appropriate amounts of pcDNA3. Approximately 48 h after transfection, cells were treated with the appropriate ligand for 6 h. For SRE-luc analysis, cells were maintained in serum-free DMEM for at least 16 h before ligand treatment. After treatment, cells were harvested and luciferase activity in cell extracts was determined using a luciferase assay system according to standard methods in a Lumat LB9501 (EG&G, Berthold, Germany). Luciferase values were normalized by the ß-galactosidase values. Transfection experiments were performed in duplicate and repeated at least three times.

cAMP Assay
Levels of cAMP were determined by measuring [3H]cAMP formation from [3H]ATP (51). HeLa cells were seeded into 12-well plates 24 h before transfection. Cells were transfected with Effectene reagent (QIAGEN), and 1 d later were labeled with 2 µCi/ml of [3H]adenine (NEN Life Science Products, Boston, MA) for 24 h. Cells were first washed in PBS and then incubated at 37 C for 20 min in serum-free DMEM containing 1 mM 1-methyl-3-isobutylxanthine and stimulated with or without 1 µM cGnRH-II for 30 min. The reactions were terminated by replacing medium by ice-cold 5% trichloroacetic acid containing 1 mM ATP and 1 mM cAMP. [3H]cAMP and [3H]ATP were separated on AG 50W-X4 resin (Bio-Rad Laboratories, Hercules, CA) and alumina columns as previously described (52). The cAMP accumulation was expressed as [3H]cAMP/([3H]ATP + [3H]cAMP) x 1000.

Measurement of IP Production
HEK 293T cells were maintained in DMEM in the presence of 10% FBS. IP production assays were performed as previously described (8, 34). HEK 293T cells were seeded into a 12-well plates 24 h before transfection and transfected the next day using Effectene reagent (QIAGEN) according to the manufacturer’s instruction. After transfection, cells were incubated in M199 medium (Life Technologies, Gaithersburg, MD) containing 2% FBS and labeled with 1 µCi/ml myo-[3H] inositol (Amersham Pharmacia Biotech, Piscataway, NJ) per well for 20 h. Medium was 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, and 1 mg/ml fatty acid-free BSA). Cells were then preincubated with buffer A containing 10 mM LiCl for 30 min, followed by addition of the 1 µM cGnRH-II at 37 C for 30 min. Replacing incubation medium by 0.5 ml of ice-cold 10 mM formic acid terminated the reaction. After 30 min at 4 C, the formic acid extracts were transferred to columns containing Dowex anion-exchange resin (AG-1-X8 resin, Bio-Rad Laboratories, Inc.). Total IPs were then eluted with 1 ml of ammonium formate/0.1 M formic acid, and radioactivity was determined.

Binding Assays
To determine receptor expression in various mutant GnRHR-expressing HeLa cells, whole-cell binding assays were carried out using the radiolabeled tracers, [125I]chicken GnRH-II and [125I]mammalian GnRH in which 1 x 105 cells were seeded per well in 12-well plates 24 h before assay. The cells were washed with PBS twice. Radioactivity was determined by resolving the cells in 1% sodium dodecyl sulfate and 0.2 M NaOH. Nonspecific binding was determined in the presence of 1 µM unlabeled (cold) chicken GnRH-II and mammalian GnRH.

Data Analysis
All assays were performed in triplicate and repeated three times; the data are presented as mean ± SEM of at least three independent experiments. Data analyses used one-way ANOVA followed by Newman-Keuls post test. P < 0.05 was considered statistically significant.


    FOOTNOTES
 
This work was supported by a grant of the Korea Health 21 Research and Development Project, Ministry of Health and Welfare (01-PJ1-PG3–20500-0035) and a grant (M103KV010004 03K2201 00410) from Brain Research Center of the 21st Century Frontier Research Program.

First Published Online November 24, 2004

Abbreviations: AC, Adenylate cyclase; AR, adrenergic receptor; C tail, C-terminal tail; CRE, cAMP-responsive element; FKN, forskolin; GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; hOTR, human oxytocin receptor; IP, inositol phosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipases C; SRE, serum-responsive element; TPA, 12-O-tetradecanoylphenol-13-acetate.

Received for publication June 1, 2004. Accepted for publication November 17, 2004.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Conn PM, Crowley WF 1994 Gonadotropin-releasing hormone and its analogs. Annu Rev Med 45:391–405[CrossRef][Medline]
  2. Reinhart JM, Mertz LM, Catt KJ 1992 Molecular cloning and expression of cDNA encoding the murine gonadotropin-releasing hormone receptor. J Biol Chem 267:21281–21284[Abstract/Free Full Text]
  3. Tsutsumi M, Shou W, Millar RP, Mellon PL, Roberts JL, Flanagan CA, Dong K, Gillo B, Sealfon SC 1992 Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor. Mol Endocrinol 6:1163–1169[Abstract]
  4. Kakar SS, Musgrove LC, Devor DC, Sellers JC, Neill JD 1992 Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor. Biochem Biophy Res Commun 189:289–295[Medline]
  5. Tensen C, Okuzawa K, Blomenröhr M, Rebers F, Leurs R, Bogerd J, Schulz R, Goos H 1997 Distinct efficacies for two endogenous ligands on a single cognate gonadoliberin receptor. Eur J Biochem 243:134–140[Abstract]
  6. Illing N, Troskie BE, Nahornlak CS, Hapgood JP, Peter RE, Millar RP 1999 Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the gold fish (Carassius auratus). Proc Natl Acad Sci USA 96:12280–12284
  7. Troskie BE, Hapgood JP, Millar RP, Illing N 2000 Complementary deoxyribonucleic acid cloning, gene expression, and ligand selectivity of a novel gonadotropin-releasing hormone receptor expressed in the pituitary and midbrain of Xenopus laevis. Endocrinology 141:1764–1771[Abstract/Free Full Text]
  8. Wang L, Bogerd J, Choi HS, Seong JY, Soh JM, Chun SY, Blomenröhr M, Troskie BE, Millar RP, Yu WH, McCann SM, Kwon HB 2001 Three distinct types of GnRH receptor characterized in the bullfrog. Proc Natl Acad Sci USA 98:361–366[Abstract/Free Full Text]
  9. Wang L, Oh DY, Bogerd J, Choi HS, Ahn RS, Seong JY, Kwon HB 2001 Inhibitory activity of alternative splice variants of the bullfrog GnRH receptor-3 on wild type receptor signaling. Endocrinology 142:4015–4025[Abstract/Free Full Text]
  10. Millar R, Lowe S, Conklin D, Pawson A, Maudsley S, Troskie B, Ott T, Millar M, Lincoln G, Sellar R, Faurholm B, Scobie G, Kuestner R, Terasawa E, Katz A 2001 A novel mammalian receptor for the evolutionarily conserved type II GnRH. Proc Natl Acad Sci USA 98:9636–9641[Abstract/Free Full Text]
  11. Neill JD, Duck LW, Sellers JC, Musgrove LC 2001 A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates. Biochem Biophys Res Commun 282:1012–1018[CrossRef][Medline]
  12. Powell JF, Zohar Y, Elizur A, Park M, Fischer WH, Craig AG, Rivier JE, Lovejoy DA, Sherwood NM 1994 Three forms of gonadotropin-releasing hormone characterized from brains of one species. Proc Natl Acad Sci USA 91:12081–12085[Abstract/Free Full Text]
  13. Fernald RD, White RB 1999 Gonadotropin-releasing hormone genes: phylogeny, structure, and functions. Front Neuroendocrinol 20:224–240[CrossRef][Medline]
  14. Heding A, Vrecl M, Bogerd J, McGregor A, Sellar R, Taylor PL, Eidne KA 1998 Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics. J Biol Chem 273:11472–11477[Abstract/Free Full Text]
  15. Willars GB, Heding A, Vrecl M, Sellar R, Blomenrohr M, Nahorski SR, Eidne KA 1999 Lack of a C-terminal tail in the mammalian gonadotropin-releasing hormone receptor confers resistance to agonist-dependent phosphorylation and rapid desensitization. J Biol Chem 274:30146–30153[Abstract/Free Full Text]
  16. Fukushima Y, Asano T, Takata K, Funaki M, Ogihara T, Anai M, Tsukuda K, Saitoh T, Katagiri H, Aihara M, Matsuhashi N, Oka Y, Yazaki Y, Sugano K 1997 Role of the C terminus in histamine H2 receptor signaling, desensitization, and agonist-induced internalization. J Biol Chem 272:19464–19470[Abstract/Free Full Text]
  17. Hukovic N, Panetta R, Kumar U, Rocheville M, Patel YC 1998 The cytoplasmic tail of the human somatostatin receptor type 5 is crucial for interaction with adenylyl cyclase and in mediating desensitization and internalization. J Biol Chem 273:21416–21422[Abstract/Free Full Text]
  18. Blomenröhr M, Bogerd J, Leurs R, Schulz RW, Tensen CP, Zandbergen MA, Goos HJ 1997 Differences in structure-function relations between nonmammalian and mammalian gonadotropin-releasing hormone receptors. Biochem Biophys Res Commun 238:517–522[CrossRef][Medline]
  19. Grosse R, Schmid A, Schoneberg T, Herrlich A, Muhn P, Schultz G, Gudermann T 2000 Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins. J Biol Chem 275:9193–9200[Abstract/Free Full Text]
  20. Berridge MJ 1993 Inositol triphosphate and calcium signaling. Nature 361:315–325[CrossRef][Medline]
  21. Arora KK, Krsmanovic LZ, Mores N, O’Farrell H, Catt KJ 1998 Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor. J Biol Chem 273:25581–25586[Abstract/Free Full Text]
  22. Liu F, Usui I, Evans LG, Austin DA, Mellon PL, Olefsky JM, Webster NJG 2002 Involvement of both Gq/11 and Gs proteins in gonadotropin-releasing hormone receptor-mediated signaling in LßT2 cells. J Biol Chem 277:32099–32108[Abstract/Free Full Text]
  23. Oh DY, Wang L, Ahn RS, Park JY, Seong JY, Kwon HB 2003 Differential G protein coupling preference of mammalian and nonmammalian gonadotropin-releasing hormone receptors. Mol Cell Endocrinol 205:89–98[CrossRef][Medline]
  24. Seifert R, Wenzel-Seifert K, Lee TW, Gether U, Sanders-Bush E, Kobilka BK 1998 Different effects of Gs{alpha} splice variants on ß2-adrenoreceptor-mediated signaling. The ß2-adrenoreceptor coupled to the long splice variant of Gs{alpha} has properties of a constitutively active receptor. J Biol Chem 273:5109–5116[Abstract/Free Full Text]
  25. Ku CY, Qian A, Wen Y, Anwer K, Sanborn BM 1995 Oxytocin stimulates myometrial guanosine triphosphatase and phospholipase-C activities via coupling to G{alpha}q/11. Endocrinology 136:1509–1515[Abstract]
  26. Hedin KE, Duerson K, Calpharm DE 1993 specificity of receptor-G protein interactions: searching for the structure behind signal. Cell Signal 5:505–518[CrossRef][Medline]
  27. Wong SKF, Parker EM, Ross EM 1990 Chimeric muscarinic cholinergic: ß-adrenergic receptors that activate Gs in response to muscarinic agonists. J Biol Chem 265:6219–6224[Abstract/Free Full Text]
  28. Wong SKF, Ross EM 1994 Chimeric muscarinic cho-linergic: ß-adrenergic receptors that are functionally promiscuous among G proteins. J Biol Chem 269:18968–18976[Abstract/Free Full Text]
  29. Liggett SB, Caron MG, Lefkowitz RJ, Hnatowich M 1991 Coupling of a mutated form of the human ß2-adrenergic receptor to Gi and Gs: requirement for multiple cytoplasmic domains in the coupling process. J Biol Chem 266:4816–4821[Abstract/Free Full Text]
  30. Wess J 1997 G protein-coupled receptors: molecular mechanisms involved in receptor activation and selectivity of G protein recognition. FASEB J 11:346–354[Abstract/Free Full Text]
  31. Pin JP, Joly C, Heinemann SF, Bokaert J 1994 Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors. EMBO J 13:342–348[Abstract]
  32. Gomeza J, Joly C, Kuhn R, Knöpfel T, Bockaert J, Pin JP 1996 The second intracellular loop of the metabotropic glutamate receptor 1 cooperates with other intracellular domains to control coupling to G proteins. J Biol Chem 271:2199–2205[Abstract/Free Full Text]
  33. Wess J 1998 Molecular basis of receptor/G protein-coupling selectivity. Pharmacol Ther 80:231–264[CrossRef][Medline]
  34. Acharjee S, Maiti K, Soh JM, Im WB, Seong JY, Kwon HB 2002 Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors. Mol Cells 14:101–107[Medline]
  35. Ferguson SSG, Barak LS, Zhang J, Caron MG 1996 G protein-coupled receptor regulation: role of G protein-coupled receptor kinases and arrestins. Can J Physiol Pharmacol 74:377–384
  36. Diviani D, Lattion AL, Cotecchia S 1997 Characterization of the phosphorylation sites involved in G protein-coupled receptor kinase- and protein kinase C-mediated desensitization of the {alpha}1B-adrenergic receptor. J Biol Chem 272:28712–28719[Abstract/Free Full Text]
  37. Hanyaloglu AC, Vrecl M, Kroeger KM, Miles LEC, Qian H, Thomas WG, Eidne KA 2001 Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to ß-arrestin-dependent internalization. J Biol Chem 276:18066–18074[Abstract/Free Full Text]
  38. Alblas J, van Etten I, Khanum A, Moolenaar WH 1995 C-terminal truncation of the neurokinin-2 receptor causes enhanced and sustained agonist-induced signaling: role of receptor phosphorylation in signal attenuation. J Biol Chem 270:8944–8951[Abstract/Free Full Text]
  39. Preisser L, Ancellin N, Michaelis L, Creminon C, Morel A, Corman B 1999 Role of the carboxyl-terminal region, di-leucine motif and cysteine residues in signaling and internalization of vasopressin V1a receptor. FEBS Lett 460:303–308[CrossRef][Medline]
  40. Parker EM, Ross EM 1991 Truncation of the extended carboxyl-terminal domain increases the expression and regulatory activity of the avian ß-adrenergic receptor. J Biol Chem 266:9987–9996[Abstract/Free Full Text]
  41. Ida-Klein A, Guo J, Xie LY, Juppner H, Potts Jr JT, Kronenberg HM, Bringhurst FR, Abou-Samra AB, Segre GV 1995 Truncation of the carboxyl-terminal region of the rat parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of adenylyl cyclase but not phospholipase C. J Biol Chem 270:8458–8465[Abstract/Free Full Text]
  42. Pankevych H, Korkhov V, Freissmuth M, Nanoff C 2003 Truncation of the A1 adenosine receptor reveals distinct roles of the membrane-proximal carboxyl terminus in receptor folding and G protein coupling. J Biol Chem 278:30283–30293[Abstract/Free Full Text]
  43. Hasegawa H, Negishi M, Ichikawa A 1996 Two isoforms of the prostaglandin E receptor EP3 subtypes different in agonist independent constitutive activity. J Biol Chem 271:1857–1860[Abstract/Free Full Text]
  44. El Far O, Bofill-Cardona E, Airas JM, O’Connor V, Boehm S, Freissmuth M, Nanoff C, Betz H 2001 Mapping of calmodulin and Gß{gamma} binding domains within the C-terminal region of the metabotropic glutamate receptor 7A. J Biol Chem 276:30662–30669[Abstract/Free Full Text]
  45. Herlitze S, Garcia DE, Mackie K, Hille B, Scheuer T, Catterall WA 1996 Modulation of Ca2+ channels by G-protein ß {gamma} subunits. Nature 380:258–262[CrossRef][Medline]
  46. Ikeda SR 1996 Voltage-dependent modulation of N-type calcium channels by G-protein ß {gamma} subunits. Nature 380:255–258[CrossRef][Medline]
  47. O’Connor V, El Far O, Bofill-Cardona E, Nanoff C, Freissmuth M, Karschin A, Airas JM, Betz H, Boehm S 1999 Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling. Science 286:1180–1184[Abstract/Free Full Text]
  48. Fredericks ZL, Pitcher JA, Lefkowitz RJ 1996 Identification of the G protein-coupled receptor kinase phosphorylation sites in the human ß2-adrenergic receptor. J Biol Chem 271:13796–13803[Abstract/Free Full Text]
  49. Oakely RH, Laporte SA, Holt JA, Barak LS, Caron MG 2001 Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-ß-arrestin complexes after receptor endocytosis. J Biol Chem 276:19452–19460[Abstract/Free Full Text]
  50. Klausen C, Chang JP, Habibi HR 2002 Multiplicity of gonadotropin-releasing hormone signaling: a comparative perspective. Prog Brain Res 141:111–128[Medline]
  51. Marsh SR, Grishina G, Wilson PT, Berlot CH 1998 Receptor-mediated activation of Gs{alpha}: evidence for intramolecular signal transduction. Mol Pharmacol 53:981–990[Abstract/Free Full Text]
  52. Wedegaertner PB, Chu DH, Wilson PT, Levis MJ, Bourne HR 1993 Palmitoylation of required for signal functions and membrane attachment of Gq {alpha} and Gs {alpha}. J Biol Chem 268:25001–25008[Abstract/Free Full Text]