Rat Follicle-stimulating Hormone Receptor Research Articles

Roles of N-linked and O-linked glycosylation sites in the activity of equine chorionic gonadotropin in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and follicle-stimulating hormone receptor

BackgroundEquine chorionic gonadotropin (eCG), which comprises highly glycosylated α-subunit and β-subunit, is a unique member of the glycoprotein hormone family as it elicits both follicle-stimulating hormone (FSH)-like and luteinizing hormone (LH)-like responses in non-equid species. To examine the biological function of glycosylated sites in eCG, the following glycosylation site mutants were constructed: eCGβ/αΔ56, substitution of Asn56 of α-subunit with Gln; eCGβ-D/α, deletion of the O-linked glycosylation site at the carboxyl-terminal peptide (CTP) region of the β-subunit; eCGβ-D/αΔ56, double mutant. The recombinant eCG (rec-eCG) mutants were expressed in Chinese hamster ovary suspension (CHO-S) cells. The FSH-like and LH-like activities of the mutants were examined using CHO-K1 cells expressing rat lutropin/CG receptor (rLH/CGR) and rat FSH receptor (rFSHR).ResultsBoth rec-eCGβ/α and rec-eCGβ/αΔ56 were efficiently secreted into the CHO-S cell culture medium on day 1 post-transfection. However, the secretion of eCGβ-D/α and eCGβ-D/αΔ56, which lack approximately 12 O-linked glycosylation sites, was slightly delayed. The expression levels of all mutants were similar (200–250 mIU/mL) from days 3 to 7 post-transfection. The molecular weight of rec-eCGβ/α, rec-eCGβ/αΔ56 and rec-eCG β-D/α were in the ranges of 40–45, 37–42, and 34–36 kDa, respectively. Treatment with peptide-N-glycanase F markedly decreased the molecular weight to approximately 5–10 kDa. Rec-eCGβ/αΔ56 exhibited markedly downregulated LH-like activity. The signal transduction activity of both double mutants was completely impaired. This indicated that the glycosylation site at Asn56 of the α-subunit plays a pivotal role in the LH-like activity of eCG. Similarly, the FSH-like activity of the mutants was markedly downregulated. eCGβ-D/α exhibited markedly downregulated LH-like and FSH-like activities.ConclusionsRec-eCGβ/α exhibits potent biological activity in cells expressing rLH/CGR and rFSHR. The findings of this study suggest that the LH-like and FSH-like activities of eCG are regulated by the N-linked glycosylation site at Asn56 of the eCG α-subunit and/or by the O-linked glycosylation sites of the eCG β-subunit. These findings improved our understanding of the mechanisms underlying both LH-like and FSH-like activities of eCG.

Luteinizing hormone-like and follicle-stimulating hormone-like activities of equine chorionic gonadotropin β-subunit mutants in cells expressing rat luteinizing hormone/chorionic gonadotropin receptor and rat follicle-stimulating hormone receptor

ABSTRACT To identify the specific region of eCG involved in FSH-like activity, the following mutant expression vectors were constructed targeting the amino acid residues 102–104 of the eCG β-subunit: single mutants, eCGβV102G/α, eCGβF103P/α, and eCGβR104K/α; double mutants, eCGβV102G;F103P/α, eCGβV102G;R104K/α, and eCGβF103P;R104K/α; triple mutant, eCGβV102G;F103P;R104K/α. The LH-like and FSH-like activities of eCG mutants were examined in CHO-K1 cells expressing rat LH/CG receptor and rat FSH receptor. The levels of eCGβV102G/α, eCGβR104K/α, and eCGβV102G;R104K/α in the culture supernatant were markedly lower than those of eCGβ/α-wt. The other mutants and rec-eCGβ/α-wt were efficiently secreted into the culture supernatant. The LH-like activities of eCGV104G/α, eCGβV102G;R104K/α, and eCGβF103P;R104K/α were approximately 61%, 52%, and 54%, respectively, of those of eCG-wt. The Rmax values of the mutants were 58.9%–78.8% those of eCG-wt with eCGβR104K/α exhibiting the lowest value. The FSH-like activities of single mutants were only 16%–20% of those of eCG-wt. Additionally, the FSH-like activity of double mutants was less than 10% of that of eCG-wt. In particular, the FSH-like activities of βV102G;R104K/α and βF103P;R104K/α were 2.5–2.9% of that of eCG-wt. These results suggest that the amino acid residues 102–104 of the eCG β-subunit are dispensable and that the residue 104 of the eCG β-subunit plays a pivotal role in signal transduction through the rat FSH receptor. Thus, these mutants may aid future studies on eCG interactions with mammalian FSH receptors in vitro and in vivo.

Different roles of cAMP/PKA and PKC signaling in regulating progesterone and PGE2 levels in immortalized rat granulosa cell cultures

Follicular cells from various species secrete steroids and prostaglandins, which are crucial for reproduction, in response to gonadotropins. Here, we examined prostaglandin E2 (PGE2) secretion from immortalized rat granulosa cells derived from preovulaotry follicles expressing the rat follicle stimulating hormone receptor (denoted as FSHR cells) that produce progesterone in response to gonadotropins. The cells were stimulated with a) pregnant mare’s serum gonadotropin (PMSG; a rat FSH receptor agonist), b) activators of the protein kinase A (PKA) pathway (forskolin and a cell permeable cAMP analog Dibutyryl-cAMP (DB-cAMP)) and c) protein kinase C (PKC) (12-O-tetradecanoylphorbol 13-acetate; TPA), alone and in combination for 24 h. Thereafter, PGE2 and progesterone levels in the culture media were determined. In accordance with previous studies, while PMSG and the PKA pathway activators induced progesterone accumulation in the media, TPA did not. In contrast, our data indicate that TPA, but neither PMSG, forskolin and DB-cAMP evoked PGE2 accumulation in the media. Western Blot analysis of cell lysate showed a drastic TPA induced increase of COX-2 levels, which was not seen with neither PMSG nor forskolin treatment. This association between the COX-2 and PGE2 levels suggests that the enzyme activity is the likely factor that determines the synthesis and levels of the prostaglandin in the culture media of the granulosa-derived cells. The addition of the PKA inhibitor H-89 to the FSHR cultures suppressed the gonadotropin and forskolin induction of progesterone secretion. Incubation in the presence of GF109203X (a PKC inhibitor) attenuated the TPA induced PGE2 accumulation in the culture media of the cells (a dose dependent reduction of 40–70%). In addition, while TPA inhibited the PMSG and forskolin induced-accumulation of progesterone in the media, the gonadotropin and forskolin inhibited the elevation of PGE2 levels evoked by TPA (a dose dependent decrease of 35–55%). These data suggest that cAMP/PKA and PKC signaling have opposite effects on PGE2 and progesterone synthesis in FSHR cells. We propose that this PKA and PKC interplay on progesterone and PGE2 may be advantageous for the coordination of these key mediators for successful ovulation and luteinization.

Internalization of Rat FSH and LH/CG Receptors by rec-eCG in CHO-K1 Cells.

ABSTRACTEquine chorionic gonadotropin (eCG) is a unique molecule that elicits the response characteristics of both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in other species. Previous studies from this laboratory had demonstrated that recombinant eCG (rec-eCG) from Chinese hamster ovary (CHO-K1) cells exhibited both FSH- and LH-like activity in rat granulosa and Leydig cells. In this study, we analyzed receptor internalization through rec-eCGs, wild type eCG (eCGβ/α) and mutant eCG (eCGβ/αΔ56) with an N-linked oligosaccharide at Asn56 of the α-subunit. Both the rec-eCGs were obtained from CHO-K1 cells. The agonist activation of receptors was analyzed by measuring stimulation time and concentrations of rec-eCGs. Internalization values in the stably selected rat follicle-stimulating hormone receptor (rFSHR) and rat luteinizing/chorionic gonadotropin receptor (rLH/CGR) were highest at 50 min after stimulation with 10 ng of rec-eCGβ/α. The dose-dependent response was highest when 10 ng of rec-eCGβ/α was used. The deglycosylated eCGβ/αΔ56 mutant did not enhance the agonist-stimulated internalization. We concluded that the state of activation of rFSHR and rLH/CGR could be modulated through agonist-stimulated internalization. Our results suggested that the eLH/CGRs are mostly internalized within 60 min by agonist-stimulation by rec-eCG. We also suggested that the lack of responsiveness of the deglycosylated eCGβ/ αΔ56 was likely because the site of glycosylation played a pivotal role in agonist-stimulated internalization in cells expressing rFSHR and rLH/CGR.

Pharmacologic profiling of corifollitropin alfa, the first developed sustained follicle stimulant

Corifollitropin alfa (Elonva®, MSD, previously N.V. Organon or Schering-Plough Oss, The Netherlands) is a newly developed sustained follicle stimulant composed of the α subunit of human follicle-stimulating hormone (FSH) and a hybrid β subunit formed by fusion of the human chorionic gonadotropin β subunit carboxy terminal peptide with the β subunit of human FSH. Binding characteristics of corifollitropin alfa at the rat FSH receptor and transactivation properties at the rat FSH receptor, human luteinizing hormone (LH) receptor, and human thyroid-stimulating hormone receptor (TSH receptor) were assessed in vitro. Bioactivity of corifollitropin alfa in rats was also assessed. Serum corifollitropin alfa levels in rats and dogs were used to derive the main pharmacokinetic parameters of corifollitropin alfa. Binding and transactivation profile of corifollitropin alfa to rat FSH receptor was specific and comparable to that of recombinant human FSH, with no intrinsic TSH receptor or LH receptor activation. From pharmacokinetic studies, circulating half-life of corifollitropin alfa was calculated to be 17.3h in rats and 46.9h in dogs, 1.5- to 2-fold longer than recombinant FSH. Corifollitropin alfa demonstrated a 2- to 4-fold increase in bioactivity (ovarian weight, serum estradiol and progesterone, ovulated ova) over recombinant FSH across all in vivo parameters assessed. These data demonstrate that corifollitropin alfa is a specific ligand with high affinity for FSH receptor, lacking intrinsic activity for LH receptor and TSH receptor. By virtue of its increased in vivo half-life, corifollitropin alfa can be a valuable alternative to FSH by acting as a sustained follicle stimulant.

Identification of the region 285–309 of follicle stimulating hormone receptor as a bioneutralizing epitope

Structure–function relationship studies of the follicle stimulating hormone and its receptor assume importance as this hormone is essential for folliculogenesis and spermatogenesis in females and males, respectively. The interaction between the hormone and the receptor is complex and not well understood. In vitro studies using synthetic peptides from the extracellular domain of the receptor and corresponding antipeptide antibodies have suggested that the 285–309 region is surface-oriented. Antipeptide antibodies to this region also inhibit hormone-receptor interaction in a dose-dependent manner and the mechanism of inhibition appears to be competitive in nature. To test this hypothesis in an animal model, antibodies to peptide 285–309 from rat follicle stimulating hormone receptor (FSHR) were developed and characterized. These antibodies were able to detect FSHR in rat ovaries by immunohistochemistry. Further, these antibodies were administered into adult female rats and their effect on fertility status was monitored. These antibodies were found to neutralize the biological activity of endogenous receptor, which resulted in the induction of infertility in the treated animals. Thus, bioneutralization of FSHR has been achieved by targeting its region 285–309 in an in vivo system.

Detailed analysis of the region 9–30 of rat follicle stimulating hormone receptor: Identification of peptide 20–30 as a potential hormone binding inhibitor

The extracellular domain (ECD) of the follicle stimulating hormone receptor (FSHR) has been shown to be a major determinant of hormone selectivity. The N-terminal 9–30 region, the sequence of which is unique to FSHR, has been extensively studied earlier and has been proposed to be an FSHR neutralizing epitope. In this study antipeptide antibodies specific to the peptide 9–30 were generated and used for identifying a specific immunodominant region within it. Overlapping peptides corresponding to the regions 9–19, 15–25 and 20–30 were synthesized. The ability of the antipeptide antibodies to 9–30 of FSHR to bind to different peptides was checked. The results indicated that the antibodies mainly recognized the peptide 20–30 and not the other two overlapping peptides. Further, the effect of the peptide 20–30 on the binding of radiolabeled FSH to its receptor was monitored. This peptide showed FSH-binding inhibitory activity with an IC 50 value of 0.598 × 10 −4 M and was more effective than the peptide 9–30 itself. Binding kinetics revealed that the observed effect of the peptide 20–30 is due to mixed type of inhibitory mechanism. This is the smallest peptide from the rat FSHR sequence having ability to inhibit FSH binding to its receptor by more than 90%.

Follicle-stimulating hormone (FSH) activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization

BackgroundThe follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK). However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired.MethodsHuman embryonic kidney (HEK) 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319–418) dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis.ResultsIn HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319–418) construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation.ConclusionFrom these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH.

Production of Biological Active Single Chain Bovine LH and FSH

Luteinizing hormone as other glycoprotein hormones is characterized by a heterodimeric structure composed a common α-subunit noncovalently linked to a specific β-subunit. The correct conformation of the heterodimer is important for efficient secretion, hormonal-specific post-translational modifications, receptor binding and signal transduction. To determine whether α- and β- subunits can be synthesized as a single polypeptide chain (tethered-bLH and -bFSH) and also display biological activities, the tethered- bLH and -bFSH molecules were constructed and transfected into chinese hamster ovary (CHO-K1) cells. LH and FSH activities were assayed by using the human embryonic kidney (HEK) 293 cells expressing rat LH and FSH receptor genes. The tethered-bLH and - bFSH proteins were efficiently secreted and showed a similar activity to the dimeric bovine LH and FSH α/β wild type and native purified from bovine pituitary. The tethered-molecules can be permit development of potent new analogues that stimulate ovarian development. Taken together, a single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds. These data indicate the potentiality of the single chain approach to further investigate structure- function relationships of LH and FSH. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 4 : 498-503)

Identification and characterization of a selective, nonpeptide follicle-stimulating hormone receptor antagonist.

The glycoprotein hormones (LH, FSH, and TSH) are critical to the maintenance of physiological homeostasis and control of reproduction. However, despite an obvious utility for synthetic pharmacological agents, there are few reports of selective, nonpeptide agonists or antagonists to receptors for these hormones. We have identified and characterized a novel synthetic molecule capable of inhibiting the action of FSH. This compound, 7-[4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro-(1,3,5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)-naphthalene]-2-sulfonic acid, sodium salt (compound 1), is a selective, noncompetitive inhibitor of the human (h) and rat (r) FSH receptors (FSHRs). Compound 1 selectively inhibited binding of [(125)I]hFSH with an IC(50) value of 5.4 +/- 2.3 micro M. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [(125)I]hFSH binding to BV-tFSHR with an IC(50) value of 10 +/- 2.8 micro M. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC(50) value of 3 +/- 0.6 micro M. Competition of compound 1 for binding to other glycoprotein hormone receptors and other G protein-coupled receptors demonstrated select activity for FHSRs. Compound 1 inhibited ovulation in immature and cycling adult rats. These data provide proof of concept that selective, small molecule antagonists can be designed for glycoprotein hormone receptors.

Activation of the rat follicle-stimulating hormone receptor promoter by steroidogenic factor 1 is blocked by protein kinase a and requires upstream stimulatory factor binding to a proximal E box element.

The receptor for the pituitary glycoprotein hormone FSH (FSHR) and the nuclear hormone receptor steroidogenic factor 1 (SF-1) play important roles in control of the hypothalamic-pituitary- gonadal axis. FSHR is essential for integrating the pituitary FSH signal to gonadal response, while SF-1 is an important transcriptional regulator of many genes that function within this axis and is essential for the development of gonads and adrenal glands. Given the critical role of SF-1 in regulation of the gonads and the coexpression of FSHR and SF-1 in Sertoli and granulosa cells, we examined the ability of SF-1 to regulate transcription of the FSHR gene. We found that SF-1 stimulated rat FSHR promoter activity in a dose-dependent and promoter-specific manner. Examination of various promoter deletion mutants indicated that SF-1 acts through the proximal promoter region and upstream promoter sequences. An E box element within the proximal promoter is essential for activation of the FSHR promoter by SF-1. This element binds the transcriptional regulators USF1 and USF2 (upstream stimulatory factors 1 and 2) but not SF-1, as shown by electrophoretic mobility shift assays. In addition, functional studies identified a requirement for the USF proteins in SF-1 activation of FSHR and mapped an important regulatory domain within exons 4 and 5 of USF2. Cotransfection studies revealed that activation of protein kinase A leads to inhibition of SF-1-stimulated transcription of FSHR, while it synergized with SF-1 to activate the equine LH beta-promoter (ebeta). Thus, stimulation of the cAMP pathway differentially regulates SF-1 activation of the FSHR and ebeta-promoters.

Activation of the Rat Follicle-Stimulating Hormone Receptor Promoter by Steroidogenic Factor 1 Is Blocked by Protein Kinase A and Requires Upstream Stimulatory Factor Binding to a Proximal E Box Element

Activation of the Rat Follicle-Stimulating Hormone Receptor Promoter by Steroidogenic Factor 1 Is Blocked by Protein Kinase A and Requires Upstream Stimulatory Factor Binding to a Proximal E Box Element

Mechanisms involved in the homologous down-regulation of transcription of the follicle-stimulating hormone receptor gene in Sertoli cells

The action of follicle-stimulating hormone (FSH) in spermatogenesis is regulated at a fundamental level by controlling the number of competent receptors present at the surface of Sertoli cells. By controlling the number of receptors, the cell is able to modulate the timing and magnitude of subsequent signal transduction in response to FSH. One mechanism of control is the down-regulation of the steady state levels of the FSH receptor gene after exposure to FSH or agents that stimulate or prolong the cAMP signal transduction cascade (homologous down-regulation) in Sertoli cells. The goals of this study were to examine possible mechanisms involved in the down-regulation of mRNA levels of this gene. Analysis of transcription and processing by a PCR-based assay showed that treatment of Sertoli cells with FSH caused at least a 50% reduction of hnRNA for the FSH receptor gene. Reporter genes controlled by 5′ flanking sequences of the FSH receptor gene that were transiently transfected into Sertoli cells were not down-regulated. In electrophoretic mobility shift assays (EMSA), cAMP-inducible nuclear protein complex containing c-Fos formed on the activator protein-1/cAMP responsive element-like site located at −216 to −210 in the promoter of the rat FSH receptor gene. We concluded from this study that there was no evidence for the putative role of ICER in the down-regulation of the FSH receptor promoter. In addition, the FSH-induced down-regulation of the transcription of the FSH receptor gene in Sertoli cells was prevented by the treatment of Sertoli cells with trichostatin A prior to the addition of FSH. This experiment coupled with other observations suggested that the down-regulation may be mediated by changes in chromatin structure.

The USF proteins regulate transcription of the follicle-stimulating hormone receptor but are insufficient for cell-specific expression.

Expression of the FSH receptor (FSHR) is limited to granulosa cells of the ovary and Sertoli cells of the testis. Previous studies showed that an E box in the proximal promoter of the FSHR gene is required for transcription and that the predominant E box binding proteins are the ubiquitous transcription factors, upstream stimulatory factor 1 (USF1) and USF2. Through cotransfection analysis, we have shown that both wild-type and dominant negative forms of the USF proteins regulate the rat FSHR promoter and that transcriptional activation of FSHR required several domains within the amino-terminal portion of the USF proteins. Analysis of the FSHR promoter region using in vivo genomic footprinting indicated that the E box is occupied by proteins in Sertoli cells but not in cells that fail to express the receptor, despite the presence of the USF proteins. To help delineate the regions of the rat FSHR gene required for correct spatial and temporal expression, transgenic mice harboring two constructs containing variable amounts of 5'-flanking sequence (5,000 bp and 100 bp) were generated. Examination of 16 different transgenic lines revealed varied transgene expression profiles with multiple lines having different amounts of ectopic expression and two lines failing to express the transgene. In addition, little or no expression was observed in Sertoli cells. These studies indicate that additional regulatory sequences outside the region from -5,000 to +123 bp are needed for proper expression in Sertoli cells.

The USF Proteins Regulate Transcription of the Follicle-Stimulating Hormone Receptor but Are Insufficient for Cell- Specific Expression

Expression of the FSH receptor (FSHR) is limited to granulosa cells of the ovary and Sertoli cells of the testis. Previous studies showed that an E box in the proximal promoter of the FSHR gene is required for transcription and that the predominant E box binding proteins are the ubiquitous transcription factors, upstream stimulatory factor 1 (USF1) and USF2. Through cotransfection analysis, we have shown that both wild-type and dominant negative forms of the USF proteins regulate the rat FSHR promoter and that transcriptional activation of FSHR required several domains within the amino-terminal portion of the USF proteins. Analysis of the FSHR promoter region using in vivo genomic footprinting indicated that the E box is occupied by proteins in Sertoli cells but not in cells that fail to express the receptor, despite the presence of the USF proteins. To help delineate the regions of the rat FSHR gene required for correct spatial and temporal expression, transgenic mice harboring two constructs containing variable amounts of 5′-flanking sequence (5,000 bp and 100 bp) were generated. Examination of 16 different transgenic lines revealed varied transgene expression profiles with multiple lines having different amounts of ectopic expression and two lines failing to express the transgene. In addition, little or no expression was observed in Sertoli cells. These studies indicate that additional regulatory sequences outside the region from− 5,000 to +123 bp are needed for proper expression in Sertoli cells.

Human follicle stimulating hormone receptor trafficking and hormone binding sites in the amino terminus

Previous studies of the rat follicle-stimulating hormone receptor (rFSHR) demonstrated that the amino terminus is important in FSH binding and signal transduction. To define the structure–function correlates of this region, we prepared deletion and alanine scanning mutants of amino acids (a.a.) 9–30 in human FSHR (hFSHR). The deletion mutants ΔS9-S18, ΔK19-N30 and ΔS9-N30 failed to bind 125I-hFSH. Alanine substitution in the mutants 2HHRI 5/ 2AAAA 5, 7HCSNR 11/ 7ACAAA 11, 16QES 18/ 16AAA 18 and 19KVT 21/ 19AAA 21 increased the affinity of hFSHR for hFSH with equilibrium dissociation constants two to fivefold lower than wild type (wt) values. Signal transduction in 2HHRI 5/ 2AAAA 5 and 19KVT 21/ 19AAA 21 was similar to wt values, whereas 7HCSNR 11/ 7ACAAA 11 and 16QES 18/ 16AAA 18 showed a twofold lower accumulation of cAMP in response to hFSH than wt. These results indicate that these regions play a role in hormone binding and signal transduction. In contrast, cells infected with mutants 12VFL 14/ 12AAA 14, 22EIPS 25/ 22AAPA 25 and 26DLPRN 30/ 26AAPAA 30 were incapable of binding 125I-hFSH even when solubilized with nonionic detergent. Flow cytometry indicated that hFSHR in 12VFL 14/ 12AAA 14, 22EIPS 25/ 22AAPA 25 and 26DLPRN 30/ 26AAPAA 30 was not present on the cell surface although the protein was expressed at high levels as determined by Western blotting. These results suggest that a discontinuous epitope in the N-terminus, likely stabilized by disulfide bonds and outside of the leucine-rich repeat domains, constitutes a hormone binding site, membrane localization signal or both.

Deletion of follicle-stimulating hormone (FSH) receptor residues encoded by exon one decreases FSH binding and signaling in the rat.

The rat FSH receptor (rFSHR) shares considerable homology with the rat LH receptor (rLHR), yet binds human FSH (hFSH) with high fidelity, suggesting that the binding determinant encoded by the rFSHR gene shares no homology with the analogous rLHR primary sequence, thereby affording specificity of ligand binding. Two such regions of primary sequence have been previously identified and studied by peptide challenge tests and immunoneutralization studies. We therefore implemented site-directed mutagenesis to delete the regions S9-N30 and D300-F315 of the mature rFSHR sequence. The mutant receptor (DeltarFSHR) cDNAs were expressed in insect cells. The large deletion DeltarFSHRS9-N30 and a smaller deletion, DeltarFSHRS9-S18, did not bind (125)I-hFSH. However, DeltarFSHRK19-R29 and DeltarFSHRD300-F315 bound (125)I-hFSH with an affinity indistinguishable from wild-type rFSHR. The deletion mutants DeltarFSHR S9-N30 or DeltarFSHRS9-S18 were not detectable on the cell surface by flow cytometry unless cells were sheared. Although (125)I-hFSH binding to DeltarFSHRK19-R29 was normal, this form of the receptor was defective for signal transduction whereas DeltarFSHRD300-F315 was not. Furthermore, neither region seems to be a specificity determinant, since their removal did not result in high-affinity binding of hCG to DeltarFSHR.

Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action.

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.

Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor.

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4alpha, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G alpha t, a scavenger of G betagamma. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G alpha t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.

Multiple promoter elements contribute to activity of the follicle-stimulating hormone receptor (FSHR) gene in testicular Sertoli cells.

The FSH receptor (FSHR) is expressed only in granulosa cells of the ovary and Sertoli cells of the testis. This highly specific pattern of gene expression asserts that transcriptional events unique to these two cell types are responsible for activation of the FSHR gene. We have characterized the promoter elements required for activity of the rat FSHR gene in a Sertoli cell line MSC-1, primary cultures of rat Sertoli cells, and two non-Sertoli cell lines. Transient transfection analysis of deletion and block replacement mutants identified several elements, both 5' and 3' to the transcriptional start sites, that are essential for full promoter activity in Sertoli cells. These studies confirmed the use of an important E box element (CACGTG), which had the single greatest impact on promoter function. Bases within the core CACGTG of the E box, as well as flanking sequences, were shown to be essential for its function. Electrophoretic mobility shift assays identified both upstream stimulatory factor 1 (USF1) and USF2 as primary components of the complexes binding the E box. Sequence requirements for USF binding in vitro modestly diverged from the sequence requirements for in vivo function of the element. Comparison of the E box binding proteins in different cell types revealed that similar proteins bind the E box in Sertoli and non-Sertoli cell lines. Extracts from primary cultures of rat and mouse Sertoli cells have a second E box-binding complex that cross-reacts with USF antibodies that is not present in the cell lines.