FSH Actions and Pregnancy: Looking Beyond Ovarian FSH Receptors.

By mediating estrogen synthesis and follicular growth in response to FSH, the ovarian FSH receptor (FSHR) is essential for female fertility. Indeed, ovarian stimulation via administration of FSH to women with infertility is part of the primary therapeutic intervention used in assisted reproductive technology. In physiological and therapeutic contexts, current dogma dictates that once ovulation has occurred, FSH/FSHR signaling is no longer required for successful pregnancy outcomes. However, a continued role for FSH during pregnancy is suggested by recent studies demonstrating extraovarian FSHR in the female reproductive tract. Furthermore, functional roles for FSHR in placenta and in uterine myometrium have now been demonstrated. In placenta, vascular endothelial FSHR of fetal vessels within the chorionic villi (human) or labyrinth (mouse) mediate angiogenesis, and it has further been shown that deletion of placental Fshr in mice has deleterious effects on pregnancy. In uterine myometrium, changes in the densities of FSHR in muscle fiber and stroma in the nonpregnant state, early pregnancy, and term pregnancy differentially regulate contractile activity, suggesting that signaling through myometrial FSHR may contribute to the quieting of contractile activity required for successful implantation and that the temporal upregulation of the FSHR at term pregnancy may be required for the appropriate timing of parturition. In addition, extraovarian expression of mRNAs encoding the glycoprotein hormone α subunit and the FSH ß subunit has been demonstrated, suggesting that these novel aspects of extraovarian FSH/FSHR signaling during pregnancy may be mediated by locally synthesized FSH.

Deletion of fetoplacental Fshr inhibits fetal vessel angiogenesis in the mouse placenta.

It has been shown in both human and mouse placentas that follicle stimulating hormone receptor (FSHR) is expressed in fetal vascular endothelium. There are conflicting reports, however, on the role of FSH to stimulate angiogenesis in vitro in cultured endothelial cells from umbilical veins. Therefore, in this study we undertook an in vivo approach utilizing Fshr null mice to definitively address this question. In the context where all pregnant dams have identical Fshr genotypes, we generated fetuses and associated fetal portions of placenta that were Fshr wt or Fshr null and analyzed angiogenesis within the placental labyrinths. Quantitative morphometric analyses of placentas obtained at mid-gestation revealed that the percentage of the placenta composed of labyrinth is significantly decreased in Fshr null placentas relative to wt placentas. Furthermore, data presented demonstrate that within the Fshr null labyrinths, fetal vessel angiogenesis was significantly reduced relative to wt labyrinths. The results obtained with this combination of in vivo and genetic approaches conclusively demonstrate that signaling through endothelial FSHR does indeed stimulate angiogenesis and that placental Fshr is essential for normal angiogenesis of the fetal placental vasculature.

Differential Regulation of Human and Mouse Myometrial Contractile Activity by FSH as a Function of FSH Receptor Density.

Previous studies from our laboratory revealed that the follicle-stimulating hormone receptor (FSHR) is expressed at low levels in nonpregnant human myometrium and that it is up-regulated in pregnant term nonlaboring myometrium; however, the physiological relevance of these findings was unknown. Herein, we examined signaling pathways stimulated by FSH in immortalized uterine myocytes expressing recombinant FSHR at different densities and showed that cAMP accumulation is stimulated in all cases but that inositol phosphate accumulation is stimulated only at high FSHR densities. Because an increase in cAMP quiets myometrial contractile activity but an increase in 1,4,5-triphosphoinositol stimulates contractile activity, we hypothesized that FSHR density dictates whether FSH quiets or stimulates myometrial contractility. Indeed, in human and mouse nonpregnant myometrium, which express low levels of FSHR, application of FSH resulted in a quieting of contractile activity. In contrast, in pregnant term nonlaboring myometrium, which expresses higher levels of FSHR, application of FSH resulted in increased contractile activity. Examination of pregnant mouse myometrium from different stages of gestation revealed that FSHR levels remained low throughout most of pregnancy. Accordingly, through mid-gestation, the application of FSH resulted in a quieting of contractile activity. At Pregnancy Day (PD) 16.5, FSHR was up-regulated, although not yet sufficiently to mediate stimulation of contractility in response to FSH. This outcome was not observed until PD 19.5, when FSHR was further up-regulated. Our studies describe a novel FSHR signaling pathway that regulates myometrial contractility, and suggest that myometrial FSHR levels dictate the quieting vs. stimulation of uterine contractility in response to FSH.

FSH receptor (FSHR) expression in human extragonadal reproductive tissues and the developing placenta, and the impact of its deletion on pregnancy in mice.

Expression and function of the follicle-stimulating hormone receptor (FSHR) in females were long thought to be limited to the ovary. Here, however, we identify extragonadal FSHR in both the human female reproductive tract and the placenta, and test its physiological relevance in mice. We show that in nonpregnant women FSHR is present on: endothelial cells of blood vessels in the endometrium, myometrium, and cervix; endometrial glands of the proliferative and secretory endometrium; cervical glands and the cervical stroma; and (at low levels) stromal cells and muscle fibers of the myometrium. In pregnant women, placental FSHR was detected as early as 8-10 wk of gestation and continued through term. It was expressed on: endothelial cells in fetal portions of the placenta and the umbilical cord; epithelial cells of the amnion; decidualized cells surrounding the maternal arteries in the maternal decidua; and the stromal cells and muscle fibers of the myometrium, with particularly strong expression at term. These findings suggest that FSHR expression is upregulated during decidualization and upregulated in myometrium as a function of pregnancy. The presence of FSHR in the placental vasculature suggests a role in placental angiogenesis. Analysis of genetically modified mice in which Fshr is lacking in fetal portions of the placenta revealed adverse effects on fetoplacental development. Our data further demonstrate FSHB and CGA mRNAs in placenta and uterus, consistent with potential local sources of FSH. Collectively, our data suggest heretofore unappreciated roles of extragonadal FSHR in female reproductive physiology.

Signaling through FSH receptors on human umbilical vein endothelial cells promotes angiogenesis.

CONTEXT: The FSH receptor (FSHR) is traditionally thought to play a role in female reproductive physiology solely within the context of ovarian FSHR. However, FSHR is also expressed in endothelial cells of the placental vasculature and human umbilical cord vessels, suggesting additional facets of female reproduction regulated by extragonadal FSHR. OBJECTIVE: We sought to determine the functional role of FSHR on human umbilical cord endothelial cells (HUVECs), hypothesizing that activation of the FSHR would stimulate angiogenesis. DESIGN: The ability of FSH to stimulate several angiogenic processes in HUVECs was determined. SETTING: This was a laboratory-based study using commercially prepared HUVECs. RESULTS: Tube formation, wound healing, cell migration, cell proliferation, nitric oxide production, and cell survival were stimulated in response to FSH. Quantitative comparisons between HUVECs incubated with maximally stimulatory concentrations of FSH vs vascular endothelial growth factor (VEGF), a well-characterized angiogenic factor, revealed that FSH is as efficacious as VEGF in promoting angiogenic processes. FSH did not provoke increased secretion of VEGF by HUVECs, suggesting the direct stimulation of angiogenic processes by FSH in endothelial cells. In contrast to gonadal cells, the FSHR on HUVECs did not mediate an FSH-stimulated increase in cAMP. However, increased phosphorylation of AKT in response to FSH was observed, suggesting that FSH stimulation of HUVEC FSHR stimulates the PI3K/AKT signaling pathway. CONCLUSIONS: Our studies reveal a novel role for FSHR in female reproductive physiology. Its ability to promote angiogenesis in placental endothelial cells suggests that the FSHR may have an influential role in pregnancy.

Heterodimerization between the lutropin and follitropin receptors is associated with an attenuation of hormone-dependent signaling.

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

Regulatory processes governing the cell surface expression of LH and FSH receptors.

The LH receptor (LHR) and FSH receptor (FSHR), collectively termed the gonadotropin receptors, are members of the Family A of GPCRs. The gonadotropin receptors each contain N-linked carbohydrates that are not directly involved in hormone binding, but contribute to the proper folding, and therefore, cell surface expression of the receptor. Loss-of-function mutations of an LHR or FSHR results in decreased target cell responsiveness. Most inactivating mutations cause receptor misfolding, resulting in the retention of the mutant in its immature form in the endoplasmic reticulum. A membrane-permeable allosteric agonist of the LHR has been shown to serve as a pharmacological chaperone for misfolded and intracellularly retained LHRs by promoting their cell surface expression. Wild-type LHR and FSHR each form homodimers and heterodimers while in the ER. Therefore, when wild-type receptor is co-expressed with a misfolded mutant, the misfolded receptor dimerizes with immature wild-type receptor in the ER, causing a dominant-negative effect on cell surface expression of the mature wild-type receptor. Notably, the propensity for homodimerization is not affected by the activation status of the receptor. However, within a receptor dimer, the activity of one protomer may allosterically regulate the other protomer. Therefore, the dimerization of the gonadotropin receptors appears to be an obligate process that is part of the normal itinerary for trafficking to the cell surface and, once there, the dimerized receptors allow for additional modulations of cell signaling.

Revisiting and questioning functional rescue between dimerized LH receptor mutants.

The glycoprotein hormone receptors are G protein-coupled receptors containing a large extracellular domain fused to a prototypical serpentine domain. cis-activation occurs when binding of hormone to the extracellular domain stabilizes the serpentine domain in an active conformation. Studies by others suggested that these receptors can also signal by trans-activation, where hormone binding to one receptor protomer activates the serpentine domain of an associated protomer, as documented by the partial rescue of hormone-dependent signaling when a binding defective mutant is coexpressed with a signaling defective mutant. However, our characterizations of several LH receptor (LHR) mutants used in previous studies differ markedly from those originally reported. Also, when examining a pair of LHR mutants previously shown to functionally rescue in vitro as well as in vivo, in addition to finding that the properties of the individual mutants differ significantly from those originally described, we determined that when this pair of mutants was coexpressed in vitro, quantitative analyses did not indicate functional rescue. Additional data are presented that provide a plausible alternate explanation for the apparent in vivo trans-activation that was reported. Finally, using LHR mutants that we have documented to be expressed at the cell surface but to lack human chorionic gonadotropin binding activity or to be severely impaired in their ability to activate Gs, we did not observe functional rescue of human chorionic gonadotropin-stimulated cAMP when the mutants were coexpressed, even though bioluminescence resonance energy transfer analyses confirmed that the coexpressed mutants formed dimers. Taken altogether, our data substantively question the concept of functional rescue between LHR mutants.

Rescue of expression and signaling of human luteinizing hormone G protein-coupled receptor mutants with an allosterically binding small-molecule agonist.

Naturally occurring mutations of G protein-coupled receptors (GPCRs) causing misfolding and failure to traffic to the cell surface can result in disease states. Some small-molecule orthosteric ligands can rescue such misfolded receptors, presumably by facilitating their correct folding and shuttling to the plasma membrane. Here we show that a cell-permeant, allosterically binding small-molecule agonist (Org 42599) rescues the folding and cell surface expression, and therefore target cell signaling, of mutant human luteinizing hormone (LH) receptors (A593P and S616Y) that cause Leydig cell hypoplasia in man. Both mutant receptors were retained in the cytoplasm whereas WT receptor localized at the cell membrane, and binding of LH to cells expressing the mutant receptors was markedly lower than to those expressing the WT receptor. Incubation with Org 42599 increased mutant receptor expression, cell surface localization, and the proportion of mutant receptor in the mature glycosylated form. Importantly, although LH stimulated little (S616Y) or no (A593P) activation of cells expressing mutant receptors, incubation of cells with Org 42599 facilitated rescue of expression and stimulation by the native ligand, LH. Although Org 42599 could activate these receptors, it could not displace (125)I-labeled human LH binding to the WT receptor, indicating that it acts in an allosteric manner. Here we demonstrate a small-molecule GPCR allosteric agonist that functionally rescues intracellularly retained mutant LH receptors by facilitating their cell surface expression. This approach may have application for treatment of infertile patients bearing such mutations and, more broadly, for other misfolded GPCR mutants resulting in human pathologic processes.

Constitutive activity of the lutropin receptor and its allosteric modulation by receptor heterodimerization.

The lutropin receptor (LHR) is a G protein-coupled receptor (GPCR) that mediates the actions of pituitary LH in males and females and that of placental hCG in pregnant women and, therefore, plays an essential role in reproductive physiology. Mutations of the lhcgr gene that result in constitutive activation of the LHR have been shown to be causative of gonadotropin-independent precocious puberty in young boys. Studies on constitutively active mutants (CAMs) of the LHR have been extremely informative in elucidating the roles of the LHR in reproductive physiology as well as in understanding the molecular basis underlying activation of this GPCR. The constitutive activities of hLHR CAMs can be attenuated by introducing mutations into the CAMs that stabilize the resting state of the hLHR or by coexpressing the hLHR CAMs with an hLHR mutant that is stabilized in the resting state, allowing the two forms of the hLHR to heterodimerize. This chapter describes the experimental methods and strategies underlying studies of hLHR CAMs.

Female mice expressing constitutively active mutants of FSH receptor present with a phenotype of premature follicle depletion and estrogen excess.

Strong gain-of-function mutations have not been identified in humans in the FSH receptor (FSHR), whereas such mutations are common among many other G protein-coupled receptors. In order to predict consequences of such mutations on humans, we first identified constitutively activated mutants of the mouse (m) Fshr and then expressed them under the human anti-Müllerian hormone promoter in transgenic mice or created knock-in mutation into the mouse genome. We show here that mutations of Asp580 in the mFSHR significantly increase the basal receptor activity. D580H and D580Y mutations of mFSHR bind FSH, but the activity of the former is neither ligand-dependent nor promiscuous towards LH/human choriogonadotropin stimulation. Transgenic expression of mFshr(D580H) in granulosa cells leads to abnormal ovarian structure and function in the form of hemorrhagic cysts, accelerated loss of small follicles, augmented granulosa cell proliferation, increased estradiol biosynthesis, and occasional luteinized unruptured follicles or teratomas. The most affected mFshr(D580H) females are infertile with disturbed estrous cycle and decreased gonadotropin and increased prolactin levels. Increased estradiol and prolactin apparently underlie the enhanced development of the mammary glands, adenomatous pituitary growth, and lipofuscin accumulation in the adrenal gland. The influence of the mFSHR(D580Y) mutation is milder, mainly causing hemorrhagic cysts in transgenic mFSHR(D580Y) and mFSHR(D580Y) -knock-in mice. The results demonstrate that gain-of-function mutations of the FSHR in mice bring about distinct and clear changes in ovarian function, informative in the search of similar mutations in humans.

Structural determinants underlying constitutive dimerization of unoccupied human follitropin receptors.

The human follitropin receptor (hFSHR) is a G protein-coupled receptor (GPCR) central to reproductive physiology that is composed of an extracellular domain (ECD) fused to a serpentine region. Using bioluminescence resonance energy transfer (BRET) in living cells, we show that hFSHR dimers form constitutively during their biosynthesis. Mutations in TM1 and TM4 had no effect on hFSHR dimerization, alone or when combined with mutation of Tyr(110) in the ECD, a residue predicted to mediate dimerization of the soluble hormone-binding portion of the ECD complexed with FSH (Q. Fan and W. Hendrickson, Nature 433:269-277, 2005). Expressed individually, the serpentine region and a membrane-anchored form of the hFSHR ECD each exhibited homodimerization, suggesting that both domains contribute to dimerization of the full-length receptor. However, even in the context of only the membrane-anchored ECD, mutation of Tyr(110) to alanine did not inhibit dimerization. The full-length hFSHR and the membrane-anchored ECD were then each engineered to introduce a consensus site for N-linked glycosylation at residue 110. Despite experimental validation of the presence of carbohydrate on residue 110, we failed to observe disruption of dimerization of either the full-length hFSHR or membrane-anchored ECD containing the inserted glycan wedge. Taken altogether, our data suggest that both the serpentine region and the ECD contribute to hFSHR dimerization and that the dimerization interface of the unoccupied hFSHR does not involve Tyr(110) of the ECD.

A cell surface inactive mutant of the human lutropin receptor (hLHR) attenuates signaling of wild-type or constitutively active receptors via heterodimerization.

The D405N and Y546F mutations of the human lutropin receptor (hLHR) have previously been shown to partially attenuate hCG-stimulated cAMP synthesis despite normal cell surface expression and hCG binding affinity (Min, L. and Ascoli, M. Mol. Endocrinol. 14:1797-1810, 2000). We now show that these mutations each stabilize a resting state of the hLHR. A combined mutant D405N,Y546F is similarly expressed at the cell surface and exhibits normal ligand-binding, but is profoundly signaling impaired. Introduction of hLHR(wt) into cells stably expressing the signaling inactive D405N,Y546F resulted in the attenuation of hCG-stimulated cAMP production by hLHR(wt) even if excess Gs is co-expressed. Similarly, co-expression of D405N,Y546F with hLHR constitutively active mutants (CAMs) attenuated their constitutive activity. Quantitative bioluminescence resonance energy transfer (BRET) analyses demonstrated that D405N,Y546F formed heterodimers with both wt and CAM hLHR. In contrast hLHR(D405N,Y546F) did not heterodimerize with the melanocortin 3 receptor (MC3R) and agonist-stimulated cAMP production through the MC3R was not attenuated when these two receptors were co-expressed. Taken altogether, our data demonstrate that a signaling inactive hLHR mutant (that is trafficked normally to the plasma membrane) attenuates the signaling of the cell surface localized wt or the constitutively active hLHR due to receptor heterodimerization. Our studies, therefore, suggest a novel ramification of GPCR signaling resulting from receptor dimerization.

Bioluminescence resonance energy transfer studies reveal constitutive dimerization of the human lutropin receptor and a lack of correlation between receptor activation and the propensity for dimerization.

Previous studies from our laboratory using co-immunoprecipitation techniques suggested that the human lutropin receptor (hLHR) constitutively self-associates into dimers/oligomers and that agonist treatment of cells either increased hLHR dimerization/oligomerization and/or stabilized hLHR dimers/oligomers to detergent solubilization (Tao, Y. X., Johnson, N. B., and Segaloff, D. L. (2004) J. Biol. Chem. 279, 5904-5914). In this study, bioluminescence resonance energy transfer (BRET(2)) analyses confirmed that the hLHR constitutively self-associates in living cells. After subcellular fractionation, hLHR dimers/oligomers were detected in both the plasma membrane and endoplasmic reticulum (ER). Further evidence supporting the constitutive formation of hLHR dimer/oligomers in the ER is provided by data showing homodimerization of misfolded hLHR mutants that are retained in the ER. These mutants, when co-expressed with wild-type receptor, are shown by BRET(2) to heterodimerize, accounting for their dominant-negative effects on cell surface receptor expression. Hormone desorption assays using intact cells demonstrate allosterism between hLHR protomers, indicating functional cell surface hLHR dimers. However, quantitative BRET(2) analyses in intact cells indicate a lack of effect of agonist on the propensity of the hLHR to dimerize. Using purified plasma membranes, human chorionic gonadotropin was similarly observed to have no effect on the BRET(2) signal. An examination of the propensity for constitutively active and signaling inactive hLHR mutants to dimerize further showed no correlation between dimerization and the activation state of the hLHR. Taken altogether, our data suggest that hLHR dimers/oligomers are formed early in the biosynthetic pathway in the ER, are constitutively expressed on the plasma membrane, and are not affected by the activation state of the hLHR.

Diseases associated with mutations of the human lutropin receptor.

The human lutropin receptor (LHCGR) plays an integral role in male and female reproductive physiology. In response to either placental hCG or pituitary LH, gonadal LHCGR mediates its effects primarily through Gs activation. Heterozygous mutations leading to constitutive activation of the LHCGR cause gonadotropin-independent precocious puberty in males, but have no detectable effects on prepubertal or postpubertal females. Homozygous or compound heterozygous inactivating mutations of the LHCGR cause gonadal resistance to hCG and LH, where the clinical phenotypes associated with these mutations are closely correlated with the severity of the mutation. Inactivating mutations in 46,XY individuals cause Leydig cell hypoplasia and impairments in the differentiation of male external genitalia, the development of secondary sexual characteristics and sperm production. 46,XX siblings with inactivating LHCGR mutations exhibit infertility and varying degrees of menstrual irregularities.

Follicle stimulating hormone receptor mutations and reproductive disorders.

The follicle stimulating hormone receptor (FSHR) plays a critical role in reproductive function. In the males, FSH supports spermatogenesis, whereas in females, FSH is absolutely required for ovarian follicle growth. In females, inactivating mutations in the FSHR result in ovarian dysgenesis with amenorrhea and infertility. The few males reported with severe inactivating mutations exhibited varying spermatogenic defects, but not azoospermia. While these findings may potentially suggest that FSH action is not absolutely required for spermatogenesis, it cannot be ruled out that these individuals have some residual FSHR activity. Gain-of-function mutations in the FSHR cause spontaneous ovarian hyperstimulation syndrome in females due to the inappropriate stimulation of the mutant FSHR by human choriogonadotropin.

An intracellular loop (IL2) residue confers different basal constitutive activities to the human lutropin receptor and human thyrotropin receptor through structural communication between IL2 and helix 6, via helix 3.

The human lutropin receptor (hLHR) and human TSH receptor (hTSHR) are G protein-coupled receptors that play key roles in reproductive and thyroid physiology, respectively. We show using a quantitative assessment of cAMP production as a function of cell surface receptor expression that the hTSHR possesses greater basal constitutive activity than the hLHR. Further studies were undertaken to test the hypothesis that different potential Gs-coupling motifs identified in IL2 of the hTSHR and hLHR contribute to their different basal constitutive activities. Although mutating the receptors to interchange their potential Gs-coupling motifs reversed their relative activities, we show this to be due to the swapping of one IL2 residue (Q476 in the hLHR; R531 in the hTSHR). Molecular dynamics simulations show that the effect of the hLHR(Q476R) mutation, switching the structural features of the hLHR toward those of the hTSHR, is greater than the switching effect of the hTSHR(R531Q) mutant toward the hLHR. The structural model of the hLHR(Q476R) mutant can be considered as a hybrid of wild-type (wt) hTSHR and constitutively active mutant hLHR forms. In this hLHR(Q476R) mutant, IL2 adopts a structure similar to IL2 of the wt hTSHR, but it shares with the hLHR constitutively active mutant the solvent exposure and the reciprocal arrangement of helices 3, 5, and 6, including the weakening of the wt native R3.50-D6.30 interaction. Our results suggest a H3-mediated structural connection between IL2 and the cytosolic extension of H6. Thus, IL2 contributes significantly to the inactive and active state ensembles of these G protein-coupled receptors.

Intrinsic differences in the response of the human lutropin receptor versus the human follitropin receptor to activating mutations.

In contrast to the human lutropin receptor (hLHR), very few naturally occurring activating mutations of the structurally related human follitropin receptor (hFSHR) have been identified. The present study was undertaken to determine if one aspect underlying this discrepancy might be a general resistance of the hFSHR to mutation-induced constitutive activity. Five different mutations were introduced into both the hLHR and hFSHR (four based on activating mutations of the hLHR gene, one based on an activating mutation of the hFSHR gene). Our results demonstrate that hFSHR constitutively activating mutants (CAMs) were not as active as hLHR CAMs containing the comparable mutation. Furthermore, although all hFSHR CAMs exhibited strong promiscuous activation by high concentrations of the other glycoprotein hormone receptors, hLHR CAMs showed little or no promiscuous activation. Our in vitro findings are consistent with in vivo observations of known pathophysiological conditions associated with hLHR CAMs, but not hFSHR CAMs, and with promiscuous activation of hFSHR CAMs, but not hLHR CAMs. Computational experiments suggest that the mechanisms through which homologous mutations increase the basal activity of the hLHR and the hFSHR are similar. This is particularly true for the strongest CAMs like L460(3.43)R. Disparate properties of the hLHR versus hFSHR CAMs may, therefore, be due to differences in shape and electrostatics features of the solvent-exposed cytosolic receptor domains involved in the receptor-G protein interface rather than to differences in the nature of local perturbation at the mutation site or in the way local perturbation is transferred to the putative G protein binding domains.

Evaluating the roles of follicle-stimulating hormone receptor polymorphisms in gonadal hyperstimulation associated with severe juvenile primary hypothyroidism.

CONTEXT: Rare activating mutations of the human (h)FSHR have been reported in some women with spontaneous ovarian hyperstimulation in pregnancy, where follicular growth is inappropriately stimulated by elevated concentrations of human chorionic gonadotropin acting through the hFSHR. It is not known whether ovarian hyperstimulation in peripubertal girls with untreated primary hypothyroidism is caused by hFSHR mutations and/or influenced by hFSHR allelic variants, rendering the hFSHR more sensitive to circulating TSH. OBJECTIVE: The aim of the study was to determine whether mutations of the hFSHR and/or hFSHR allelic variants are associated with greater sensitivity of the hFSHR to TSH. DESIGN: The hFSHR gene was sequenced from eight pediatric patients displaying gonadal hyperstimulation due to primary hypothyroidism. HEK293 cells expressing different hFSHR allelic combinations were studied for their responsiveness to recombinant (r)hTSH. SETTING: The study was conducted at university research centers. PATIENTS: Eight unrelated patients (seven girls and one boy) who exhibited primary hypothyroidism and gonadal hyperstimulation were included in the study. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURE: DNA sequencing of the hFSHR gene was the main outcome measure. Basal, rhFSHR- and rhTSH receptor-stimulated cAMP levels were assayed in HEK293 cells transfected with the hTSH receptor or different hFSHR allelic combinations. Cell surface receptor numbers were also determined. RESULTS: No hFSHR mutations were identified in the patient population, but we did identify two known polymorphisms. In vitro experiments demonstrated a dose-dependent and specific rhTSH-dependent increase in cAMP production in HEK293 cells expressing the wild-type hFSHR, regardless of hFSHR isoform. CONCLUSIONS: Pediatric gonadal hyperstimulation associated with severe primary hypothyroidism is likely due to the actions of the elevated concentrations of TSH on the wild-type hFSHR, and this response is not dependent upon the hFSHR isoform.

Insights learned from L457(3.43)R, an activating mutant of the human lutropin receptor.

The L457(3.43)R mutation of the hLHR was initially identified in a Brazilian boy with gonadotropin-independent precocious puberty. As would be expected, L457(3.43)R, when expressed in 293 cells, caused a marked elevation in basal cAMP levels. Interestingly, in spite of the fact that the elevated basal levels of cAMP elicited by L457R were not as great as those elicited by the wild-type hLHR when stimulated with hCG, L457(3.43)R cells were unresponsive to further hormonal stimulation. We have since determined that the L457(3.43)R mutant, as well as other constitutively active mutants of the hLHR, causes an increase in phosphodiesterase activity that attenuates the target cell to hormonal stimulation of the wild-type hLHR or other Gs-coupled GPCRs. We have also shown that the constitutive activity and lack of hormonal responsiveness of L457(3.43)R are due to the formation of a salt bridge between the introduced arginine in the mid portion of helix 3 with D578(6.44) in the mid portion of helix 6. The formation of this salt bridge results in the disruption of interactions between the cytoplasmic ends of helices 3 and 6 that are associated in general with activation of the hLHR. As such, this mutant has provided novel insights into the properties of target cells expressing activating hLHR mutants and into the structural basis for hLHR activation.