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Endocrinology Vol. 143, No. 5 1732-1740
Copyright © 2002 by The Endocrine Society

RECEPTORS

Intracellular Calcium Mobilization in Response to the Activation of Human Wild-Type and Chimeric Gonadotropin Receptors

Pearly S. N. Lee, Alison M. J. Buchan, Aaron J. W. Hsueh, Basil Ho Yuen and Peter C. K. Leung1

Departments of Obstetrics and Gynecology (P.S.N.L., B.H.Y., P.C.K.L.) and Physiology (A.M.J.B.), University of British Columbia, Vancouver, British Columbia, Canada V6H 3V5; and Division of Reproductive Biology, Stanford University Medical Center (A.J.W.H.), Stanford, California 94305

Address all correspondence and requests for reprints to: Dr. Peter C. K. Leung, Department of Obstetrics and Gynecology, University of British Columbia, 2H30-4490 Oak Street, Vancouver, British Columbia, Canada V6H 3V5. E-mail: . peleung{at}interchange.ubc.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is well established that LH action is mediated primarily by adenylate cyclase/cAMP. However, the role of inositol phosphate/calcium in LH signaling is less well established. We examined the effects of gonadotropins in primary culture human granulosa-lutein cells and in HEK293 cells transiently transfected with human wild-type or chimeric gonadotropin receptors. The intracellular free calcium concentration was measured using fura-2 microspectrofluorometric techniques. Human (h) LH (2–4 µg/ml) and CG (10 IU/ml) consistently evoked oscillatory calcium signals in HEK293 cells transfected with hLH receptor, whereas hFSH (2–4 µg/ml) failed to elicit any response. Conversely, both hLH and hFSH failed to elicit a calcium response from HEK293 cells transfected with hFSHR, indicating the specificity of the response to the LH receptor. Pretreatment of transfected HEK293 cells with pertussis toxin (100 ng/ml) attenuated all gonadotropin-evoked calcium mobilization.

Studies with chimeric LH receptor showed that the sequence of the long extracellular portion of the receptor was not critical for stimulation of PLC activity, but maintained agonist binding specificity. The C-terminal sequence of the receptor was clearly important for the generation of the basal calcium oscillations, but the precise extent of the critical sequence has yet to be identified. Although various subdivisions of this region were capable of stimulating calcium transients, an intact carboxyl-terminal third of the receptor was required for normal and sustained intracellular calcium signaling. Our study unequivocally shows that the hLH receptor is coupled to the inositol phosphate/calcium signaling pathway via a pertussis toxin-sensitive G protein-coupled receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LH and FSH regulate gonadal function and gametogenesis, and are critical for normal sexual maturation and reproductive function. These two pituitary glycoprotein hormones share chemical and structural similarities; both hormones are heterodimers composed of glycosylated subunits ({alpha} and ß) tightly bound in a noncovalent association.

The LH/hCG receptor is highly conserved, with the highest degree of conservation in the transmembrane domains and connecting loops, followed by the extracellular amino-terminal domains. The lowest degree of conservation occurs in the intracellular carboxyl-terminal cytoplasmic tails (1). The human receptor is 85% identical to the rat LH/CG receptor and 87% identical to the porcine LH/CG receptor (2).

The main difference in biological activity between hCG and LH is the more prolonged action of hCG in vivo, because of its slower metabolic clearance and its somewhat higher affinity for the LH receptor sites in the testis and ovary. LH and CG bind to the same LH/CG receptor, which belongs to the family of G protein-coupled receptors (GPCR) (2, 3, 4, 5, 6, 7). It is well established that the LH/CG receptor is coupled to the adenylate cyclase/cAMP pathway (8, 9, 10). However, it has been reported that the murine and rat LH receptors can signal via the PLC/IP3 pathway (11, 12, 13, 14). The FSH receptor has structural similarities to the LH/CG receptor (15, 16); however, the intracellular signaling pathways activated have not been comprehensively investigated.

Over the last 2 decades, it has become evident that the concentration of intracellular calcium is critical to the regulation of normal cellular activities such as secretion, division, and differentiation. The elevation in intracellular free calcium ion concentration ([Ca2+]i) may arise from the release of calcium from internal stores and/or the influx of extracellular calcium (17, 18, 19). Intracellular calcium oscillations, first described by Prince and Berridge (20), are involved in the potentiation of a ligand response (21), with the amplitudes and frequency activating different signaling pathways. Calcium oscillations have been reported after the activation of several receptors in the reproductive system, notably the P2-purinoreceptor in human ovarian cells (18, 19) and the GnRH receptor in gonadotrophs (22).

To investigate the structure-function of human gonadotropin receptors, we studied the mobilization of [Ca2+]i in response to LH and hCG treatment, in primary cultures of human granulosa-lutein cells (GLCs) and human embryonic kidney 293 (HEK293) cells expressing wild-type and chimeric human gonadotropin receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and materials
Human gonadotropic receptor cDNAs were obtained as described previously (23). Human (h) LH and FSH were obtained from the National Pituitary and Hormone Distribution Program. hCG was obtained from Sigma (St. Louis, MO). Caffeine, DMEM, HBSS, penicillin-streptomycin, thapsigargin, and trypsin were obtained from Life Technologies, Inc. (Burlington, Canada). Heat-inactivated FBS was obtained from HyClone Laboratories, Inc. (Logan, UT). Fura-2/AM was obtained from Molecular Probes, Inc. (Eugene, OR).

Human GLC culture
Human GLCs were obtained from the University of British Columbia in vitro fertilization program. The use of GLCs was approved by the University of British Columbia clinical screening committee for research and other studies involving human subjects.

Follicular development was stimulated by one of several protocols. One of the more commonly used protocols involved administering a GnRH analog to down-regulate pituitary function. Once pituitary down-regulation was achieved, human menopausal gonadotropin was administered to stimulate follicular growth. Serum E2 levels and ultrasound measurements of follicular size and number were used as indicators of oocyte maturity. Once at least three follicles exceeded 17 mm in diameter, 10,000 IU hCG were administered, and oocyte retrieval was performed 34–36 h later.

Human GLCs were harvested from the follicular aspirate collected during oocyte retrieval. Harvested human GLCs were centrifuged (1,000 x g, 5 min) and resuspended in DMEM containing 2% penicillin-streptomycin (vol/vol). The cell suspension was then layered onto a Percoll/HBSS (40:60, vol/vol) column and centrifuged (1,000 x g, 5 min). After centrifugation, cells on the surface of the Percoll/HBSS column were collected and suspended in DMEM. This suspension was centrifuged (1,000 x g, 5 min) and resuspended in DMEM containing 5% FBS and 2% penicillin-streptomycin (DMEM/FBS). Cell viability was determined to be approximately 95% by trypan blue exclusion.

The cells were seeded onto 22-mm circular glass coverslips and incubated for 3–4 d with medium 199/FBS at 37 C in humidified air with 5% CO2 before microfluorometric experiments.

Transfection
hLH receptor cDNA was subcloned into the pcDNA3 vector and transiently transfected into HEK293 cells by the calcium phosphate method (24). The HEK293 cells were cultured until 80% confluent, then trypsinized (0.0625% in calcium- and magnesium-free HBSS) and reseeded at a density of 1 x 106 cells/100-mm culture dish. The HEK293 cells were incubated with DMEM containing 5% FBS and 2% penicillin-streptomycin (DMEM/FBS) at 37 C in humidified air with 5% CO2 for 24 h before transfection. Thirty minutes before transfection, the HEK293 cells were incubated at 37 C in humidified air with 3% CO2. Twenty micrograms of cDNA per 100-mm culture dish were used. All HEK293 cells were cotransfected with lacZ cDNA. Transfection efficiency was estimated to be 50–80%, as determined by X-galactosidase staining.

Microspectrofluorimetry
Intracellular calcium levels were measured using established single cell fluorometric techniques (18). All fura-2 ratio measurements were performed using the Attoflour Digital Fluorescence Microscopy System (Atto Instruments, Rockville, MD). The temperature-controlled perifusion chamber was connected to a six-channel system with a flow rate of 1–2 ml/min. All experiments were completed using the Carl Zeiss x40 Fluar oil immersion objective lens (New York, NY). The cells were illuminated alternately with light at 340 and 380 nm. Measurements of [Ca2+]i were collected at 1- to 2-sec intervals. All data presented have been corrected for background fluorescence, as determined by cell-free regions of the coverglass. Changes in the fluorescence ratio recorded at 340 and 380 nm correspond to changes in [Ca2+]i.

Experiments were performed 50–80 h posttransfection. The transfected HEK293 cells were incubated with fura-2/AM loading buffer (2.5 µM) for 30 min at 37 C in humidified air with 5% CO2. The coverglass was mounted onto the temperature-controlled perifusion chamber and equilibrated for 10 min before the start of the experiment. Fura-2-loaded cells were perifused with a balanced salt solution (137 mM NaCl, 5.36 mM KCl, 1.26 mM CaCl2, 0.81 mM MgSO4·7H2O, 0.34 mM Na2HPO4·7H2O, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 10 mM HEPES, and 2.02 mM glucose, pH 7.4).

The data presented are representative of the changes in [Ca2+]i and are reported as the total number of cells imaged (n) and the number of transfections (#) for each protocol. Approximately 50–75% of the selected cells responded to treatments.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In the first set of experiments the coupling of the human LH receptor to the PLC/IP3 pathway was established by assessing mobilization of intracellular calcium via single cell, dual excitation microspectrofluorometry. The data presented are representative of the changes in [Ca2+]i and are reported as the total number of cells imaged (n) and the number of patients or transfections (#) for each protocol. Four control groups (HEK293, HEK293/ß-gal, HEK293/pcDNA3, and HEK293/ß-gal/pcDNA3) were used to demonstrate that the intracellular calcium response was indeed due to activation of the transfected gonadotropic receptors.

Treatment of primary cultures with 10 IU/ml hCG evoked calcium oscillations in human GLCs (Fig. 1Go). Treatment with hFSH (40 µg/ml) failed to elicit a calcium response from the primary culture human GLCs (data not shown). Significant differences in the responses were observed from the different preparations, although all cells were studied after the same interval in culture following isolation. This heterogeneity could be due to a number of factors, including the pharmacological doses of hCG given to stimulate oocyte maturation, to the possible underlying endocrinological causes of infertility in the woman. In view of the heterogeneity of the responses in primary cells, further experiments were conducted using HEK293 cells transfected with either human wild-type or chimeric receptors. These experiments also allowed determination of the importance of specific receptor sequences for intracellular signaling.



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  		Figure 1. Effects of hCG on human GLCs. Single cell microfluorometric studies demonstrated that hCG successfully evoked mobilization of intracellular calcium in human GLCs (n = 26; # = 6). The cells were loaded with fura-2/AM and perifused with a balanced salt solution. All microfluorometric studies were conducted in a temperature-controlled (37 C) chamber. The agonist was administered at a concentration of 10 IU/ml for a duration of 180 sec.

 
Effects of gonadotropic hormones on wild-type gonadotropin receptors
The specificity of the gonadotropin receptor was confirmed by treating HEK293 cells, transfected with wild-type hLH/hCG receptors, with either hFSH or hLH at a dose of 4 µg/ml for a duration of 180 sec (Fig. 2Go). hFSH failed to elicit a calcium response from the transfected cells (n = 42; # = 2). Under the same conditions, hLH consistently evoked oscillatory calcium signals (n = 42; # = 2). The onset of the [Ca2+]i oscillations was rapid, well within 15 sec of the LH treatment. The calcium oscillations lasted throughout the entire treatment period and persisted for 25 min after the cessation of LH treatment.



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  		Figure 2. Effects of gonadotropins on hLH receptors expressed in HEK293 cells. Only hLH was capable of eliciting an intracellular calcium response. The cells were loaded with fura-2/AM and perifused with a balanced salt solution. All microfluorometric studies were conducted in a temperature-controlled (37 C) chamber. The transfected cells were treated with both hFSH (4 µg/ml) and hLH (4 µg/ml) for a duration of 180 sec.

 
The concentration-response relationship between hCG and [Ca2+]i is shown in Fig. 3Go. hCG was administered at 1, 5, and 10 IU/ml for a duration of 180 sec. At 1 and 5 IU/ml (n = 54 and 10, respectively; # = 2 and 1, respectively), hCG elicited baseline calcium oscillations that were sustained even after treatment withdrawal. At 10 IU/ml, hCG evoked a rise in [Ca2+]i, with oscillations superimposed on the sustained plateau phase (n = 81; # = 3). The cessation of the oscillations is probably due to the depletion of internal calcium stores.



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  		Figure 3. Concentration-response between hCG and intracellular calcium.

 
To determine the relative contribution of calcium influx vs. calcium mobilization of cytosolic stores in the initiation and maintenance of the gonadotropic response, hCG was administered in the absence of extracellular calcium. In the presence of 1 mM EGTA, hCG increased [Ca2+]i, consistent with the mobilization of calcium stores, but the response was transient (n = 64; # = 2; Fig. 4Go). To identify the calcium source, transfected HEK293 cells were pretreated with 1 µM thapsigargin (n = 83; # = 2) to empty IP3-gated calcium stores; subsequent perfusion with hCG failed to elicit a calcium response (Fig. 5Go). The role of intracellular IP3-sensitive calcium stores was confirmed by the ability of 20 mM caffeine to block calcium oscillations, returning [Ca2+]i to baseline levels (Figs. 6Go, A and B).



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  		Figure 4. Involvement of extracellular calcium on hCG-evoked calcium mobilization in HEK293 cells transiently transfected with the wild-type hLH receptor. The cells were loaded with fura-2/AM and perifused with a balanced salt solution. All microfluorometric studies were conducted in a temperature-controlled (37 C) chamber. The agonist was administered at a concentration of 10 IU/ml for a duration of 180 sec.

 


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  		Figure 5. Effects of thapsigargin (TPG) on HEK293 cells transiently transfected with the wild-type hLH receptor. Thapsigargin is a plant-derived lactone whose mode of action appears to result from the emptying of intracellular calcium stores by inhibiting sequestration pathways (43 ). Thapsigargin specifically inhibits all members of the endoplasmic and sarcoplasmic reticulum calcium pump family (44 ). The cells were loaded with fura-2/AM and perifused with a balanced salt solution. All microfluorometric studies were conducted in a temperature-controlled (37 C) chamber. The agonist was administered at a concentration of 10 IU/ml for a duration of 180 sec.

 


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  		Figure 6. A, Effects of caffeine (20 mM) on hCG-evoked intracellular calcium mobilization in HEK293 cells transiently transfected with the wild-type hLH receptor. The cells were loaded with fura-2/AM and perifused with a balanced salt solution. All microfluorometric studies were conducted in a temperature-controlled (37 C) chamber. The agonist was administered at a concentration of 10 IU/ml for a duration of 180 sec. B, Effects of caffeine (20 mM), under calcium-free conditions, on hCG-evoked intracellular calcium mobilization in HEK293 cells transiently transfected with the wild-type hLH receptor. The cells were loaded with fura-2/AM and perifused with a balanced salt solution. All microfluorometric studies were conducted in a temperature-controlled (37 C) chamber. The agonist was administered at a concentration of 10 IU/ml for a duration of 180 sec.

 
To determine whether the hLH/hCG receptor is coupled to calcium signaling through the G{alpha}i/o, transfected HEK293 cells were pretreated for 16 h with pertussis toxin (100 ng/ml). After pertussis toxin treatment, hCG failed to elicit a calcium response (n = 163; # = 4; data not shown).

Wild-type LH receptors in both primary cultured human GLCs and transfected HEK293 cells couple to intracellular calcium mobilization; however, the closely related FSH receptors failed to modulate intracellular calcium levels. The close structural homology between the LH and FSH receptors allowed us to use chimeric receptors to establish the precise structural requirements of the LH receptor conferring coupling to intracellular calcium mobilization. We examined 14 chimeric receptors, of which 5 failed to show a calcium response to hCG (Table 1Go).


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  		Table 1. Wild-type and chimeric human gonadotropin receptor schematics and detectability of [Ca2+]i mobilization

 
Structure-function analysis
To investigate the involvement of the extracellular binding region of the hLH/hCG receptor in LH/CG-induced calcium mobilization, the FLR chimeric was generated, wherein the extracellular region of the hFSH receptor was substituted for that of the hLH/hCG receptor. Thus, this chimeric comprised the extracellular region of the hFSH receptor and the transmembrane and intracellular portions of the hLH/hCG receptor. Although activation of the FLR chimeric receptor resulted in an increase in [Ca2+]i, there was a high degree of variability in the level of the responses compared with that of the wild-type human receptor. Marked hysteresis was observed in these FSH-induced calcium profiles (Fig. 7Go; n = 292; # = 5). As expected, hCG (10 IU/ml) failed to elicit a calcium response (data not shown).



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  		Figure 7. Effects of gonadotropin treatment on HEK293 cells transfected with the chimeric human gonadotropin receptor FLR. Porcine FSH (40 µg/ml) and hCG (10 IU/ml) were administered for a duration of 180 sec. The cells were loaded with fura-2/AM and perifused with a balanced salt solution. All microfluorometric studies were conducted in a temperature-controlled (37 C) chamber.

 
To determine whether the first half of the transmembrane region of the hLH/hCG receptor was required for calcium mobilization, the F(1-4)LR chimeric was generated. In this chimeric, the sequence encoding extracellular loop 2 through to the end of the carboxyl-terminus of the hFSH receptor was substituted with that of the hLH/hCG receptor. Stimulation of HEK293 cells expressing this chimeric human receptor with porcine FSH (40 µg/ml) elicited a calcium profile similar to that normally observed with LH/CG activation of the wild-type hLH/hCG receptor (Fig. 8Go; n = 143; # = 3).



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  		Figure 8. Effects of gonadotropin treatment on HEK293 cells transfected with the chimeric human gonadotropin receptor F(1-4)LR. Porcine FSH was administered at a concentration of 40 µg/ml for a duration of 180 sec. The cells were loaded with fura-2/AM and perifused with a balanced salt solution. All microfluorometric studies were conducted in a temperature-controlled (37 C) chamber.

 
To determine whether the calcium-mobilizing effect of the hLH/hCG receptor was generated through its carboxyl-terminus tail region, the FL(C)R was generated. In this chimeric, the intracellular carboxyl-terminus of the hFSH receptor was substituted with that of the hLH/hCG receptor. Porcine FSH (40 µg/ml) elicited a single calcium spike (Table 1BGo; n = 156; # = 3). Neither calcium oscillations nor sustained calcium mobilization were evident after activation of the FL(C)R receptor.

The data from the previous chimeric indicated that additional regions of the receptor were required for complete calcium mobilization. The next chimeric investigated, FL(7-C)R, comprised the third extracellular loop, the seventh transmembrane segment, and the carboxyl-terminus of the hLH/hCG receptor. Stimulation of HEK293 cells with FSH (40 µg/ml) still elicited a calcium response, but the sustained oscillatory pattern was not achieved (Table 1AGo; n = 97; # = 2). Some oscillations were observed, but were not sustained upon removal of the agonist. Marked hysteresis was observed in the calcium responses of the FL(7-C)R receptors.

To investigate whether the central regions of the hLH/hCG receptor were critical for the generation of calcium oscillations, the fifth and sixth transmembrane segments and the third intracellular loop of the hLH/hCG receptor were inserted into the hFSH receptor, FL(V-VI)R. The ligand-induced calcium mobilization profile resembled that obtained with the wild-type hLH/hCG receptor in the absence of extracellular calcium (Table 1BGo; n = 167; # = 3), indicating that this region of the receptor is coupled to the mobilization of intracellular calcium stores.

Previous studies have indicated that the third intracellular loop is responsible for coupling receptors, such as muscarinic receptors (25, 26), to the GPCR. To test whether this was the case for the hLH/hCG receptor, the fifth and sixth transmembrane segments of the hLH/hCG receptor were inserted into the hFSH receptor. Addition of FSH (40 µg/ml) elicited attenuated calcium transients from HEK293 cells transfected with this FL(V/VI)R chimeric receptor; however, oscillations were not observed, and there was a marked desensitization to the second application of the agonist (Table 1BGo; n = 173; # = 3). These data reinforce the importance of the third intracellular loop, but indicate that the fifth and sixth transmembrane segments are also involved in calcium mobilization.

The next chimeric, FL(V-i3)FR, was the hFSH receptor with part of the second extracellular loop, the fifth transmembrane segment, and the third intracellular loop of the hLH/hCG receptor. Addition of FSH (40 µg/ml) elicited calcium transients; however, these were severely attenuated without oscillations, and there was essentially no response to the second addition of the agonist (Table 1BGo; n = 174; # = 3).

The final chimeric that showed evidence of coupling to mobilization of intracellular calcium was the hLH/hCG receptor with the carboxyl-terminal tail region of the hFSH receptor. Activation of the LF(C)R chimeric receptor resulted in the loss of calcium oscillations, and the increase in [Ca2+]i was only sustained for the duration of the gonadotropin treatment (Table 1CGo; n = 131; # = 3), resembling the agonist-induced response of the wild-type receptor in the absence of extracellular calcium (Fig. 6BGo). Porcine FSH (40 µg/ml) failed to elicit a calcium response from this chimeric receptor.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Previous studies have reported LH/CG-induced calcium elevations in Xenopus oocytes (12) and in HEK293 cells transfected with the rat LH/CG receptor (27). The type of calcium response elicited by activation of the rat LH receptor was dependent upon the hormone concentration and the presence of extracellular calcium. Lakkakorpi et al. (27) observed calcium oscillations in 72% of the cells in the presence of extracellular calcium and in only 33% of cells in the absence of extracellular calcium. We observed hCG-induced calcium oscillations in all HEK293 cells expressing the hLH receptor, with differences in the pattern and frequency of oscillation rather than in the absence/presence of calcium oscillations. Hirsch et al. (23) previously reported that HEK293 cells transiently transfected with the hLH/hCG receptor do exhibit hLH-induced elevations in intracellular IP3 and cAMP.

It has long been established that LH action is mediated primarily via the adenylate cyclase signaling pathway (8, 9, 10). However, recent reports have raised the possibility that the PLC signal transduction pathway is also involved in LH action (11, 13, 14, 28). Although the mechanism underlying the bifurcating signal transduction remains unknown, it has been shown that in bovine corpora lutea and L cells stably expressing the murine LH receptor, LH stimulation can couple to both Gs and Gi, and the {alpha}ß-subunits released from either G protein contribute to the stimulation of PLCß isoforms (13). Gudermann et al. (12) have shown that L cells stably expressing murine LH receptors respond to hCG with an increase in their rate of phosphoinositide hydrolysis and an increase in [Ca2+]i.

It has been suggested that a single LH/CG receptor can couple to both adenylate cyclase and PLC, and that the ability of LH/CG to activate PLC is independent of cAMP accumulation (11). The concentrations of LH and hCG used in the present study were in the same range as those employed to induce IP3 accumulation associated with the hLH receptor (23). Interestingly, it appears that higher concentrations of LH are required to activate the PLC pathway than that which is required for the adenylate cyclase pathway. In light of the data reported by Zhu et al. (29), activation of the PLC pathway seems to be associated with events surrounding ovulation and pregnancy, when circulating levels of LH and hCG are high. This concept is corroborated by the well known increase in the number of LH receptors during follicular maturation (30). Moreover, recent studies have shown that the ability of LH to induce ovulation is impaired by PKC inhibitors (31), further supporting a role for the PLC pathway in LH action during the periovulatory period.

Our study shows that the hLH/CG receptor is specific for LH and hCG. Although purified hFSH failed to elicit calcium signals, hLH and hCG consistently evoked calcium oscillations that were sustained even after treatment withdrawal. The initial phase of the hCG-evoked increase in [Ca2+]i results from the mobilization of cytosolic calcium stores and is then sustained by an influx of extracellular calcium. The intracellular calcium stores in question are probably the intracellular IP3-sensitive calcium stores in the endoplasmic reticulum. The hLH/hCG receptor also appears to be coupled to calcium signaling through the Gi protein, as is the case for the murine LH receptor (13). Taken together with recent report of increased IP3 accumulation after activation of the hLH/hCG receptor (13), the present results support the concept that in addition to adenylate cyclase, activation of PLC is an alternative signaling pathway coupled to the hLH/hCG receptor.

Calcium oscillations have been reported after the activation of several receptors in the reproductive system, notably the P2-purinoreceptor in human ovarian cells (18, 19) and the GnRH receptor in gonadotrophs (22). GnRH and endothelin-1 induce biphasic [Ca2+]i transients in gonadotroph cell suspensions, but oscillatory [Ca2+]i responses in single gonadotrophs (22), which resemble the LH-induced [Ca2+]i oscillations observed in the present study.

Several investigators have suggested that calcium oscillations encode hormone signals. Because oscillation frequencies can vary with agonist concentrations, calcium transients might be part of a frequency-encoded signaling system (17, 32). Calcium oscillations probably encode information to be detected over a broad range of hormone levels. Prolonged exposure to low concentrations of some PLC-linked hormones can induce biological responses such as changes in gene expression (33, 34). Meyer et al. (35) suggested that the kinetic behavior of the calmodulin/calcium-calmodulin-dependent protein kinase interaction can detect and respond to calcium oscillations; thus, calmodulin may form the link between calcium oscillations and changes in gene expression.

The data presented in this study clearly indicate that the binding of LH/CG to the hLH/hCG receptor activates the PLC pathway. IP3 stimulates the release of calcium from intracellular stores by binding to and opening the IP3 receptor, a calcium release channel in the endoplasmic reticulum. These receptors are sensitive not only to intracellular IP3 concentrations, but also to intracellular calcium concentrations (36) and function as calcium-induced calcium release channels in the continued presence of IP3 (37). The IP3-induced rate of calcium release depends on a feed-forward mechanism whereby the initial calcium released through the channel stimulates the opening of additional IP3 receptors and a further increase in calcium levels (38, 39). The LH/CG-induced calcium oscillations observed were probably generated by the process of calcium-induced calcium release involving IP3 receptors.

To investigate the segments of the hLH receptor involved in signal transduction, we studied the response of intracellular calcium concentrations to gonadotropin treatment in HEK293 cells expressing wild-type and chimeric human gonadotropin receptors. The advantage of studying gonadotropic receptors is that binding and activation are interrelated, but separate, phenomena (40, 41). Ryu et al. (42) reported that hCG binding at the high affinity site in the amino-terminal half of the receptor induces conformational adjustments. This leads to low affinity secondary contacts of the complex of the hCG amino-terminal end of the receptor with the carboxyl-terminal end of the receptor. This low affinity secondary contact is responsible for activating the receptor. This property allows generation of chimeric receptors with alterations in the signal-transducing transmembrane domains without perturbation of ligand binding.

Replacing the extracellular domain of the hLH receptor with that of the hFSH receptor did not alter receptor activation. Apart from the delay in the onset of the calcium response, the intracellular calcium profile elicited by the binding of FSH to the human chimeric gonadotropin FLR receptor is very similar to that evoked by LH stimulation of the hLH receptor. This suggests that the extracellular domain of the receptor is important for ligand binding, but is not involved in activation of the intracellular signaling pathways. It has previously been reported that receptor activation results in a conformational change in the LH receptor that promotes binding of the cytoplasmic tail to the main body of the receptor. The data obtained using FLR chimeric receptors indicate that switching the external binding site to that of the FSH receptor resulted in FSH binding initiating the conformational change required for C-terminal attachment.

Alterations in the transmembrane regions of the hLH receptor resulted in perturbations of the agonist-induced intracellular calcium profile. In all cases this resulted in ablation of the basal calcium oscillations. Our findings suggest that transmembrane regions V–VII are critical in retaining a normal intracellular calcium profile for gonadotropin-induced activation. Any alteration to these regions resulted in a significantly perturbed calcium profile. These data would be consistent with the conclusion that the carboxyl-terminal third of the hLH receptor mediates the activation of PLC. Although the chimeric receptors containing transmembrane regions V and VI were capable of initiating a transient increase in intracellular calcium levels, these were not equivalent to the sustained basal oscillations produced by stimulation of the native receptor. These results indicate that the intact carboxyl-terminal third of the receptor is required to achieve the normal intracellular calcium oscillation profile.

Activation of the hLH receptor by LH or hCG results in the stimulation of at least two signal transduction pathways. The data presented concerning intracellular calcium dynamics in stimulated cells were consistent with stimulation of the PLC pathway in addition to the established linkage with adenylate cyclase. The studies with the chimeric receptors indicated that the sequence of the long extracellular portion of the receptor was not critical for stimulation of PLC activity, but maintained the specificity of agonist binding. The C-terminal sequence of the receptor is clearly important for generation of the normal calcium oscillatory pattern. These data represent the initial report of a direct linkage between the C-terminal sequence of a GPCR and the extent and duration of the calcium oscillations evoked in response to agonist binding.


   Acknowledgments
 
The authors thank Dr. Lynn A. Raymond for her assistance with the transfection protocol.


   Footnotes
 
This work was supported by Grant MT7711 of the Canadian Institutes of Health Research (to P.C.K.L.) and NIH Grant HD-23273 (to A.J.W.H.).

1 Distinguished Scholar of the Michael Smith Foundation for Health Research. Back

Abbreviations: [Ca2+]i, Intracellular free calcium ion concentration; GLC, granulosa-lutein cells; GPCR, G protein-coupled receptor; h, human; HEK, human embryonic kidney; #, number of transfections.

Received January 29, 2001.

Accepted for publication December 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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