Activation of extracellular-regulated kinase pathways in ovarian granulosa cells by the novel growth factor type 1 follicle-stimulating hormone receptor. Role in hormone signaling and cell proliferation.

Follicle-stimulating hormone (FSH) regulated growth and function of the ovarian follicle was previously thought to be mediated solely through activation of G(s)-coupled receptors. In this study, we show for the first time that this function is predominantly mediated through the alternatively spliced and novel growth factor type 1 receptor (oFSH-R3) that is also present in the ovary. Immortalized granulosa cells lacking endogenous FSH receptors, when transfected with either oFSH-R3 cDNA (JC-R3) or the G(s)-coupled oFSH-R1 (JC-R1), expressed the corresponding glycosylated receptor. In JC-R3 or JC-R1 cells labeled with bromodeoxyuridine or [(3)H]thymidine, FSH stimulated the cells to progress through S-phase and divide. The growth promoting effect of recombinant FSH in JC-R3 cells was preceded by the rapid activation of ERK1 and ERK2. This effect was hormone-specific and transient. In JC-R3 cells inhibitors like calphostin C, PD98059, Ag 18, or calcium chelators EGTA or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM inhibited both mitogen-activated protein kinase activation and bromodeoxyuridine incorporation. FSH induced phosphorylation of the FSH-R3 receptor was blocked by pretreating cells with calphostin C. There was no cAMP induction by FSH in JC-R3 cells. The cAMP independent growth promoting effect of FSH is mediated by activation of Ca(2+) and mitogen-activated protein kinase-dependent pathways. Thus, alternative splicing of a G-protein coupled receptor creates the expression of a novel receptor motif that can mediate a widely recognized function of the glycoprotein hormone.


INTRODUCTION
The ovarian follicle is among the most prolific of normal tissues undergoing rapid cellular proliferation and differentiation to accommodate the development and maturation of the ovum that is vital for propagation of the species. Two dimeric pituitary gonadotropins, follicle stimulating hormone or follitropin (FSH) and luteinizing hormone or lutropin (LH) that belong to the complex glycoprotein hormone family are critically involved in the mechanisms regulating follicle status and development. The actions of gonadotropic hormones are mediated by binding to high affinity receptors present on plasma membranes of target gonadal cells. FSH regulates a large number of genes encoding nuclear, cytoplasmic, membrane associated (1), and secreted proteins (2). The full length FSH receptor (hereafter called FSH-R1) belongs to a super family of Gprotein coupled receptors, which interact with intracellular effector system through seven transmembrane domains (3)(4)(5). This FSH-R1 receptor like LH and TSH (thyroid stimulating hormone-thyrotropin) receptors has a large extracellular (EC) amino terminal domain comprised of more than 300 amino acid residues. The EC domains of these receptors are encoded by multiple exons and contain leucine rich repeat sequences that are thought to be important for ligand binding. According to present evidence, the FSH receptor is coded by a single large gene (80-100 kb), in various species (3,6). Alternative pre m-RNA splicing is a wide spread theme for gene regulation and for generating isoform variants as this mechanism will ensure molecular diversity for cellular regulation in instances where only one gene exists (7). There are many reports on the identification of various alternatively spliced transcripts for most glycoprotein hormone receptors including that of FSH in the ovary and testis, the two exclusive targets of the hormone action (4,5,8,9). In our previous investigations, we have reported the cloning of 5 MAP kinase activation were reported in primary granulosa cell cultures in response to FSH (18)(19)(20) and some of the actions are apparently mediated by cAMP independent pathways (19,20).
This raises the critical question whether all FSH actions on target cells are mediated by one type of receptor (R1), the heptahelical transmembrane form that is coupled to the activation of adenylyl cyclase producing cyclic AMP as the second messenger? As the FSH receptor gene undergoes extensive alternative splicing (3,6) and most of the earlier observations in literature are made with primary cultures of target cells that may express heterogeneous populations of FSH receptors, it is conceivable that other FSH receptor motifs may be implicated in signaling events that contribute to cell proliferation. In view of its novel structural features, the alternatively spliced receptor 39 kDa FSH-R3 identified in the developing ovary (12) that is distinctly different from R1 becomes a good candidate. Like other growth factor type I receptors, the FSH-R3 has a single transmembrane domain and undergoes dimerization in response to the action of FSH (10). We have also shown that this R3 type receptor but not the Gs coupled R1 receptor mobilizes Ca 2+ influx into the cell through L-Voltage Ca 2+ channels (21). The experiments presented herein were designed to test for the potential role of this growth factor type I receptor in FSH mediated MAP kinase signaling in target granulosa cell proliferation. The availability of a granulosa cell line (JC-410) (22,23) that lost innate expression of gonadotropin receptors during spontaneous immortalization but retained its steroidogeneic capability has provided us an ideal system to understand the role of the alternatively spliced FSH receptor in hormone signaling. The results of the current study provide the first direct evidence strongly implicating a role for the novel growth factor type I receptor in activating extracellular regulated kinase pathways by mechanisms that are independent of the cAMP pathway. by guest on  http://www.jbc.org/ Downloaded from 8 staining buffer (PBS containing 1% bovine serum albumin) and resuspended in 100 µl of staining buffer containing the FSH-R antisera (1:100) raised in rabbits and incubated at RT for 1 h. They were again washed and incubated with 100 µl of staining buffer containing fluorosceinated goat anti-rabbit IgG (1:50 dilution) in dark for 30 min at RT. After one more final wash they were resuspended in 1 ml staining buffer. The green fluorescence intensity of cells was measured in a Coulter flow cytometer at 530 nm. Control cells were processed in the same manner excluding the primary antibody. The antibodies used were characterized previously and shown to react with FSH receptor in cells or gonadal tissues (12,24,25).
Deglycosylation of oFSH-R3 in JC-R3 Cells -The cells were scraped in cold PBS and collected after centrifugation at 800Xg for 10 min at 4 o C. They were suspended in lysis buffer and left on ice for 30 min to solubilize membrane proteins. The solubilized receptor was deglycosylated using N-Glycosidase F (Boehringer-Mannheim), which cleaves all N-linked glyco moieties of the FSH receptor (26). Approximately 200 µg of membrane protein was suspended in 0.1M Tris-HCl buffer pH 7.4 with 0.1 mM PMSF and incubated with 50U/ml of N-Glycosidase F.
After incubation for 4 hours at 30 o C, 100µg equivalent protein was reduced in 1X SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting using the FSH-R3 specific peptide antibody (12).
Western Blot Analysis -Granulosa cells expressing the cloned oFSH-R3 or oFSH-R1 were stimulated in serum free medium as described in figures. In preliminary studies, we used natural and highly purified hFSH and hLH or hCG prepared in our laboratory. In subsequent experiments all data reported herein were derived from the use of recombinant human glycoprotein hormones (see methods). Cells collected by scraping in cold PBS, were washed and homogenized in lysis buffer (20mM Tris HCl pH 7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 2.5 9 mM sodium pyrophosphate, 1mM Na 3 VO 4 and 0.1mM PMSF). Protein content in the samples was estimated by the Bio-Rad protein assay. For verifying the expression of FSH-R3 ~100 µg protein was used whereas for MAP kinases 5 µg of total solubilized protein was adequate.
Bound antibody was visualized using the Luminol (Boehringer-Mannheim) chemiluminescent detection system.

Quantification of Granulosa Cell Proliferation by BrdU Incorporation and Dual Parameter
Flow Cytometry -The quantification of BrdU-positive cells by flow cytometry can be considered as a measure of actively dividing S-phase cells (27). Quantification of BrdU-labeled cells was done according to the procedure described earlier (28). Granulosa cells transfected with/without FSH-R3 or FSH-R1 receptor were growth arrested by serum-starvation for 16 Granulosa cells (JC-R3, JC-R1 or JC-Vector) were plated at a density of 2.5x10 4 cm 2 in 24 well tissue culture plates and grown for 2 days without a medium change. They were rinsed once with serum-free DMEM and serum starved for 16 hrs by incubating with DMEM. The medium was changed to DMEM containing varying concentrations of rFSH plus 1 µCi/well of [ 3 H] thymidine (specific activity 79 Ci/mmol). The cells were incubated at 37 o C for 24 hrs and the amount of [ 3 H] thymidine incorporated in to DNA was determined as described previously (10).

Measurement of [ 3 H]Thymidine Incorporation-
Phosphorylation of Intact JC-R3 Cells and Immunoprecipitation. JC-R3 cells plated in 100 mm dishes were biosynthetically labeled in phosphate free DMEM medium with 100µci/ml of 32 P orthophosphate for 3 hours at 37 o C under 5%CO 2 as described by others (29)  and FSH-R1 (lower panel), whereas these antibodies did not show any specific binding with JC-membranes (12). The apparent increase in the molecular weight that is different from the value predicted from the cDNA sequence (12) is suggestive of the covalent modification of one or both of the two potential N-glycosylation sites in FSH-R3. Previously published data suggest that in both FSH-R and LH/hCG receptors, glycosylation patterns influence the migration in SDS-PAGE and these receptors are sensitive to N-glycosidase F treatment (26,31,32). Accordingly, we tested for the presence of glycosylation sites (at Asn 174 and 182) of the receptor in transfected cells as described earlier with slight modifications (33). Digestion of soluble cell extracts expressing FSH-R3 with N-Glycosidase F led to a decrease in the Mr by ~ 8-10 kDa, which was clearly evident by the downward shift in protein mobility. Thus, it appears that the N-linked oligosacharides of expressed R3 receptor are accessible to the action of N-Glycosidase F. The results suggest that recombinantly expressed FSH-R3 in the granulosa cells is a glycoprotein that is N-glycosylated at both sites as predicted by its cDNA structure. size only in extracts of +/+ mouse ovary. These data confirm for the first time that a receptor equivalent to FSH-R3 is also present in the mouse ovary. Fig. 3, when JC-R3 or JC-R1 and vector control cells were stimulated with different concentrations of rFSH, there was no accumulation of cyclic AMP in JC-R3 cells despite the presence of the phosphodiesterase inhibitor. These results, verified several times, suggest that the R3 receptor expressed in the granulosa cells is not coupled to the activation of cAMP unlike FSH-R1 type of receptor. In JC-R1 cells expressing the Gs coupled receptor, cAMP increase was evident in presence of 1ng/ml FSH. (Fig. 3). The latter is consistent with numerous reports showing that FSH-R1 in HEK 293 cells is efficiently coupled to this signaling pathway (3,5,11). Forskolin, a non-hormonal adenylyl cyclase activator, promptly increased cAMP production in JC-R3 cells in a concentration dependent manner from 1-10 µM indicating that following FSH-R3 cDNA transfection the Gprotein coupled signaling apparatus is fully intact.

FSH-Induced Phosphorylation -
The results presented in Fig. 4 show that cells expressing oFSH-R3 respond to rFSH (10ng/ml) or PMA (200 nM) with a 4 fold increase in receptor phosphorylation compared to unstimulated cells. The FSH induced phosphorylation was partially blocked by pretreating the FSH-R3 expressing cells with PKC inhibitor Calphostin C, suggesting that FSH-R3 receptor phosphorylation is mediated by either PKC or one or more other kinases.

Regulation of MAP Kinase Pathway in JC-R3 Cells -The regulation of MAP kinase pathway
in JC-R3 cells was monitored by measuring the total phosphorylation pattern and activation of two MAP kinase isoforms, ERK1 and ERK2. Cells were maintained in serum free medium for 12 hours to arrest growth and then stimulated with the hormone. The cells were harvested at different time periods and subjected to Western blot analysis using antibody that specifically However, repeated experiments confirmed that FSH is unable to activate other kinases including both P38 and SAPK/JNK in JC-R3 cells (data not shown). This is probably because these two pathways are primarily associated with cellular stress responses, a signaling mechanism that may not be mediated by the FSH-R3 in the granulosa cell. Fig. 5B shows the concentration (FSH) dependent increase in ERK1 and ERK2 activation in JC-R3 cells. It may be noted that a very low concentration of the hormone FSH (0.1ng/ml ≈3 X 10 -12 M) is sufficient to double ERK1 and ERK2 activation, and this effect is enhanced with increasing FSH up to 100 ng/ml.
As it is known that PKC's are involved with activation of ERK MAP kinase pathway, we studied the effect of both PKC inhibitor (Calphostin C) and MEK inhibitor (PD98059) on FSH action in JC-R3 cells (Fig. 5A). Pretreatment of cells either with PD98059 or Calphostin-C at a concentration of 10 -5 mole/L for 30 min prior to FSH stimulation blocked the activation of ERK1 and ERK2 indicating the potential role of PKC pathways in the activation of MAP kinases.
However, as the compound H89, which is a PKA inhibitor did not produce a comparable effect significant involvement of cAMP pathways in the activation of MAP kinases is unlikely in JC-R3 cells. This result is in agreement with the lack of cAMP production by FSH in JC-R3 cells (Fig.   3). The specificity of FSH induced activation of ERK1 and ERK2 in JC-R3 cells is supported by data shown in Fig. 5C. The JC-R3 cells stimulated with 10% serum for 10 min served as positive control, whereas the JC-410 or vector transfected cells stimulated under identical conditions with FSH did not show any activation of ERK1 and ERK2. Similarly the homologous hormone, rhCG, did not have any effect on activation of MAP kinases in JC-R3 cells (Fig. 5C). Compared to the two-fold activation with 0.1 ng/ml FSH (Fig. 5B) rhCG at 100 ng/ml was without effect. Results were similar using highly purified natural hCG (data not shown). In the light of these novel observations, we compared the effects of the hormone in JC-R1 cells designed to express the G scoupled receptor. It was already shown (Fig. 3) (Fig. 7). This suggests that calcium influx plays a major role in the FSH controlled events that may induce cell proliferation. We also tested whether activation of PKC will have any effect on MAP kinases in these cells and as seen in  (34,35), we examined the activation of Akt (protein kinase), a downstream regulatory molecule in PI-3 pathway in JC-R3 cells. FSH did not activate Akt phosphorylation (Fig. 8A). However, in cells treated with serum Akt phosphorylation was clearly evident. When we pretreated JC-R3 cells with the specific PI-3 kinase inhibitor LY294002 there was no effect on ERK1/2 activation (Fig.   8B). Together these data suggest that FSH has no influence on PI-3 activation and this pathway is not involved in the activation of ERK1/2 in JC-R3 cells.  10B) show that blocking MEK activity by PD98509 or inhibiting intra cellular calcium has no effect on BrdU labeling but a partial reduction is seen with PKA inhibitor H89. In sum these data suggest the presence of potential alternative pathways in granulosa cell proliferation.

Role of MAP Kinases in the Growth
Steroidogenic Response in Granulosa Cells -JC-R3 cells were stimulated in minimal medium with rhFSH in presence or absence of 0.1µM androstenedione, which serves as the substrate for the enzyme aromatase. This enzyme converts the androgen to the phenolic steroid estradiol-17β. reproducible, the differences in the estradiol production induced by these two receptors may be attributable to differences in their mode of action during steroidogenesis. Irrespective of the type 20 of FSH-R present in the transfected granulosa cell, addition of the non-hormonal agent forskolin resulted in the same level of activation of steroidogenesis (Fig. 11B).

DISCUSSION
During the growth and maturation of ovarian follicles, the granulosa cells, which are the major cell type in this structure that also contains the developing ovum, proliferate rapidly and undergo differentiation in a precisely ordered sequence. This process includes the acquisition of receptors for the glycoprotein hormone FSH in the early stages of growth. It is known that FSH, a major regulator of receptor(s) induction, increases FSH receptor mRNA and protein levels both in vivo and in vitro (37,38). The rapid increase in FSH induced DNA synthesis in the immature ovary well before cAMP production (39), increased uptake of ( 3 H)-thymidine in preantral follicles (40), imply that growth has been initiated by allowing the cells to enter the cell cycle. Subsequently, check points of the cell cycle like cyclin D2 (41) and other control mechanisms determine the path of the granulosa cells towards further development and differentiation (42). However, the precise molecular nature of FSH receptors or the signaling mechanisms that mediate hormonal induction of such mitotic activities during the early stages of follicular growth have remained a mystery. According to the present dogma in the glycoprotein hormone receptor field, the diverse action(s) of the glycoprotein hormones is assumed to be mediated solely by the G-protein coupled heptahelical transmembrane receptor entity (43). Having cloned novel structural motifs of the functional FSH receptor that differ from this topography and documented expression of the corresponding protein in the ovary (12), we launched a systematic attempt to define the role of the growth factor type I receptor in ovarian cells to identify its signaling pathways. While this report focuses on revealing the properties of the novel receptor FSH-R3, we have included some studies comparing the Gs coupled receptor providing data on relative differences in signaling. The growth factor receptor type I motif for FSH was initially cloned from the sheep testis (14) and ovary (12). Expression of the corresponding protein has also been verified (12). The universal participation of such alternatively spliced receptors in signaling processes requires confirmation in other species. Thus, our first demonstration that the R3 receptor also exists in the mouse ovary ( Fig. 2) is significant. The specificity of our detection system for R3 protein was evident in the Western blots because only the wild type +/+ mouse ovary extracts showed the expression of this receptor protein with a migration very similar to that of the receptor in JC-R3 transfected cells.
The complete absence of a protein band corresponding to this as well as R1 in the ovaries of FSH receptor knockout (-/-) mouse also proves that the entire repertoire of FSH receptor motifs were eliminated in our knockout strategy causing sterility in females (16).
Based on recent x-ray crystallographic evidence, the glycoprotein hormone FSH and other members of this family have been modeled (47) as having structural features that include the cystine knot motif that are also present in many ligands that act as growth factors. The receptors for these ligands and their signaling properties are different from the G-protein coupled receptors.
The discovery of a growth factor type receptor R3 for FSH is consistent with the structural predictions for the hormone and is strongly supported by the data described in this study which show that signaling mechanisms other than the G-proteins are also utilized by the hormone. Thus, its linkage to the activation of MAP kinase pathways as demonstrated here now provides a new perspective in understanding glycoprotein hormone action. The MAP kinase signaling cascade, an important regulator of cell cycle progression, has been used as a biochemical marker to evaluate the status of hormones and growth factors as mitogens (48).
The FSH-R3 receptor itself consists near its carboxyl terminus the sequence PVILSP (10,12,14), which represents a potential consensus motif (PXnS/TP where X is basic or neutral residue) for phosphorylation by MAP kinases (49). Because this motif appears only after alternative splicing at the 8th exon of the FSH receptor gene (10,14) we can argue that the change must have structural and functional significance. Structurally, the change led to the appearance of a single transmembrane domain for FSH-R3 as well as the other accompanying motifs that couple to alternative signaling pathways. This consensus site is present in the analogous hTSH receptor variant in the thyroid where this transcript is also generated by alternative splicing at exon 8 (50).
As both FSH and TSH are mitogenic hormones and stimulate cell proliferation in their respective target tissues, presence of the R3 type of motif may have great significance in receptor function and dynamics. FSH induced receptor phosphorylation (Fig. 4) in JC-R3 cells suggest that some of the serine residues in C-terminal end may be involved in the modification. However, the mechanisms by which the receptor is phosphorylated remain to be clarified. Receptor autophosphorylation is unlikely because the C-terminus lacks intrinsic kinase domains. The effects of gonadotropin receptor phosphorylation in general have recently been reviewed (51) and other investigators studying the R1 type FSH receptor in transfected HEK 293 cells (52) have shown that phosphorylation modulates receptor uncoupling and internalization.
Gonadotropins are known to increase the MAP kinases in ovarian granulosa cells collected from intact animals (18,19). The differences in the activation of ERK1/ERK2 by cAMP and gonadotropins (LH and FSH) may suggest that gonadotropins activate these enzymes via pathways that operate, at least in part independent of cAMP (19). However, the precise receptor mechanisms identifying how FSH may regulate MAP kinase activation and its subsequent role in granulosa cell proliferation was unknown. It has long been appreciated that a significant elevation of intracellular cAMP levels may potently inhibit cell proliferation and division (53) but at lower concentration may have opposite effect (54). However, FSH induced cell proliferation in ovarian follicles is a major effect of the hormone, that may be incompatible with elevation of cAMP, a second messenger that is clearly elevated by the R1 receptor (3,36,45,46). Based on the data presented in Figs. 3, 5, 6, 8 and 9, we may conclude that FSH signaling through the R3 receptor is a major mechanism of MAP kinase activation and cell proliferation independent of the cAMP pathway. The absence of ERK1/2 activation by forskolin (Fig. 6B) an agent that promptly elevated cAMP in JC-R3 cells, and the complete abrogation of hormone-induced cell proliferation by PD98059, a specific inhibitor of MAP kinase activity, clearly justifies such a conclusion. Thus, our data provides a mechanistic explanation for inferences drawn from studies on FSH action in primary cultures (19). FSH action in JC-R3 cells increased BrdU labeling of granulosa cell nuclei, in a hormone specific manner. The biphasic nature of this response and maximal stimulation obtained at low concentration ( Fig. 9) although intriguing, is typical of the behavior exhibited by growth promoting hormones. This is further confirmed by long term growth promoting effects of intracellular calcium had no effect on proliferation in JC-R1 cells (Fig. 10).
It has been well established that growth factors activate MAP kinases in a variety of cell types (48) and that inhibitors of the activation of the MAPK cascade block the mitogenic action of the growth factors (56). These observations have been confirmed and extended in the present study with the novel glycoprotein hormone receptor motif FSH-R3. As both H89 and K57032, that are PKA inhibitors did not significantly influence the FSH induced proliferation and activation of MAP kinase in FSH-R3 transfected cells, our conclusion that the FSH-R3 receptor utilizes other pathways to activate MAP kinase instead of cAMP/PKA appears justified. This is further supported by the absence of cAMP production by FSH in FSH-R3 transfected cells (Fig. 3). Our investigations, designed solely to understand the signaling properties of the novel FSH-R3 in an individual setting in cells that express only one type of receptor does not preclude a role for the Gs coupled FSH-R1 and the PKA pathway in cell proliferation. Our results indeed support that FSH-R1 may also coupled to cell proliferation through other mechanisms which need to be clarified in more detail. In addition to the differences noted above, the phenotypic characteristics of granulosa cells bearing one or the other FSH receptor appears to be different. While JC-R1 cells that produce cAMP in response to FSH promptly show the cell rounding phenomenon as reported in other studies (36), the JC-R3 cells that activate ERK1/2 do not display this behavior (data not shown). Therefore, it is possible that the preponderance of one or the other form of FSH receptors varying as the ovarian granulosa cells mature might dictate the selection of different signaling pathways and cross talk that is likely to occur during rapid developmental changes.
The relation between hormone action, Ca 2+ influx, MAP kinase activation, and cell proliferation and steroidogenesis in JC-R3 cells can be integrated by our investigations. In a previous study, we reported that, FSH induces Ca 2+ influx in FSH-R3 transfected HEK 293 cells.
through L-voltage dependent pathways (21). As the hormone induces Ca 2+ in primary granulosa or Sertoli cells but not in R1 transfected cells (see 21,57), participation of a different receptor motif is likely. Taken together with the results of the present study, the implication is clear that FSH induces calcium influx in target granulosa cells via the FSH-R3 and the increased Ca 2+ in the cytosol in turn leads to MAP kinase activation. Ca 2+ influx is well known to activate MAP kinase in cultured cells by several mechanisms (58,59) including PKC activation of Ras (60). Since FSH-induced ERK activation in JC-R3 cells is inhibited by a PKC inhibitor, the involvement of PKC pathways upstream of ERK signaling is by guest on July 27, 2018 http://www.jbc.org/ Downloaded from 27 most likely. This conclusion is further supported by data showing inhibition of ERK activation by intracellular or extracellular Ca 2+ chelators. Recent reports that PI-3 kinase can be involved in the regulation of ERK kinase pathway and PI-3 kinase can also be activated by Ca 2+ influx (59) have led to the implication of PI-3 kinase in down stream control of Ras in some cellular systems (34,35,(61)(62)(63)(64). More importantly the PI-3 kinase activity is also able to modulate the ERK/MAP kinase pathway (34,35). Since we did not observe any effect of the hormone on phosphorylation of Akt and furthermore a PI-3 kinase inhibitor did not influence the MAP kinase activation, we suggest that mediation of FSH effects by PI-3 kinase is unlikely in the JC-R3 cells.
Another important new observation emerging from the current study is that the growth factor type I receptor of FSH is also coupled to steroidogenic machinery albeit weakly. Therefore, a second pathway in addition to the classical Gs-coupled signaling mechanism may also be operative in granulosa cells. A hallmark of FSH action in the ovarian granulosa cell is the synthesis of estrogen, which subsequently activates transcription of numerous genes via its nuclear receptors.
Based on the data shown in Fig. 11, the conclusion is inescapable that cells expressing FSH-R3 indeed secreted estradiol-17β into the medium in response to the hormone. However, under identical conditions the full length FSH-R1, which activates the Gs-signaling systems is more efficient in inducing steroidogenesis. The identification of two different pathways for hormone signaling including steroidogenesis may represent a back up mechanism utilized by a dynamic system such as the granulosa cell that has to perform diverse functions.
In conclusion, these results for the first time demonstrate that a novel type of FSH receptor (R3) exhibiting features of a growth factor type I receptor is primarily responsible activation of the ERK pathway in ovarian granulosa cells. This was essentially independent of cAMP/PKA mediated events but dependent upon Ca 2+ influx and PKC pathway. The identification of putative       The cells were lysed in lysis buffer as described in methods, 5 µg of soluble cell protein was separated on 10% SDS-PAGE and transferred to PVDF membrane. The blot was probed with phospho specific ERK1/2 antibody (1:1000) and same blot was stripped and reprobed with ERK1/2 antibody to evaluate equal loading of protein (see bottom).