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Constitutively Active Mutations of G Protein-Coupled Receptors
Archives of Medical Research 30 (1999) 501–509
REVIEW ARTICLE
Constitutively Active Mutations of G Protein-Coupled Receptors: The Case of the Human Luteinizing Hormone and Follicle-Stimulating Hormone Receptors Verena Nordhoff, Jörg Gromoll and Manuela Simoni Institute of Reproductive Medicine of the University, Münster, Germany Received for publication September 9, 1999; accepted September 10, 1999 (99/158).
Activating mutations of the luteinizing hormone receptor (LHR) and the follicle-stimulating hormone receptor (FSHR) have been known for several years. These activating mutations permanently stimulate, in the absence of their cognate ligand, the receptor signaling pathways. In the case of the LHR, the induced chronic stimulation causes sporadic and familial pseudoprecocious puberty, a phenotype observed only in males. The absence of a female phenotype is probably due to the requirement for FSH in the induction of LHR expression. For the FSHR, one activating mutation was found in a patient with normal spermatogenesis without detectable gonadotropins. Whether activating mutations of the gonadotropin receptors are involved in tumor development is not yet clear. Activating mutations of the FSHR were supposedly involved but not found in ovarian tumors. For the LHR, only one patient with a seminoma and an activating mutation was described. The different occurrence of activating mutations of the LHR compared to the FSHR is surprising, since the two genes are adjacently located on chromosome 2 and should therefore be affected by a similar mutation rate. It might well be that mutations occur with the same frequency, but that activating mutations of the FSHR do not result in any particular phenotype. © 2000 IMSS. Published by Elsevier Science Inc. Key Words: Luteinizing hormone receptor, Follicle-stimulating hormone receptor, Constitutively active receptor, Mutation.
Introduction Hormonal Control of Sexual Differentiation and Maturation In the human, sex differentiation is determined by two major switches. The genetic sex of the individual is determined by the presence of a testis-determining factor, the sex-determining factor on the Y-chromosome (SRY), directing male determination during embryonic development. The second major switch in sex differentiation is hormonal control of the gonads and secondary sex characteristics. Hypothalamic gonadotropin-releasing hormone (GnRH) and the two pituitary gonadotropins, luteinizing hormone (LH) and folliclestimulating hormone (FSH), are already detected at around week 10 of fetal development. The hypophyseal portal system matures until midgestation and GnRH, LH, and FSH
Address reprint requests to: Professor Manuela Simoni, Institute of Reproductive Medicine of the University, Domagkstrasse 11, 48149 Münster, Germany. Tel: (149) (251) 835-6444; Fax: (149) (251) 835-2736; Email: [email protected]
levels increase, stimulating gonadal maturation and hormone production. In the male fetal testis, differentiation and development of the Wolffian duct are grossly dependent on the presence of the maternal hCG, which stimulates testosterone production by the Leydig cells. Ovarian steroidogenesis is also active early in development, but is not necessary for female genital differentiation. At birth, gonadotropin and sex steroid levels are high, but decline during the first few days of life (1). During the following weeks, serum LH and FSH levels rise to a level higher than that observed during the rest of childhood in both sexes, FSH being higher in females than in males. After a peak, at 2–3 months of life, gonadotropin levels drop and persist as low for several years. The pituitary and the gonads remain quiescent until puberty, when GnRH is released in a pulsatile fashion, stimulating synthesis and secretion of gonadotropins. Presence of LH and FSH results in an increasing secretion of steroids, such as testosterone or estrogens, leading to the development of secondary sexual characteristics, acceleration of growth, and progressive gain of fertility. Adult levels of LH and FSH are
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regulated by steroid hormones through a negative feedback effect in the male. In the female, both negative and positive feedback mechanisms operate during the different stages of the cycle (2). The gonadotropin receptors. LH and FSH exert their action through specific receptors (3). These receptors, the LH receptor (LHR) and the FSH receptor (FSHR), belong to the G protein-coupled receptor family and are characterized by a very large extracellular domain to which the hormones bind specifically. They share, together with the thyroid-stimulating hormone receptor (TSHR), the common feature of seven-transmembrane helices inserted into the plasma membrane and an intracellular domain for signal transduction. The LH receptor. The LHR gene is located on chromosome 2p21 and consists of 11 exons spanning at least 60 kbp (4,5). Exons 1–10 encode the extracellular domain, involved in hormone binding, whereas exon 11 bears seven membrane-spanning domains—characteristic of G proteincoupled receptors—and one intracellular domain. In the male, the LHR is predominantly expressed in the Leydig cells. Expression begins during the late fetal period and continues at low levels until puberty. Thereafter, LHR expression is up-regulated (3). In the female, LHR expression can be detected from the second postnatal week onward. The LHR is expressed by the theca and granulosa cells of the ovary. During follicle maturation, which begins at puberty, expression of the LHR is induced by FSH. Upon stimulation of LH, theca cells produce androgens, which are converted under FSH stimulation into estrogens by the granulosa cells. Expression of LH receptors at midcycle in the dominating follicle prepares the follicle for the LH surge, which will induce ovulation. In addition, LH receptors are necessary for the production of progesterone by the corpus luteum. Later in the cycle, estradiol stimulates LHR density and activity. If FSH stimulation is lacking, the number of FSHR and LHR declines, and the granulosa cells die (6). The FSH receptor. The FSHR gene is, like the LHR gene, located on chromosome 2p21 and consists of 10 exons spanning 54 kbp (7,8). Exons 1–9 encode the extracellular domain, and exon 10 encodes the transmembrane and intracellular domain. In the male, FSHR expression is exclusively confined to the Sertoli cells. FSHR expression in the rat is first detectable around week 3 of fetal development and persists until adulthood. In the testis, the FSHR is expressed stage-dependently during spermatogenesis (9). In the female, FSHR expression is restricted to granulosa cells and begins first during week 1 postpartum. During puberty, the FSHR expression is induced and presumably upregulated during the follicular phase. After ovulation, the FSHR mRNA is down-regulated, indicating its importance
only for follicular growth and maturation. After luteinization, FSH binding is undetectable, as in postmenopausal women (10). Signal transduction through the LH receptor and FSH receptor. Gonadotropins act mainly through stimulation of intracellular cyclic adenosine-monophosphate (cAMP). In the inactive state, the receptor is bound to a Gs protein. Upon binding of the hormone to the receptor, the Gsa-subunit with a bound guanosine-triphosphate (GTP) dissociates and activates adenylyl cyclase, which leads to the synthesis of cAMP. Protein kinase A (PKA) is activated by cAMP, which causes the dissociation of the catalytic subunit from the regulatory subunit. The activated catalytic subunit is then able to activate other proteins by phosphorylation. Action of the LHR can also be mediated through production of inositol triphosphate (IP3), leading to an increase in Ca21 concentrations. Activation of protein kinase C (PKC) leads to phosphorylation of other proteins, such as structural proteins, enzymes and transcriptional activators or repressors (2).
Activating Mutations of the LHR and FSHR Mutations of receptor genes can result in gain-of-function, due to an activating mutation, or in loss-of-function, as a result of an inactivating mutation. Activating mutations produce a phenotype even when heterozygous, while inactivating mutations need to be homozygous to display a phenotype. In the case of familial diseases, underlying mutations are genomic and inherited. Somatic mutations are also described as occurring, for example, only in the tumor and not in the surrounding tissue (11). Activating mutations of the LHR. The clinical picture of testotoxicosis, also called familial male-limited pseudoprecocious puberty (FMPP), has been known for years. Puberty is considered precocious if secondary sexual characteristics appear before 8 years of age in girls and 9 years of age in boys. True precocious puberty is defined by premature sustained activation of the hypothalamic GnRH pulse generator. GnRH-independent precocious puberty is due to a primary sex-steroid secretion of the gonads, which can initiate development of secondary sexual characteristics. In girls, this can be due to granulosa-theca cell tumors (12,13). In boys, gonadotropin-independent pseudoprecocious puberty or testotoxicosis is a rare autosomal-dominant disorder in which Leydig cell hyperplasia and germ cell maturation proceed in the absence of stimulation by gonadotropins (14). The treatment is based on either medroxyprogesterone acetate (15), cyproterone acetate (16) or ketoconazole (17). The antifungal drug ketoconazole inhibits C17-20 lyase activity and leads to significant reduction of testosterone levels (17). After 1–3 months of treatment, an escape is often observed, triggering a true central precocious puberty. A
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combination of ketoconazole and GnRH analogs is then necessary to reduce pituitary and gonadal hormone concentrations to the upper range of normal for prepubertal children (17). In 1991, FMPP was associated by Manasco et al. (18) with a putative testis-stimulating factor. Earlier investigations with the plasma of these patients did not show evidence of stimulation of testosterone secretion from rat Leydig cells, indicating that this condition was not related to a circulating Leydig cell activator (19). Other studies confirmed the lack of measurable gonadotropins and gonadotropin-like factors in serum, and revealed a prepubertal serum gonadotropin response to stimulation with GnRH but no change in testosterone secretion in response to administration of a long-acting GnRH agonist (15,20,21). On the other hand, in another study the plasma of boys with FMPP was tested in a murine Leydig cell assay and an LH-like factor was postulated (22). These different results led Manasco et al. (18) to develop an in vivo bioassay for testosteronestimulating activity in adult cynomolgous monkeys. The authors reported data indicating that boys with FMPP had a factor in their plasma that stimulated monkey testes to secrete testosterone (18). These data, however, were not confirmed by other investigators. Shortly thereafter, Shenker et al. (23) found that an activating mutation of the LHR was responsible for the gonadotropin-independent activation of Leydig cells. After some years of molecular analysis of FMPP patients, the occurrence of activating mutations of the LHR is now being accepted as causative for this phenotype. Shenker et al. (23) screened the LHR gene of young boys from eight different FMPP families (see Figure 1). These authors found a heterozygous mutation in codon 578, located in the sixth transmembrane domain, which leads to a transposition of glycine for aspartate. Functional studies in COS-7 cells revealed that the mutated LHR in the unstimulated state increased the production of cAMP to 4.5-fold when compared with the wild-type receptor. This indicated a constitutive activation in the absence of the natural ligand. The mutated receptor was capable of responding to increasing concentrations of hCG with increasing amounts of cAMP, comparable to levels produced by the wild-type receptor (23). This was the first activating mutation described. In the meantime, several groups of researchers found other amino acid substitutions in the LHR also leading to constitutive activation (Figure 1). To date, activating mutations have been found in the first transmembrane domains (TM 1) (24) and in the second transmembrane domain (TM 2) (25–27). Most mutations, however, are found in the region covering transmembrane domains 5 and 6 (TM 5, TM 6) and the connecting intracytoplasmic loop 3 (ICL 3) (23,28–41) (Figure 1, Table 1). Within this region, the mutation of codon 578 seems to be the most frequent (28–32) (Figure 1, Table 1). Based on the distribution of mutations, a hot-spot region was postulated, crucial for LHR signal transduction. Frequent occur-
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rence of activating mutations in this region suggests that TM 5, TM 6 and ICL 3 play an important role in Gs protein coupling. This hypothesis was recently tested by Schulz et al. (42), who reported constitutive activation in vitro when deleting parts of ICL 3 in the LHR and the thyroid-stimulating hormone receptor (TSHR) (42). Deletion of nine amino acids (558–566) resulted in a 2.6-fold increase in basal cAMP levels, but a lower response when stimulated with hCG, compared to the wild-type. Deletion of four conserved amino acids (563–566) produced the same findings. After deleting single amino acids at these positions, the authors concluded that loss of aspartate at position 564 was responsible for the constitutive activity (42). Several other groups produced amino acid substitutions in vitro, leading to receptor activation. Kosugi et al. (43) mutated amino acid position 578, known to be involved in receptor activation, and replaced it with several others. A proline or asparagine at this crucial position showed no activation, while replacement with glycine, serine, leucine, tyrosine, or phenylalanine caused a 4.9–5.6-fold stimulation of basal cAMP (43). It seems that not only the location of the amino acid but the characteristics of the inserted amino acid are of importance. The explanation could be that a properly positioned hydrogen bond acceptor is necessary for maintaining the receptor in an inactive state. Yano et al. (44) also mutated in vitro a critical amino acid (phenylalanine 576) to study the influence on the secondary structure of ICL 3. A change to the amino acid isoleucine or glycine showed constitutive activation without changes in IP3 signaling. When phenylalanine at position 576 was substituted by a tyrosine, the resulting LHR showed constitutive activity with decreased hCGinduced cAMP and IP3 signaling. Affinity was higher, while capacity of hCG binding was lower (44). A change to the amino acid glutamate resulted in no cAMP response (44). Therefore, the third intracellular loop seems to be the most important and crucial region in the LHR for activating mutations, because it regulates coupling to the Gs protein. Location of the mutations is compatible with the current model of signal transduction of glycoprotein hormones. Several groups of researchers have described a mutation in codon 578 that leads most frequently to an amino acid change from aspartate to glycine. Others found a substitution of tyrosine (30,40) or glutamate (41). All these mutations lead to increasing amounts of cAMP and therefore to an activation of the LHR. One additional activating mutation was detected in codon 581, resulting in an amino acid change from cysteine to arginine (30). Not all cases of activating mutations of the LHR identified thus far involve FMPP. Yano et al. (28) described a spontaneous case in which the parents of the patient were normal, and Latronico et al. (33) found two unrelated Brazilian boys with two different heterozygous mutations. One boy had a sporadic mutation in codon 457 and his parents were normal. The mutation in the other boy, in codon 568,
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Figure 1. Naturally occurring activating mutations of the FSHR and the LHR. Only the transmembrane domain of both receptors is indicated.
was also found in his normal sister, suggesting the inherited form of FMPP in the boy (33). Additionally, Kremer et al. (45) reported sporadic cases of precocious puberty in which an activating mutation of the LHR is involved. The investigators screened eight familial cases and nine cases with a negative family history of LH-independent precocious puberty and found seven different mutations (all previously described) in 12 patients (45). All patients with true FMPP showed an activating mutation. Interestingly, in no case did the mutation of codon 578 involve the amino acid glycine, which seems to be the most frequent amino-acid change, at least in the U.S. In the sporadic cases, these mutations were found in only a few patients (45), but often the parents were not examined for inheritance. Variability in the age of onset could also not be correlated to specific mutations.
Martin et al. (39) identified an adult male with increased serum testosterone, prepubertal concentrations of FSH and LH, Leydig cell hyperplasia, and a testicular seminoma. Thirty-five years previously, the patient was diagnosed with precocious puberty, later determined as FMPP. This is the first case of a testicular germ cell tumor in an FMPP patient (39). The authors suggest the possibility of a potential harmful effect of high testosterone concentrations in early life upon the cellular components of the testes (39). However, this is the only FMPP patient to present with a seminoma. This suggests the importance of clinical observations of boys with FMPP during maturation and later in adulthood. Aside from this case, all boys affected by FMPP due to a genomic mutation in the LHR seem to have normal gonadal function in adulthood. In addition to the short stature of the
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Table 1. Naturally occurring mutations of the LH receptor Nucleotide change GCC to GCT ATG to ACG CTC to CGC ATT to CTT GAT to GGT GCT to GTT ATG to ATA GCA to GTA ATC to CTC ACC to ATC GAT to GGT GAT to TAT GAT to GAA TGC to CGC
Amino acid position
Amino acid change
Location
373 398 457 542 564 568 571 572 575 577 578 578 578 581
A–V M–T L–R I–L D–G A–V M–I A–V I–L T–I D–G D–Y D–E C–R
TM 1 TM 2 TM 3 TM 5 ICL 3 ICL 3 TM 6 TM 6 TM 6 TM 6 TM 6 TM 6 TM 6 TM 6
affected patients, due to premature closure of the epiphyses induced by testosterone, it appears that activated LHR induces a pathological phenotype only during early life until puberty. It might well be that constitutively active LHR is differently regulated from the wild-type receptor. For example, mechanisms such as receptor down-regulation could be involved, resulting in normal testosterone production in adulthood. Only Martin et al. (39) described the follow-up of an FMPP patient. It will be interesting to analyze FMPP patients in adulthood with different LHR mutations to obtain greater insight into regulation of activated LHR. Interestingly, females do not show a phenotype. This is mainly due to the differences in male and female sex differentiation. In females, pubertal maturation requires both LH and FSH. FSH stimulates follicle growth and maturation during puberty, the follicles enlarge, and granulosa cells convert androgens (produced by theca cells on LH stimulus) to estrogens. An activating mutation of the LHR could theoretically lead to early production of androgens. Because of low aromatase activity in the absence of FSH, androgens cannot be converted into estrogens. However, because FSH is needed for the expression of the LHR, an activating mutation of the LHR has no effect until the receptor expression is stimulated. This different regulatory system compared to males could explain the lack of phenotype in females. Theoretically, after puberty, when FSH secretion begins and an activated form of the LHR is expressed, production of androgens in excess could lead to polycystic ovary syndrome (PCOS), but there is no evidence to date for such a phenotype in women carrying activating mutations of LHR (32). In transgenic mice, overexpression of LH b-subunit leads to an overproduction of LH and causes ovarian hyperandrogenism (46). Rosenthal et al. (32) identified two brothers with FMPP in whom the amino acid aspartate in codon 578 was mutated to glycine. Interestingly, the mutation was inherited from the mother. Analysis of her steroid levels in comparison with normal female volunteers revealed no differences, but responses to dexamethasone, na-
IP3 Low
Additional notes
Higher hCG binding High hCG affinity Ligand-unresponsive Normal affinity
High Low hCG binding normal affinity Normal
Normal binding
Ligand-unresponsive
Reference (24) (25–27) (33) (30) (30) (33,34) (29,35) (36) (37) (29,38) (28–32,39) (30,40) (41) (30)
fareline, and leuprolide acetetate were at the lower end of normal range. No ovarian dysfunction could be observed (32). Expression of the LHR is differentially regulated during the cycle. Regulation of activated LHR could be tighter, to minimize the effect of the chronic stimulation and maintain normal cyclicity. The result of this tighter regulation could be normal ovarian function. In in vitro experiments, the mutated LHR has a lower expression level compared to the wild-type (42). If this is also true for the in vivo situation, the number of LHR in the membrane could be lower than for the wild-type. This could result in lower cAMP response and lower stimulation and could be the reason for the lack of phenotype. Moreover, cAMP production in vitro by mutated LHR on gonadotropin stimulation is lower or absent in comparison to normal LHR (33,45). If this also holds true in vivo, it might contribute to the absence of a phenotype in both males and females in adulthood. Activating mutation of the FSHR. To date, one activating mutation of the FSHR has been identified in a patient hypophysectomized due to a pituitary tumor (47). Although this operation led to a loss of all pituitary hormones, and especially to undetectable levels of LH and FSH, the patient was fertile under testosterone substitution therapy alone. Normally, testosterone treatment sustains androgenization, but is incapable of maintaining spermatogenesis in hypophysectomized patients (48). To gain normal fertility, substitution of both LH and FSH is necessary (48,49). Surprisingly, the patient had normal spermatogenesis and fathered three children. After molecular analysis of the FSHR gene, a heterozygous mutation was found, which led to the substitution of glycine for aspartate at position 567. Functional studies on the mutated receptor revealed a slight-but-consistent 1.5fold increase in basal cAMP production, indicating constitutive activation (47). Re-analysis of these results in an immortalized mouse Sertoli cell line, a target cell closer to the in vivo situation, revealed a 3-fold increase in cAMP levels (9). This Sertoli cell
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line had lost its own endogenous FSHR (50), but still contained the machinery for signal transduction. It could be that this cell line represents a more adequate model for studying the constitutive activation of the mutated FSHR. Three-fold increase of cAMP levels obtained after transient transfection of the mutated receptor, in comparison to the wild-type, gives a hint of the greater sensitivity of this Sertoli cell line (9). The activating mutation is localized in the third intracytoplasmatic loop between the fifth and the sixth transmembrane domains, a region highly conserved among glycoprotein hormone receptors. Position 567 in the FSHR is homologous to codon 619 in the TSHR and codon 564 in the LHR, a location in which mutations leading to constitutive activation of these receptors have been described (30,51). In 1996, Kudo et al. (52) produced chimeric LH/FSH receptors with this crucial amino acid substitution. An LHR with FSHR TM 5 and TM 6, with the activating mutation at position 567 in ICL 3, produced no differences in cAMP response, in comparison to the nonmutated chimera (52). A FSHR with LHR TM 5 and 6 with the activating mutation at position 564 in the ICL 3 produced higher basal levels of cAMP when compared with the non-mutated chimera (52). The authors suggested the existence of stabilizing interactions between TM 5 and TM 6 in the FSHR, exceeding those in the LHR. In the LHR, this leads to a state in which mutations in the ICL 3 could be responsible for constitutive activation, while in the FSHR the activation would be blocked by the constrained state (52). It would be of interest to repeat these experiments in a Sertoli cell line that, in our opinion, might be better-suited for experiments concerning the FSHR. In other cell lines, the receptor might be coupled to unusual signal transduction components or be differently processed and may not, therefore, properly reflect constitutive activity. Further studies with constitutively active LH and FSH receptors in gonadal and nongonadal cell lines should afford further insights into putative cell-specific elements involved in gonadotropin receptor signal transduction. Recently, Schulz et al. (42) produced deletions in the ICL connecting TM 5 and TM 6 of the FSHR, and tested them in vitro in COS-7 cells. A receptor lacking amino acids 567–569 resulted in drastically reduced membrane expression of binding-competence and responded only mildly to stimulation with FSH, indicating the importance of the ICL for signal transduction. The deletion of amino acid 567 resulted in a receptor that did not constitutively couple to the Gs/adenylyl cyclase system. Substitution of glycine for aspartate in codon 567 showed slightly elevated basal cAMP levels (42). Taking into account the lower membrane expression of 60% compared to the wild-type, functional characteristics of the mutation suggest some degree of constitutive activity (42). Moreover, this experiment indicates the importance of the ICL 3 for activity, not only in the LHR but in the FSHR. The Asp567Gly mutation is the only activating mutation of the FSHR published to date. A female phenotype has not
yet been detected. It could be that constitutive activity of the FSHR leads to an increased number of follicles to maturation. A consequence could be a higher incidence of dizygotic twins (53–55). However, no mutations of genes involved in reproductive function, such as FSHb, CGb, inhibin bB, and GnRH could be detected in mothers of dizygotic twins (56), but to our knowledge a mutational analysis of the FSHR in such a population has not yet been published. It could be that naturally occurring activating mutations remain asymptomatic under normal physiological conditions. Sertoli cells in vitro progressively lose the capacity to respond to gonadotropin stimulation. In vivo, the FSHR is down-regulated by stimulation with FSH in the rat (57). It appears that the cells protect themselves from overstimulation by losing receptor expression. There are no data in vivo on down-regulation of constitutively active gonadotropin receptors. Transgenic mice overexpressing a constitutively active stimulatory Gas-protein in pancreatic beta-cells did not show a peculiar phenotype. However, treatment with a phosphodiesterase inhibitor induced a 2-fold increase of cAMP (58). A similar case is known for the b2-adrenergic receptor. A transgenic mouse overexpressing a constitutively activated b2-adrenergic receptor in the heart had only a mild phenotype (59). Pharmacological treatment with b-adrenergic receptor ligands resulted in a 50-fold increase of receptor expression, because of a stabilizing effect of the ligand. Receptor up-regulation led to an increase in adenylyl cyclase activity and a clear phenotype (59). By analogy, transgenic mouse models with a constitutive activated FSHR will be helpful for explaining the phenotype of such mutations. Neither the authors nor our colleagues (60,61) have found somatic FSHR mutations in granulosa cell tumors. It is well known that somatic constitutively activating mutations of the TSHR are found in thyroid adenomas (62,63), while in the LHR, only one case of a genomic mutation in a man with a testicular seminoma has been described (39). It is hypothesized that, in most cases, a constitutively activated G protein-coupled receptor is neither sufficient for, nor has a direct pathogenic role in, the development of tumors. This was recently confirmed by the lack of mutations in the GnRH receptor gene in gonadotrope adenomas (64). The case of a testicular seminoma associated with FMPP and an activating mutation of the LHR also remains an isolated observation (39). Whether seminoma is related to mutation or other factors is unknown.
Conclusions Activating mutations of the LHR cause familial or sporadic pseudoprecocious puberty that affects only boys. This is due mainly to the fact that in the male, contrary to the female, FSH is not required for expression of the LHR. Except for short body stature, affected subjects are normal in adulthood and constitutive activity of the LHR becomes clinically silent. Activating mutations of the FSHR seem to have no di-
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rect phenotype, although FSH activity is crucial for follicle maturation and spermatogenesis. Why is there such a great difference in the incidence of activating mutations between the LHR and the FSHR? It is known that genes for the FSHR and the LHR are localized very closely together on chromosome 2. According to current theory, the two gonadotropin receptors might have evolved by chromosome duplication. Thus, one would reasonably expect the same rate of occurrence of mutations in both receptors. Due to the lack of a female phenotype, and a normal phenotype in adult males with an activating mutation of the LHR, we must conclude that, as a rule, activating mutations of the LHR do not have clinical consequences except in prepubertal boys. Tight regulation of receptor expression and turnover could explain the lack of phenotype in otherwise normal men and women with activating mutation of the FSHR. If this is the case, it will be very difficult to detect new activating mutations of the FSHR, even if they occur naturally at a frequency similar to that of LHR mutations. Note Added in Proofs While this paper was edited, Liu et al. (65) published evidence that somatic activating mutations of the LHR are found in Leydig cell adenomas. The authors reported about a G to C transition at nucleotide position 1732 of the LHR, exchanging an aspartic acid residue with histidine at amino acid position 578, in three cases of Leydig cell tumor. The same mutation was previously found, at genomic level, in some cases of testotoxicosis (Table 1). The paper of Liu et al. represents the first convincing indication that somatic activating mutations of gonadotropin receptors may be involved in gonadal tumorigenesis. Acknowledgments We are grateful to Professor E. Nieschlag, FRCP, for his continuous, stimulating support. The language editing of T.G.C. Cooper, Ph.D., is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschunggemeinschaft Confocal Research Group “The Male Gamete: Production, Maturation, Function”.
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5. Atger M, Misrahi M, Sar S, LeFlem A, Dessen P, Milgrom E. Structure of the human luteinizing hormone choriogonadotropin receptor gene: unusual promoter and 59 non-coding regions. Mol Cell Endocrinol 1995;111:113. 6. Segaloff DL, Ascoli M. The lutropin/choriogonadotropin receptor—4 years later. Endocr Rev 1993;14:324. 7. Gromoll J, Ried T, Holtgreve-Grez H, Nieschlag E, Gudermann T. Localization of the human FSH receptor to chromosome 2 p21 using a genomic probe comprising exon 10. J Mol Endocrinol 1994;12:265. 8. Gromoll J, Pekel E, Nieschlag E. The structure and organization of the human follicle-stimulating hormone receptor (FSHR) gene. Genomics 1996 15;35:308. 9. Simoni M, Gromoll J, Nieschlag E. The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr Rev 1997;18:739. 10. Vihko KK Gonadotropins and ovarian gonadotropin receptors during the perimenopausal transition period. Maturitas 1996;23 (Suppl):S19. 11. Tonacchera M, Vitti P, Agretti P, Giulianetti B, Mazzi B, Cavaliere R, et al. Activating thyrotropin receptor mutations in histologically heterogeneous hyperfunctioning nodules of multino dular goiter. Thyroid 1998;8:559. 12. Eberlein VVR, Bongiovanni AM, Jones IT, et al. Ovarian tumors and cysts associated with sexual precocity. J Pediatr 1960;57:484. 13. Ein SH, Darte JM, Stephens CA. Cystic and solid ovarian tumors in children: a 44-year review. J Pediatr Surg 1970;5:148. 14. Egli CA, Rosenthal SM, Grumbach MM, Montalvo JM, Gondos B. Pituitary gonadotropin-independent male-limited autosomal dominant sexual precocity in nine generations familial testotoxicosis. J. Pediatr 1985;106:33. 15. Rosenthal SM, Grumbach MM, Kaplan SL. Gonadotropin-independent familial sexual precocity with premature Leydig and germinal cell maturation (familial testotoxicosis): effects of a potent luteinizing hormone-releasing factor agonist and medroxyprogesterone acetate therapy in four cases. J. Clin Endocrinol Metab 1983;57:571. 16. Itoh k, Nakada T, Kubota T, Suzuki H, Ishigo M, Izumi T. Testotoxicosis proved by immunohistochemical analysis and successfully treated with cyproterone acetate. Urol Int 1996;57:199. 17. Holland FJ, Kirsch SE, Selby R. Gonadotropin-independent precocious puberty (”testotoxicosis”): influence of maturational status on response to ketoconazole. J Clin Endocrinol Metab 1987; 64:328. 18. Manasco PK, Girton ME, Diggs RL, Doppman JL, Feuillan PP, Barnes KM, et al. A novel testis-stimulating factor in familial male precocious puberty. N Engl J Med 1991;324:227. 19. Schedewie HK, Reiter EO, Beitins IZ, Seyed S, Wooten VD, Jimenez JF, et al. Testicular Leydig cell hyperplasia as a cause of familial sexual precocity. J Clin Endocrinol Metab 1981;52:271. 20. Reiter EO, Brown RS, Longcope Longcope C, Beitins IZ. Male-limited familial precocious puberty in three generations. Apparent Leydig-cell autonomy and elevated glycoprotein hormone alpha subunit. N Engl J Med 1984;311:515. 21. Wierman ME, Beardsworth DE, Mansfield MJ, Badger TM, Crawford JD, Crigler JF Jr, et al. Puberty without gonadotropins. A unique mechanism of sexual development. N Engl J Med 1985;312:65. 22. Holland FJ, Kirsch S, Wielgosz G, Weick R. Bioassayable “LH-like” factor in familial precocious puberty (FPP): linkage studies using 32PDNA probes to b-subunit LH/CG gene locus. Abstract. Pediatr Res 1988;23(Suppl):278A. 23. Shenker A, Laue L, Kosugi S, Merendino JJ Jr, Minegishi T, Cutler GB Jr. A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 1993;365:652. 24. Gromoll J, Partsch CJ, Simoni M, Nordhoff V, Sippell WG, Nieschlag E, et al. A mutation in the first transmembrane domain of the lutropin receptor causes male precocious puberty. J Clin Endocrinol Metab 1998;83:476. 25. Kraaij R, Post M, Kremer H, Milgrom E, Epping W, Brunner HG, et
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Nordhoff et al. / Archives of Medical Research 30 (1999) 501–509 idence of a role for mutations or polymorphisms of the follicle-stimulating hormone receptor in ovarian granulosa cell tumors. J Clin Endocrinol Metab 1998;83:274. 61. Ligtenberg MJ, Siers M, Themmen AP, Hanselaar TG, Willemsen W, Brunner HG. Analysis of mutations in genes of the follicle-stimulating hormone receptor signaling pathway in ovarian granulosa cell tumors. J Clin Endocrinol Metab 1999;84:2233. 62. Russo D, Arturi F, Schlumberger M, Caillou B, Monier R, Filetti S, et al. Activating mutations of the TSH receptor in differentiated thyroid carcinomas. Oncogene 1995;11:1907.
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REVIEW ARTICLE
Constitutively Active Mutations of G Protein-Coupled Receptors: The Case of the Human Luteinizing Hormone and Follicle-Stimulating Hormone Receptors Verena Nordhoff, Jörg Gromoll and Manuela Simoni Institute of Reproductive Medicine of the University, Münster, Germany Received for publication September 9, 1999; accepted September 10, 1999 (99/158).
Activating mutations of the luteinizing hormone receptor (LHR) and the follicle-stimulating hormone receptor (FSHR) have been known for several years. These activating mutations permanently stimulate, in the absence of their cognate ligand, the receptor signaling pathways. In the case of the LHR, the induced chronic stimulation causes sporadic and familial pseudoprecocious puberty, a phenotype observed only in males. The absence of a female phenotype is probably due to the requirement for FSH in the induction of LHR expression. For the FSHR, one activating mutation was found in a patient with normal spermatogenesis without detectable gonadotropins. Whether activating mutations of the gonadotropin receptors are involved in tumor development is not yet clear. Activating mutations of the FSHR were supposedly involved but not found in ovarian tumors. For the LHR, only one patient with a seminoma and an activating mutation was described. The different occurrence of activating mutations of the LHR compared to the FSHR is surprising, since the two genes are adjacently located on chromosome 2 and should therefore be affected by a similar mutation rate. It might well be that mutations occur with the same frequency, but that activating mutations of the FSHR do not result in any particular phenotype. © 2000 IMSS. Published by Elsevier Science Inc. Key Words: Luteinizing hormone receptor, Follicle-stimulating hormone receptor, Constitutively active receptor, Mutation.
Introduction Hormonal Control of Sexual Differentiation and Maturation In the human, sex differentiation is determined by two major switches. The genetic sex of the individual is determined by the presence of a testis-determining factor, the sex-determining factor on the Y-chromosome (SRY), directing male determination during embryonic development. The second major switch in sex differentiation is hormonal control of the gonads and secondary sex characteristics. Hypothalamic gonadotropin-releasing hormone (GnRH) and the two pituitary gonadotropins, luteinizing hormone (LH) and folliclestimulating hormone (FSH), are already detected at around week 10 of fetal development. The hypophyseal portal system matures until midgestation and GnRH, LH, and FSH
Address reprint requests to: Professor Manuela Simoni, Institute of Reproductive Medicine of the University, Domagkstrasse 11, 48149 Münster, Germany. Tel: (149) (251) 835-6444; Fax: (149) (251) 835-2736; Email: [email protected]
levels increase, stimulating gonadal maturation and hormone production. In the male fetal testis, differentiation and development of the Wolffian duct are grossly dependent on the presence of the maternal hCG, which stimulates testosterone production by the Leydig cells. Ovarian steroidogenesis is also active early in development, but is not necessary for female genital differentiation. At birth, gonadotropin and sex steroid levels are high, but decline during the first few days of life (1). During the following weeks, serum LH and FSH levels rise to a level higher than that observed during the rest of childhood in both sexes, FSH being higher in females than in males. After a peak, at 2–3 months of life, gonadotropin levels drop and persist as low for several years. The pituitary and the gonads remain quiescent until puberty, when GnRH is released in a pulsatile fashion, stimulating synthesis and secretion of gonadotropins. Presence of LH and FSH results in an increasing secretion of steroids, such as testosterone or estrogens, leading to the development of secondary sexual characteristics, acceleration of growth, and progressive gain of fertility. Adult levels of LH and FSH are
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regulated by steroid hormones through a negative feedback effect in the male. In the female, both negative and positive feedback mechanisms operate during the different stages of the cycle (2). The gonadotropin receptors. LH and FSH exert their action through specific receptors (3). These receptors, the LH receptor (LHR) and the FSH receptor (FSHR), belong to the G protein-coupled receptor family and are characterized by a very large extracellular domain to which the hormones bind specifically. They share, together with the thyroid-stimulating hormone receptor (TSHR), the common feature of seven-transmembrane helices inserted into the plasma membrane and an intracellular domain for signal transduction. The LH receptor. The LHR gene is located on chromosome 2p21 and consists of 11 exons spanning at least 60 kbp (4,5). Exons 1–10 encode the extracellular domain, involved in hormone binding, whereas exon 11 bears seven membrane-spanning domains—characteristic of G proteincoupled receptors—and one intracellular domain. In the male, the LHR is predominantly expressed in the Leydig cells. Expression begins during the late fetal period and continues at low levels until puberty. Thereafter, LHR expression is up-regulated (3). In the female, LHR expression can be detected from the second postnatal week onward. The LHR is expressed by the theca and granulosa cells of the ovary. During follicle maturation, which begins at puberty, expression of the LHR is induced by FSH. Upon stimulation of LH, theca cells produce androgens, which are converted under FSH stimulation into estrogens by the granulosa cells. Expression of LH receptors at midcycle in the dominating follicle prepares the follicle for the LH surge, which will induce ovulation. In addition, LH receptors are necessary for the production of progesterone by the corpus luteum. Later in the cycle, estradiol stimulates LHR density and activity. If FSH stimulation is lacking, the number of FSHR and LHR declines, and the granulosa cells die (6). The FSH receptor. The FSHR gene is, like the LHR gene, located on chromosome 2p21 and consists of 10 exons spanning 54 kbp (7,8). Exons 1–9 encode the extracellular domain, and exon 10 encodes the transmembrane and intracellular domain. In the male, FSHR expression is exclusively confined to the Sertoli cells. FSHR expression in the rat is first detectable around week 3 of fetal development and persists until adulthood. In the testis, the FSHR is expressed stage-dependently during spermatogenesis (9). In the female, FSHR expression is restricted to granulosa cells and begins first during week 1 postpartum. During puberty, the FSHR expression is induced and presumably upregulated during the follicular phase. After ovulation, the FSHR mRNA is down-regulated, indicating its importance
only for follicular growth and maturation. After luteinization, FSH binding is undetectable, as in postmenopausal women (10). Signal transduction through the LH receptor and FSH receptor. Gonadotropins act mainly through stimulation of intracellular cyclic adenosine-monophosphate (cAMP). In the inactive state, the receptor is bound to a Gs protein. Upon binding of the hormone to the receptor, the Gsa-subunit with a bound guanosine-triphosphate (GTP) dissociates and activates adenylyl cyclase, which leads to the synthesis of cAMP. Protein kinase A (PKA) is activated by cAMP, which causes the dissociation of the catalytic subunit from the regulatory subunit. The activated catalytic subunit is then able to activate other proteins by phosphorylation. Action of the LHR can also be mediated through production of inositol triphosphate (IP3), leading to an increase in Ca21 concentrations. Activation of protein kinase C (PKC) leads to phosphorylation of other proteins, such as structural proteins, enzymes and transcriptional activators or repressors (2).
Activating Mutations of the LHR and FSHR Mutations of receptor genes can result in gain-of-function, due to an activating mutation, or in loss-of-function, as a result of an inactivating mutation. Activating mutations produce a phenotype even when heterozygous, while inactivating mutations need to be homozygous to display a phenotype. In the case of familial diseases, underlying mutations are genomic and inherited. Somatic mutations are also described as occurring, for example, only in the tumor and not in the surrounding tissue (11). Activating mutations of the LHR. The clinical picture of testotoxicosis, also called familial male-limited pseudoprecocious puberty (FMPP), has been known for years. Puberty is considered precocious if secondary sexual characteristics appear before 8 years of age in girls and 9 years of age in boys. True precocious puberty is defined by premature sustained activation of the hypothalamic GnRH pulse generator. GnRH-independent precocious puberty is due to a primary sex-steroid secretion of the gonads, which can initiate development of secondary sexual characteristics. In girls, this can be due to granulosa-theca cell tumors (12,13). In boys, gonadotropin-independent pseudoprecocious puberty or testotoxicosis is a rare autosomal-dominant disorder in which Leydig cell hyperplasia and germ cell maturation proceed in the absence of stimulation by gonadotropins (14). The treatment is based on either medroxyprogesterone acetate (15), cyproterone acetate (16) or ketoconazole (17). The antifungal drug ketoconazole inhibits C17-20 lyase activity and leads to significant reduction of testosterone levels (17). After 1–3 months of treatment, an escape is often observed, triggering a true central precocious puberty. A
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combination of ketoconazole and GnRH analogs is then necessary to reduce pituitary and gonadal hormone concentrations to the upper range of normal for prepubertal children (17). In 1991, FMPP was associated by Manasco et al. (18) with a putative testis-stimulating factor. Earlier investigations with the plasma of these patients did not show evidence of stimulation of testosterone secretion from rat Leydig cells, indicating that this condition was not related to a circulating Leydig cell activator (19). Other studies confirmed the lack of measurable gonadotropins and gonadotropin-like factors in serum, and revealed a prepubertal serum gonadotropin response to stimulation with GnRH but no change in testosterone secretion in response to administration of a long-acting GnRH agonist (15,20,21). On the other hand, in another study the plasma of boys with FMPP was tested in a murine Leydig cell assay and an LH-like factor was postulated (22). These different results led Manasco et al. (18) to develop an in vivo bioassay for testosteronestimulating activity in adult cynomolgous monkeys. The authors reported data indicating that boys with FMPP had a factor in their plasma that stimulated monkey testes to secrete testosterone (18). These data, however, were not confirmed by other investigators. Shortly thereafter, Shenker et al. (23) found that an activating mutation of the LHR was responsible for the gonadotropin-independent activation of Leydig cells. After some years of molecular analysis of FMPP patients, the occurrence of activating mutations of the LHR is now being accepted as causative for this phenotype. Shenker et al. (23) screened the LHR gene of young boys from eight different FMPP families (see Figure 1). These authors found a heterozygous mutation in codon 578, located in the sixth transmembrane domain, which leads to a transposition of glycine for aspartate. Functional studies in COS-7 cells revealed that the mutated LHR in the unstimulated state increased the production of cAMP to 4.5-fold when compared with the wild-type receptor. This indicated a constitutive activation in the absence of the natural ligand. The mutated receptor was capable of responding to increasing concentrations of hCG with increasing amounts of cAMP, comparable to levels produced by the wild-type receptor (23). This was the first activating mutation described. In the meantime, several groups of researchers found other amino acid substitutions in the LHR also leading to constitutive activation (Figure 1). To date, activating mutations have been found in the first transmembrane domains (TM 1) (24) and in the second transmembrane domain (TM 2) (25–27). Most mutations, however, are found in the region covering transmembrane domains 5 and 6 (TM 5, TM 6) and the connecting intracytoplasmic loop 3 (ICL 3) (23,28–41) (Figure 1, Table 1). Within this region, the mutation of codon 578 seems to be the most frequent (28–32) (Figure 1, Table 1). Based on the distribution of mutations, a hot-spot region was postulated, crucial for LHR signal transduction. Frequent occur-
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rence of activating mutations in this region suggests that TM 5, TM 6 and ICL 3 play an important role in Gs protein coupling. This hypothesis was recently tested by Schulz et al. (42), who reported constitutive activation in vitro when deleting parts of ICL 3 in the LHR and the thyroid-stimulating hormone receptor (TSHR) (42). Deletion of nine amino acids (558–566) resulted in a 2.6-fold increase in basal cAMP levels, but a lower response when stimulated with hCG, compared to the wild-type. Deletion of four conserved amino acids (563–566) produced the same findings. After deleting single amino acids at these positions, the authors concluded that loss of aspartate at position 564 was responsible for the constitutive activity (42). Several other groups produced amino acid substitutions in vitro, leading to receptor activation. Kosugi et al. (43) mutated amino acid position 578, known to be involved in receptor activation, and replaced it with several others. A proline or asparagine at this crucial position showed no activation, while replacement with glycine, serine, leucine, tyrosine, or phenylalanine caused a 4.9–5.6-fold stimulation of basal cAMP (43). It seems that not only the location of the amino acid but the characteristics of the inserted amino acid are of importance. The explanation could be that a properly positioned hydrogen bond acceptor is necessary for maintaining the receptor in an inactive state. Yano et al. (44) also mutated in vitro a critical amino acid (phenylalanine 576) to study the influence on the secondary structure of ICL 3. A change to the amino acid isoleucine or glycine showed constitutive activation without changes in IP3 signaling. When phenylalanine at position 576 was substituted by a tyrosine, the resulting LHR showed constitutive activity with decreased hCGinduced cAMP and IP3 signaling. Affinity was higher, while capacity of hCG binding was lower (44). A change to the amino acid glutamate resulted in no cAMP response (44). Therefore, the third intracellular loop seems to be the most important and crucial region in the LHR for activating mutations, because it regulates coupling to the Gs protein. Location of the mutations is compatible with the current model of signal transduction of glycoprotein hormones. Several groups of researchers have described a mutation in codon 578 that leads most frequently to an amino acid change from aspartate to glycine. Others found a substitution of tyrosine (30,40) or glutamate (41). All these mutations lead to increasing amounts of cAMP and therefore to an activation of the LHR. One additional activating mutation was detected in codon 581, resulting in an amino acid change from cysteine to arginine (30). Not all cases of activating mutations of the LHR identified thus far involve FMPP. Yano et al. (28) described a spontaneous case in which the parents of the patient were normal, and Latronico et al. (33) found two unrelated Brazilian boys with two different heterozygous mutations. One boy had a sporadic mutation in codon 457 and his parents were normal. The mutation in the other boy, in codon 568,
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Figure 1. Naturally occurring activating mutations of the FSHR and the LHR. Only the transmembrane domain of both receptors is indicated.
was also found in his normal sister, suggesting the inherited form of FMPP in the boy (33). Additionally, Kremer et al. (45) reported sporadic cases of precocious puberty in which an activating mutation of the LHR is involved. The investigators screened eight familial cases and nine cases with a negative family history of LH-independent precocious puberty and found seven different mutations (all previously described) in 12 patients (45). All patients with true FMPP showed an activating mutation. Interestingly, in no case did the mutation of codon 578 involve the amino acid glycine, which seems to be the most frequent amino-acid change, at least in the U.S. In the sporadic cases, these mutations were found in only a few patients (45), but often the parents were not examined for inheritance. Variability in the age of onset could also not be correlated to specific mutations.
Martin et al. (39) identified an adult male with increased serum testosterone, prepubertal concentrations of FSH and LH, Leydig cell hyperplasia, and a testicular seminoma. Thirty-five years previously, the patient was diagnosed with precocious puberty, later determined as FMPP. This is the first case of a testicular germ cell tumor in an FMPP patient (39). The authors suggest the possibility of a potential harmful effect of high testosterone concentrations in early life upon the cellular components of the testes (39). However, this is the only FMPP patient to present with a seminoma. This suggests the importance of clinical observations of boys with FMPP during maturation and later in adulthood. Aside from this case, all boys affected by FMPP due to a genomic mutation in the LHR seem to have normal gonadal function in adulthood. In addition to the short stature of the
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Table 1. Naturally occurring mutations of the LH receptor Nucleotide change GCC to GCT ATG to ACG CTC to CGC ATT to CTT GAT to GGT GCT to GTT ATG to ATA GCA to GTA ATC to CTC ACC to ATC GAT to GGT GAT to TAT GAT to GAA TGC to CGC
Amino acid position
Amino acid change
Location
373 398 457 542 564 568 571 572 575 577 578 578 578 581
A–V M–T L–R I–L D–G A–V M–I A–V I–L T–I D–G D–Y D–E C–R
TM 1 TM 2 TM 3 TM 5 ICL 3 ICL 3 TM 6 TM 6 TM 6 TM 6 TM 6 TM 6 TM 6 TM 6
affected patients, due to premature closure of the epiphyses induced by testosterone, it appears that activated LHR induces a pathological phenotype only during early life until puberty. It might well be that constitutively active LHR is differently regulated from the wild-type receptor. For example, mechanisms such as receptor down-regulation could be involved, resulting in normal testosterone production in adulthood. Only Martin et al. (39) described the follow-up of an FMPP patient. It will be interesting to analyze FMPP patients in adulthood with different LHR mutations to obtain greater insight into regulation of activated LHR. Interestingly, females do not show a phenotype. This is mainly due to the differences in male and female sex differentiation. In females, pubertal maturation requires both LH and FSH. FSH stimulates follicle growth and maturation during puberty, the follicles enlarge, and granulosa cells convert androgens (produced by theca cells on LH stimulus) to estrogens. An activating mutation of the LHR could theoretically lead to early production of androgens. Because of low aromatase activity in the absence of FSH, androgens cannot be converted into estrogens. However, because FSH is needed for the expression of the LHR, an activating mutation of the LHR has no effect until the receptor expression is stimulated. This different regulatory system compared to males could explain the lack of phenotype in females. Theoretically, after puberty, when FSH secretion begins and an activated form of the LHR is expressed, production of androgens in excess could lead to polycystic ovary syndrome (PCOS), but there is no evidence to date for such a phenotype in women carrying activating mutations of LHR (32). In transgenic mice, overexpression of LH b-subunit leads to an overproduction of LH and causes ovarian hyperandrogenism (46). Rosenthal et al. (32) identified two brothers with FMPP in whom the amino acid aspartate in codon 578 was mutated to glycine. Interestingly, the mutation was inherited from the mother. Analysis of her steroid levels in comparison with normal female volunteers revealed no differences, but responses to dexamethasone, na-
IP3 Low
Additional notes
Higher hCG binding High hCG affinity Ligand-unresponsive Normal affinity
High Low hCG binding normal affinity Normal
Normal binding
Ligand-unresponsive
Reference (24) (25–27) (33) (30) (30) (33,34) (29,35) (36) (37) (29,38) (28–32,39) (30,40) (41) (30)
fareline, and leuprolide acetetate were at the lower end of normal range. No ovarian dysfunction could be observed (32). Expression of the LHR is differentially regulated during the cycle. Regulation of activated LHR could be tighter, to minimize the effect of the chronic stimulation and maintain normal cyclicity. The result of this tighter regulation could be normal ovarian function. In in vitro experiments, the mutated LHR has a lower expression level compared to the wild-type (42). If this is also true for the in vivo situation, the number of LHR in the membrane could be lower than for the wild-type. This could result in lower cAMP response and lower stimulation and could be the reason for the lack of phenotype. Moreover, cAMP production in vitro by mutated LHR on gonadotropin stimulation is lower or absent in comparison to normal LHR (33,45). If this also holds true in vivo, it might contribute to the absence of a phenotype in both males and females in adulthood. Activating mutation of the FSHR. To date, one activating mutation of the FSHR has been identified in a patient hypophysectomized due to a pituitary tumor (47). Although this operation led to a loss of all pituitary hormones, and especially to undetectable levels of LH and FSH, the patient was fertile under testosterone substitution therapy alone. Normally, testosterone treatment sustains androgenization, but is incapable of maintaining spermatogenesis in hypophysectomized patients (48). To gain normal fertility, substitution of both LH and FSH is necessary (48,49). Surprisingly, the patient had normal spermatogenesis and fathered three children. After molecular analysis of the FSHR gene, a heterozygous mutation was found, which led to the substitution of glycine for aspartate at position 567. Functional studies on the mutated receptor revealed a slight-but-consistent 1.5fold increase in basal cAMP production, indicating constitutive activation (47). Re-analysis of these results in an immortalized mouse Sertoli cell line, a target cell closer to the in vivo situation, revealed a 3-fold increase in cAMP levels (9). This Sertoli cell
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line had lost its own endogenous FSHR (50), but still contained the machinery for signal transduction. It could be that this cell line represents a more adequate model for studying the constitutive activation of the mutated FSHR. Three-fold increase of cAMP levels obtained after transient transfection of the mutated receptor, in comparison to the wild-type, gives a hint of the greater sensitivity of this Sertoli cell line (9). The activating mutation is localized in the third intracytoplasmatic loop between the fifth and the sixth transmembrane domains, a region highly conserved among glycoprotein hormone receptors. Position 567 in the FSHR is homologous to codon 619 in the TSHR and codon 564 in the LHR, a location in which mutations leading to constitutive activation of these receptors have been described (30,51). In 1996, Kudo et al. (52) produced chimeric LH/FSH receptors with this crucial amino acid substitution. An LHR with FSHR TM 5 and TM 6, with the activating mutation at position 567 in ICL 3, produced no differences in cAMP response, in comparison to the nonmutated chimera (52). A FSHR with LHR TM 5 and 6 with the activating mutation at position 564 in the ICL 3 produced higher basal levels of cAMP when compared with the non-mutated chimera (52). The authors suggested the existence of stabilizing interactions between TM 5 and TM 6 in the FSHR, exceeding those in the LHR. In the LHR, this leads to a state in which mutations in the ICL 3 could be responsible for constitutive activation, while in the FSHR the activation would be blocked by the constrained state (52). It would be of interest to repeat these experiments in a Sertoli cell line that, in our opinion, might be better-suited for experiments concerning the FSHR. In other cell lines, the receptor might be coupled to unusual signal transduction components or be differently processed and may not, therefore, properly reflect constitutive activity. Further studies with constitutively active LH and FSH receptors in gonadal and nongonadal cell lines should afford further insights into putative cell-specific elements involved in gonadotropin receptor signal transduction. Recently, Schulz et al. (42) produced deletions in the ICL connecting TM 5 and TM 6 of the FSHR, and tested them in vitro in COS-7 cells. A receptor lacking amino acids 567–569 resulted in drastically reduced membrane expression of binding-competence and responded only mildly to stimulation with FSH, indicating the importance of the ICL for signal transduction. The deletion of amino acid 567 resulted in a receptor that did not constitutively couple to the Gs/adenylyl cyclase system. Substitution of glycine for aspartate in codon 567 showed slightly elevated basal cAMP levels (42). Taking into account the lower membrane expression of 60% compared to the wild-type, functional characteristics of the mutation suggest some degree of constitutive activity (42). Moreover, this experiment indicates the importance of the ICL 3 for activity, not only in the LHR but in the FSHR. The Asp567Gly mutation is the only activating mutation of the FSHR published to date. A female phenotype has not
yet been detected. It could be that constitutive activity of the FSHR leads to an increased number of follicles to maturation. A consequence could be a higher incidence of dizygotic twins (53–55). However, no mutations of genes involved in reproductive function, such as FSHb, CGb, inhibin bB, and GnRH could be detected in mothers of dizygotic twins (56), but to our knowledge a mutational analysis of the FSHR in such a population has not yet been published. It could be that naturally occurring activating mutations remain asymptomatic under normal physiological conditions. Sertoli cells in vitro progressively lose the capacity to respond to gonadotropin stimulation. In vivo, the FSHR is down-regulated by stimulation with FSH in the rat (57). It appears that the cells protect themselves from overstimulation by losing receptor expression. There are no data in vivo on down-regulation of constitutively active gonadotropin receptors. Transgenic mice overexpressing a constitutively active stimulatory Gas-protein in pancreatic beta-cells did not show a peculiar phenotype. However, treatment with a phosphodiesterase inhibitor induced a 2-fold increase of cAMP (58). A similar case is known for the b2-adrenergic receptor. A transgenic mouse overexpressing a constitutively activated b2-adrenergic receptor in the heart had only a mild phenotype (59). Pharmacological treatment with b-adrenergic receptor ligands resulted in a 50-fold increase of receptor expression, because of a stabilizing effect of the ligand. Receptor up-regulation led to an increase in adenylyl cyclase activity and a clear phenotype (59). By analogy, transgenic mouse models with a constitutive activated FSHR will be helpful for explaining the phenotype of such mutations. Neither the authors nor our colleagues (60,61) have found somatic FSHR mutations in granulosa cell tumors. It is well known that somatic constitutively activating mutations of the TSHR are found in thyroid adenomas (62,63), while in the LHR, only one case of a genomic mutation in a man with a testicular seminoma has been described (39). It is hypothesized that, in most cases, a constitutively activated G protein-coupled receptor is neither sufficient for, nor has a direct pathogenic role in, the development of tumors. This was recently confirmed by the lack of mutations in the GnRH receptor gene in gonadotrope adenomas (64). The case of a testicular seminoma associated with FMPP and an activating mutation of the LHR also remains an isolated observation (39). Whether seminoma is related to mutation or other factors is unknown.
Conclusions Activating mutations of the LHR cause familial or sporadic pseudoprecocious puberty that affects only boys. This is due mainly to the fact that in the male, contrary to the female, FSH is not required for expression of the LHR. Except for short body stature, affected subjects are normal in adulthood and constitutive activity of the LHR becomes clinically silent. Activating mutations of the FSHR seem to have no di-
Nordhoff et al. / Archives of Medical Research 30 (1999) 501–509
rect phenotype, although FSH activity is crucial for follicle maturation and spermatogenesis. Why is there such a great difference in the incidence of activating mutations between the LHR and the FSHR? It is known that genes for the FSHR and the LHR are localized very closely together on chromosome 2. According to current theory, the two gonadotropin receptors might have evolved by chromosome duplication. Thus, one would reasonably expect the same rate of occurrence of mutations in both receptors. Due to the lack of a female phenotype, and a normal phenotype in adult males with an activating mutation of the LHR, we must conclude that, as a rule, activating mutations of the LHR do not have clinical consequences except in prepubertal boys. Tight regulation of receptor expression and turnover could explain the lack of phenotype in otherwise normal men and women with activating mutation of the FSHR. If this is the case, it will be very difficult to detect new activating mutations of the FSHR, even if they occur naturally at a frequency similar to that of LHR mutations. Note Added in Proofs While this paper was edited, Liu et al. (65) published evidence that somatic activating mutations of the LHR are found in Leydig cell adenomas. The authors reported about a G to C transition at nucleotide position 1732 of the LHR, exchanging an aspartic acid residue with histidine at amino acid position 578, in three cases of Leydig cell tumor. The same mutation was previously found, at genomic level, in some cases of testotoxicosis (Table 1). The paper of Liu et al. represents the first convincing indication that somatic activating mutations of gonadotropin receptors may be involved in gonadal tumorigenesis. Acknowledgments We are grateful to Professor E. Nieschlag, FRCP, for his continuous, stimulating support. The language editing of T.G.C. Cooper, Ph.D., is gratefully acknowledged. This work was supported by a grant from the Deutsche Forschunggemeinschaft Confocal Research Group “The Male Gamete: Production, Maturation, Function”.
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