Luteinizing Hormone and Human Chorionic Gonadotropin

Molecular and Cellular Endocrinology 383 (2014) 203–213

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Molecular and Cellular Endocrinology

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Review Luteinizing hormone and human chorionic gonadotropin: Origins of difference q ⇑ Janet Choi a, , Johan Smitz b,1 a The Center for Women’s Reproductive Care at Columbia University, New York, NY, United States b UZ Brussel, Vrije Universiteit Brussel, Brussels, Belgium article info abstract

Article history: Luteinizing hormone (LH) and human chorionic gonadotropin (hCG) are widely recognized for their roles Received 25 September 2013 in ovulation and the support of early pregnancy. Aside from the timing of expression, however, the dif- Received in revised form 6 December 2013 ferences between LH and hCG have largely been overlooked in the clinical realm because of their similar Accepted 12 December 2013 molecular structures and shared receptor. With technologic advancements, including the development of Available online 21 December 2013 highly puriﬁed and recombinant gonadotropins, researchers now appreciate that these hormones are not as interchangeable as once believed. Although they bind to a common receptor, emerging evidence sug- Keywords: gests that LH and hCG have disparate effects on downstream signaling cascades. Increased understanding Gonadotropin of the inherent differences between LH and hCG will foster more effective diagnostic and prognostic Lutropin Luteinizing hormone assays for use in a variety of clinical contexts and support the individualization of treatment strategies Human chorionic gonadotropin for conditions such as infertility. Luteinizing hormone/choriogonadotropin Ó 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved. receptor Luteinizing hormone receptor

Contents

1. Introduction ...... 204 2. Materials and methods ...... 204 3. Molecular structure ...... 204 3.1. Gonadotropin variants ...... 204 4. Circulating forms ...... 205 4.1. Luteinizing hormone ...... 205 4.2. Human chorionic gonadotropin ...... 205 5. Metabolism...... 206 6. Receptor binding ...... 207 6.1. Luteinizing hormone/choriogonadotropin receptor...... 207 6.2. Signaling pathways ...... 210 6.3. Extragonadal LHCGR ...... 211 7. Conclusions...... 211 Disclosure statement ...... 211 Acknowledgements ...... 211 References ...... 211

q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⇑ Corresponding author. Address: The Center for Women’s Reproductive Care at Columbia University, 1790 Broadway, 2nd Floor, New York, NY 10019, United States. Tel.: +1 646 756 8282; fax: +1 646 756 8280. E-mail addresses: [email protected] (J. Choi), [email protected] (J. Smitz). 1 Address: UZ Brussel, Vrije Universiteit Brussel, Laarbeeklaan 101, 1090 Brussels, Belgium. Tel.: +32 2 477 50 52; fax: +32 2 477 50 60.

0303-7207/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.mce.2013.12.009 204 J. Choi, J. Smitz / Molecular and Cellular Endocrinology 383 (2014) 203–213

1. Introduction Table 1 Characteristics of hLHa and hCGb.

It is well established that luteinizing hormone (LH) and human hLH hCG chorionic gonadotropin (hCG) play key roles in the reproductive Molecular weight (Da) 30,000 36,000c cycle. Textbooks recognize the disparate endogenous functions of a Subunit these hormones, underscoring the role of LH in follicular matura- Gene location 6q12–q21 6q12–q21 tion and ovulation induction, while acknowledging the essential Size of mature subunit (no. of amino acids) 92 92 role of hCG in early pregnancy survival. During early pregnancy, b Subunit hCG is vital to support secretion of progesterone by the corpus lut- Gene location 19q13.32 19q13.32 eum, without which a pregnancy cannot persist (Fritz and Speroff, Size of mature subunit (no. of amino acids) 121 145 2011; Mesiano, 2009). Nonetheless, LH and hCG are frequently de- hLH, human luteinizing hormone; hCG, human chorionic gonadotropin. picted as interchangeable, with one contemporary text stating that a Bulun (2011). the ‘b subunits [of human LH (hLH) and hCG] confer identical bio- b Cole (2010). c logic activities when associated with the a subunit’ (Bulun, 2011). Molecular weight of hyperglycosylated form is 40,000–41,000 Da. Although similar in structure and function, LH and hCG are distinct molecular entities with divergent patterns of expression and physiologic functions. LH and hCG share a common receptor, yet The high degree of sequence conservation among CGB genes, each hormone triggers a unique cascade of events following recep- combined with the fact that CG is detected only in equine and pri- tor binding (Casarini et al., 2012; Gupta et al., 2012). Moreover, mate species (whereas LH is found in all vertebrates) suggests that within the same hormone family, individual isoforms exhibit un- CG is a relatively recent evolutionary derivative of LH. The amino ique characteristics regarding half-life and biologic functions. acid sequences of human LH (hLH) and hCG are highly conserved, Emerging data suggest that these differences between LH and sharing 82% homology (Bulun, 2011). hCG retains the full hCG have functional significance. Identifying the unique roles of 145Àamino-acid complement in its b-subunit. In contrast, the LH and hCG is key to understanding both normal physiologic pro- b-subunit of LH undergoes cleavage of its 24-amino acid leader se- cesses (e.g., reproduction, placentation) and dysfunctional states quence to generate its final 121-amino acid sequence. (e.g., infertility, gestational and non-gestational neoplasms). Ad- As a result of structural differences and post-translational mod- vances in gonadotropin purification and recombinant technology ifications, hCG is more stable and has a longer circulating half-life have helped to differentiate the varied actions of LH and hCG. In than LH. The half-lives of these molecules typically are expressed turn, this has improved the diagnosis and treatment of human dis- as a range (on the order of minutes for LH and hours for hCG), ease, most significantly in the context of infertility. reflecting the heterogeneity of circulating isoforms. The shorter half-life of LH is physiologically important, as it allows for the production of LH pulses. The longer half-life of hCG and its greater 2. Materials and methods receptor binding affinity make it more biologically active than hLH (Rahman and Rao, 2009; Rao, 1979). A comprehensive literature review of LH and hCG was con- ducted, focusing on differentiation in terms of hormone structure, expression, modification, receptor activation, and clinical use. Sci- 3.1. Gonadotropin variants entific literature was identified via interrogation of the MEDLINE database using relevant search terms, including but not limited In the endogenous state, LH and hCG consists of isohormone to: ‘gonadotropin’, ‘human chorionic gonadotropin’, ‘luteinizing mixtures that result from (1) post-translational modification of hormone’, ‘luteinizing hormone/choriogonadotropin receptor’, the native proteins; (2) metabolism to form truncated or nicked ‘lutropin’, and ‘lutropin receptor’. Results were limited to articles intermediates; and (3) natural sequence variants (Bergendah and published in English. Additional resources were extracted from Veldhuis, 2001; Cole, 2009; Stanton et al., 1993). Post-translational clinical texts and review articles. Article inclusion was predicated modification chiefly consists of the addition of various carbohy- on relevance to the topic without use of an objective set of criteria, drate side chains, including sialic and sulfonic acid moieties, thus given that the review is descriptive rather than systematic in creating a variety of isoforms (upwards of 30 for LH and 15 for nature. hCG) that differ with respect to half-life, bioactivity, and signaling properties (Arey and Lopez, 2011; Bergendah and Veldhuis, 2001; Cole, 2009; Stanton et al., 1993). Obviously, the number of isoform 3. Molecular structure classes for each hormone is determined by our capacity for analyt- ical discrimination. Because glycosylation affects the size and con- Both LH and chorionic gonadotropin (CG) are heterodimeric gly- formation of the molecule as well as access to binding sites, coproteins comprised of a and b subunits. Along with thyroid- differences in interactions with their cognate receptor are to be ex- stimulating hormone (TSH) and follicle-stimulating hormone pected (Arey and Lopez, 2011). (FSH), they share a common 92-amino acid a subunit, whose gene Naturally occurring variants of both LH and hCG have been resides on chromosome 6q12–q21 in humans (Bulun, 2011; Naylor characterized. A variant of LH that has an additional glycosylation et al., 1983). The unique functions and receptor-binding capacity of site compared with wild-type LH is relatively common within cer- each of these hormones stem from differences among the b sub- tain populations (Lamminen and Huhtaniemi, 2001; Nilsson et al., units (Table 1). Transcription of the b-subunit is the rate-limiting 1997). Data have been collected showing that variant LH differs step in LH and CG production (Nagirnaja et al., 2010). The genes from normal LH in its biological effects (Haavisto et al., 1995). This for the LH and hCG b subunits are located within a cluster of seven LH variant has been associated with unexplained infertility and similar sequences on human chromosome 19q13.32 (Boorstein subfertility due to ovulatory dysfunction in females, and with slo- et al., 1982; Jameson and Hollenberg, 1993). One of those se- wed pubertal progression in males (Raivio et al., 1996; Takahashi quences encodes the b-subunit of LH (LHB); four are hCG b-subunit et al., 1998, 1999). Preliminary data also suggest that the variant coding genes (CGB, CGB5, CGB7, and CGB8), and the remaining two LH is more prevalent among women who demonstrate resistance are pseudogenes (CGB1, CGB2). to ovarian stimulation with recombinant human FSH, compared J. Choi, J. Smitz / Molecular and Cellular Endocrinology 383 (2014) 203–213 205 with those who exhibit moderate or strong responses (Alviggi pin-releasing hormone (GnRH), estradiol, and testosterone et al., 2011). Men with one or two copies of the variant allele have (Bergendah and Veldhuis, 2001; Lambert et al., 1998; Wide and lower circulating LH levels compared with homozygotes for the Bakos, 1993). wild-type allele (Huhtaniemi et al., 2010). However, the ratio of Variations in LH isoform composition are also observed during biological-to-immunological activity (a measure of relative LH the reproductive life cycle. In general, LH isoforms with shorter biopotency) for variant LH is greater than that of wild-type LH half-lives but increased biopotency are present in younger post- (Haavisto et al., 1995). pubertal women, whereas longer-lived LH species are the predom- A less common LH variant resulting in a single amino acid sub- inant form detected in post-menopausal women (Reader et al., stitution in the b subunit (serine for glycine at amino acid 102) has 1983; Wide, 1985). LH in the latter group also has an increased ra- been associated with menstrual disorders and male infertility tio of biological-to-immunological activity (Marrama et al., 1983). (Hashad et al., 2012; Ramanujam et al., 1999, 2000). Little is known Interestingly, women with polycystic ovarian syndrome (PCOS)— about how this rare variant affects the functionality of LH. Based on an endocrine condition associated with reduced fertility—appear in vivo experiments, Wide et. al. showed that an extra glycosylation to predominantly secrete LH isoforms that have a high ratio of bio- site on variant LH compared to the wild type, allows for greater logical-to-immunological activity (Ding and Huhtaniemi, 1991; sialylation and results in a longer half-life in serum. Granulosa Ropelato et al., 1999). Molecular analysis of LH in women with cells are triggered typically by distinct pulses of LH; however, PCOS has revealed a substantial decrease in sulfonation at midcy- because of its prolonged residence in serum, granulosa cells are cle, accompanied by a slight increase in sialylation compared with exposed to decreased pulse amplitudes of variant LH. Thus, Wide, non-affected individuals (Wide et al., 2007). The net result is an et. al. proposed that the longer half-life of variant LH may be a increase in basic LH isoforms relative to women without PCOS contributing factor to the reproductive disorders observed (Wide (Ropelato et al., 1999; Wide et al., 2007). It is unclear what influ- et al., 2010). ence, if any, these changes have on the half-life of LH. It is known Although hCG polymorphisms with functional significance are that that basic LH isoforms are, in general, cleared more rapidly generally rare, variants in the b subunit of hCG with putative than acidic isoforms, yet based on findings regarding the specific protective or detrimental effects on recurrent miscarriage risk have effects of sialylation vs. sulfonation on LH half-life (Burgon et al., been identified (Nagirnaja et al., 2012; Rull et al., 2008). The se- 1996; Wide et al., 2009), the changes observed in women with quence changes associated with increased risk of recurrent miscar- PCOS would be expected to increase LH residence time in circula- riage occur in the most commonly transcribed hCG b-subunit tion. This question is made more complex by the apparent effect of genes, CGB5 and CGB8, and result in conformational changes to body mass index (BMI) on the LH profile of women with PCOS the protein’s structure (Nagirnaja et al., 2012). One of the b-subunit (Srouji et al., 2007). Current dogma posits that increased basal missense mutations in particular-hCG b p.Val56Leu adversely im- secretion of LH plays a greater role than altered post-translational pacts its ability to bind efficiently with its a-subunit with only modification in the elevated serum LH concentrations that have 10% forming the complete hCG heterodimer. However, additional been observed among women with PCOS. studies show increased bioactivity of this resultant variant hCG In men, LH stimulates testosterone production by Leydig cells of (possibly due to subtle conformational changes) when bound to the testis. The degree of LH sialylation and the ratio of biological- LHCGR, increasing the impact of this more sparsely produced var- to-immunological activity of LH are generally greater in men than iant hCG. The paucity of polymorphisms that have been identified in pre-menopausal women (Bergendah and Veldhuis, 2001; Wide in hCG imply that only modest changes in CGB5 and CGB8 are et al., 2007). Unlike women, however, the biological-to-immuno- compatible with a successful pregnancy outcome. logical activity ratio of LH decreases with age in men (Marrama et al., 1984; Warner et al., 1985). A shift toward more LH species that have a shorter serum half-life has been described in obese 4. Circulating forms men, which may be a source of the reduced testosterone levels reported in those individuals (Castro-Fernandez et al., 2000). 4.1. Luteinizing hormone 4.2. Human chorionic gonadotropin Gonadotrophic cells of the anterior pituitary produce LH, which regulates ovulation. Over the course of a normal menstrual cycle, a At least four physiologically important isoforms of hCG have surge in LH spurs ovulation from the dominant follicle. This pro- been described, including regular hCG, hyperglycosylated hCG (h- cess initiates during the mid-follicular phase, when LH stimulates hCG), hyperglycosylated free hCG b subunit, and pituitary hCG. Dif- androgen production in thecal cells. These androgens are aroma- ferent cell types produce these isoforms, which have unique, tized in the granulosa cells to estrogen. Prolonged, sustained estro- although sometimes overlapping, functions. Each of the four vari- gen levels generate positive feedback on the pituitary gland and ants shares a common protein sequence (at least for the b subunit), the subsequent LH surge that results in ovulation (Liu and Yen, but each is modified differently, resulting in isoforms with dispa- 1983) Post-ovulation, LH promotes progesterone production, sup- rate half-lives and biologic functions. The distribution and quantity porting development of the corpus luteum (Van de Wiele et al., of hCG isoforms varies over the course of pregnancy and in the con- 1970). text of aberrant development or disease. For example, in early The composition of LH isoforms, their longevity, and their bio- pregnancy, an abnormally low proportion of h-hCG to total hCG activity vary throughout the menstrual cycle. The ratio of biologi- (<50%) indicates a failing pregnancy, (Kovalevskaya et al., 2002; cal-to-immunological activity is lowest during the luteal phase, Sasaki et al., 2008), while decreased first trimester serum free b- rising slowly during the follicular phase to a peak at mid-cycle hCG levels have been associated with ectopic pregnancies (Borrelli (Anobile et al., 1998). This corresponds with the pinnacle of LH et al., 2003). Conversely, elevated h-hCG can indicate choriocarci- sialylation and a marked trough in sulfonation (Wide and Eriksson, noma or Down syndrome pregnancy (Cole et al., 1999; Elliott 2013). Earlier studies had established that sialylation prolongs LH et al., 1997; Palomaki et al., 2007). half-life, whereas sulfonation hastens protein elimination (Burgon Regular hCG is secreted by fused and differentiated syncytio- et al., 1996; Wide et al., 2009). Changes in the extent of glycosyla- trophoblasts. It is the predominant hCG species measured in serum tion during the menstrual cycle appear to be regulated by the during pregnancy, after the initial early surge of h-hCG (Cole, pituitary gland, under the influence of such factors as gonadotro- 2010). The chief role of hCG historically has been the promotion 206 J. Choi, J. Smitz / Molecular and Cellular Endocrinology 383 (2014) 203–213

Table 2 Production and biological functions of hCG isoforms.

Isoform (source) Function(s)

hCG (syncytiotrophoblast cells) Promotes corpus luteum progesterone secretion Stimulates angiogenesis within pregnant uterus Differentiates cytotrophoblasts and may stimulate cytotrophoblast metalloproteinases, facilitating pregnancy implantation Inhibits immune response and macrophage phagocytosis at utero-placental junction Relaxes uterine muscle contraction and permits uterine growth during pregnancy May promote differentiation and development of umbilical cord and fetal organs Blastocyst cross-talk with endometrium prior to implantation LHCG receptors found in sperm and fallopian tubes suggesting pre-pregnancy communication roles hCG receptors in hippocampus, hypothalamus and brain stem suggests its role in hyperemesis gravidarum Hyperglycosylated hCG (cytotrophoblast cells – normal and Promotes implantation via cytotrophoblast invasion; also inhibits apoptosis and stimulates invasion malignant cells) by choriocarcinoma cells Free b-hCG (non-trophoblastic malignancies) Inhibits non-trophoblastic malignancies apoptosis; promotes cancer cell growth Pituitary hCG (gonadotroph cells of the anterior pituitary) Mimics LH functions during the normal menstrual cycle hCG, human chorionic gonadotropin; LH, luteinizing hormone. Adapted from Cole (2010).

of progesterone secretion by the corpus luteum in early pregnancy. Hyperglycosylated free hCG b subunits are found at low levels However, as noted by Cole, hCG levels continue to increase after during normal pregnancy and are also produced by non-tropho- hCG is no longer needed for progesterone production, suggesting blastic malignancies (Butler et al., 2000; Cole, 2009; Cole et al., other functions for this hormone during pregnancy (Cole, 2009). 1988; Iles et al., 1990). Akin to h-hCG, the hyperglycosylated free It is now believed that hCG is involved in placentation through b subunit is characterized by N- and O-linked oligosaccharides activities such as maintaining angiogenesis of the uterine vascula- containing a large number of sugar residues. Free b subunits are ture and promoting differentiation of cytotrophoblasts into syncy- not only a biomarker of malignancy but are also believed to play tiotrophoblasts (Rao and Alsip, 2001; Shi et al., 1993; Zygmunt an active role in cancer development by preventing apoptosis et al., 2002). Emerging evidence also suggests roles for regular and promoting carcinogenesis (Butler et al., 2000; Cole and Butler, hCG in fostering implantation, preventing fetal rejection, coordi- 2008). Indeed, elevated levels of free b-hCG in urine samples of pa- nating uterine and fetal growth, and, potentially, growth and tients with malignancies increase the likelihood for metastasis and development of fetal organs (Cole, 2010). The varieties of estab- mortality (Butler et al., 2000; Iles et al., 1996). The anti-apoptotic lished and putative functions for regular hCG are listed in Table 2. effects of free b-hCG may result from antagonistic inhibition of Secreted by root or extravillous cytotrophoblasts, h-hCG con- other growth factor receptors, rather than interaction with the centrations peak early in the first trimester. The percentage of canonical receptor (Butler and Iles, 2004). Because it lacks the a hCG in the form of h-hCG subsequently declines, becoming a minor subunit, free b-hCG cannot bind to the lutropin receptor (Cole, component of total hCG measurement during the last two trimes- 2009). ters. The key differentiator between regular hCG and h-hCG is the Pituitary hCG is secreted by gonadotrophic cells of the anterior complexity of the oligosaccharide side chains: h-hCG tends toward pituitary. The higher number of sulfonated side chains of pituitary oligosaccharides with a greater number of sugar residues. Conse- hCG gives it a shorter half-life than its placental counterpart (Bir- quently, the molecular weight of h-hCG is in the range of ken et al., 1996). Pituitary hCG is present in all adults, including 40,000–41,000 Da, whereas that of regular hCG is approximately men and pre-/post-menopausal women, and is suppressed with 36,000 Da. The additional sugar moieties prevent normal protein GnRH agonist treatment (Odell and Griffin, 1987, 1989). During a folding, thus exposing alternative receptor binding sites and alter- regular menstrual cycle, pituitary hCG secretion parallels that of ing the hormone’s functionality (Cole, 2010). Indeed, results of re- LH, although the half-life of pituitary hCG is shorter than that of cent experiments by Berndt and colleagues indicate that h-hCG- LH. As much as one-third of follicular-stage LH activity may derive mediated angiogenesis may occur independent of activation of its from hCG during a natural menstrual cycle (Cole, 2010). The func- cognate receptor (Berndt et al., 2013). tion of pituitary hCG is generally not known, but its concomitant The association between pregnancy loss and low h-hCG levels temporal appearance with LH during the menstrual cycle and com- supports a key role for this variant in implantation (Kovalevskaya mon receptor suggest that its functionality may mimic LH—stimu- et al., 2002; Sasaki et al., 2008). The importance of h-hCG in lating follicular maturation, inducing ovulation, and supporting implantation/invasion is also evident in the context of choriocarci- progesterone production during the luteal phase (Cole, 2010). In noma, as a pathologic process (Cole, 2010). Current data suggest contrast, expression of the LHB gene is down-regulated in preg- that h-hCG and hCG act in conjunction to forewarn the endome- nancy (Rull and Laan, 2005) suggesting that while hCG might have trium of impending implantation, to foster growth and differentia- a functional role during the normal menstrual cycle, LH cannot tion of trophoblast cells, and to establish placental villous substitute for hCG in supporting pregnancy. structures. During pregnancy, the amount of total and h-hCG in serum and urine demonstrates a high degree of inter-individual variability 5. Metabolism (Cole, 2010). This endogenous variation may be normalized at the receptor level via downregulation in the presence of high Gonadotropins undergo a series of metabolic conversions be- hCG concentrations, or through the ‘spare receptor’ concept, which fore ultimately being excreted in urine (Braunstein, 2011; Cole, generates the same response regardless of the proportion of recep- 2009; O’Connor et al., 1998). This process has been well character- tors activated. The end result is that, unless the hCG level is insuf- ized for hCG. One of the initial steps in hCG degradation is proteo- ficient to support its basic functionality, variation is compatible lytic cleavage of the b subunit (possibly by human leukocyte with normal pregnancy. elastase), which generates a ‘nicked’ form of the protein. Nicked J. Choi, J. Smitz / Molecular and Cellular Endocrinology 383 (2014) 203–213 207 hCG rapidly dissociates into its component a and b subunits. that occur within certain clinical contexts may generate erroneous (Alternately, hCG that has not been nicked may slowly dissociate results. It is important, therefore, to be aware of the limitations for prior to enzymatic cleavage.) Therefore, h-hCG dissociates more a particular assay when interpreting laboratory findings. readily than regular hCG in this context. The nicked b subunit is further degraded, predominantly in the kidneys, resulting in a b- core fragment. A comparable LH b-core fragment has been isolated 6. Receptor binding from human pituitary and urine samples (O’Connor et al., 1998). Given the array of glycosylated hCG forms and the potential for 6.1. Luteinizing hormone/choriogonadotropin receptor nicked and dissociated varieties therein, a multitude of hCG degradation products are detected in the urine of pregnant women, of LH and hCG bind to and activate a common receptor, the LH/ which the b-core fragment is most prevalent from 7 weeks’ gesta- choriogonadotropin receptor (LHCGR), also known as the LH or tion onward (Cole, 2009). In total, more than 30 hLH isoforms and lutropin receptor (LHR) (Ascoli et al., 2002; Puett et al., 2007). This 15 variants of hCG have been identified from serum and urine sam- 675-amino-acid, G-protein-coupled receptor is a member of the ples (Cole, 2009; Stanton et al., 1993). rhodopsin subfamily of glycoprotein hormone receptors, as are The extent of gonadotropin glycosylation dictates molecular the FSH and TSH receptors. Like all G-protein coupled receptors, charge, which, in turn, governs clearance rate, such that more acidic LHCGR is anchored within the cell membrane by seven transmem- isoforms have a longer half-life in vivo (Lambert et al., 1998). Inves- brane domains. The unusually large extracellular domain of LHCGR tigators have also noted that the sialic acid content of LH positively is characterized by leucine-rich repeats and multiple sites for gly- correlates with metabolic clearance rate (Burgon et al., 1996). In cosylation (Fig. 1). The human LHCGR gene is located on chromo- other words, a greater degree of sialylation increases LH retention some 2p21, in the same general region as the FSH receptor in circulation. This observation holds true in the context of GnRH (Rousseau-Merck et al., 1993, 1990). receptor blockade as well. In response to GnRH receptor blockade, Consistent with its role in female reproduction, LHCGR is ex- LH levels decrease and the overall glycosylation profile shifts: the pressed by multiple cell types in the ovary, including thecal, luteal, mean number of sialic acid residues per molecule increases and interstitial, and differentiated granulosa cells (Ascoli et al., 2002). the number of sulfonated residues decreases (Wide et al., 2009). Expression of LHCGR in these cell types during the ovarian cycle The more sialylated and less sulfonated LH isoforms have longer is dynamic rather than constitutive, reflecting changes in the hor- circulating half-lives. A similar effect occurs naturally via an LH monal milieu (Menon and Menon, 2012; Menon et al., 2010). polymorphism that results in an extra site for glycosylation. This LHCGR is downregulated transiently after the pre-ovulatory LH LH variant has an increased number of sialic acid residues per mol- surge, reaches a maximum at mid-luteal phase, and decreases with ecule, resulting in a circulating half-life approximately 40% longer corpus luteum regression. This pattern is the inverse of what has than wild-type LH in humans (Wide et al., 2010). been observed for bioactive LH isoforms, wherein peak biologic- hCG glycosylation also affects its longevity in circulation. Meta- to-immunologic activity occurs at midcycle and reaches its lowest bolic clearance rates of deglycosylated, b-core fragment, and desi- point during the luteal phase (Anobile et al., 1998). The mecha- alylated hCG are all faster relative to hCG, with the greatest degree nisms that govern temporal changes in LHCGR expression are not of acceleration observed for desialylated hCG (Liu et al., 1989; Rosa yet fully elucidated but are generally believed to involve transcrip- et al., 1984). The effects of naturally occurring hCG polymorphisms tional as well as post-transcriptional regulation (Ascoli et al., 2002; on metabolic clearance rate or protein half-life have yet to be Menon and Menon, 2012). Induction of LHCGR expression in differ- described. entiating granulosa cells in response to FSH is an example of pre- As a valuable early marker of pregnancy as well as various dis- dominantly transcriptional control, whereas desensitization of ease states, the ability to accurately measure hCG in its myriad LHCGR in response to LH or hCG exposure includes an element of forms is particularly important. Beyond pregnancy detection, post-transcriptional regulation. Agonist-mediated desensitization hCG measurement is useful for identifying gestational trophoblas- of LHCGR involves a reduction in cell surface receptor concentra- tic disease, risk of failing or abnormal pregnancy, and non-gesta- tion, as well as diminished mRNA abundance. The decrease in tional malignancies. The predominant hCG species characteristic mRNA has been attributed to increased transcript degradation of each of these conditions differs. In early pregnancy and gesta- rather than suppressed transcription (Lu et al., 1993). tional trophoblastic disease, h-hCG is the dominant form, whereas Through a series of studies involving rat and human ovarian elevated free b-subunit production is characteristic of certain models, Menon and colleagues identified a binding protein, meva- cancers (Cole et al., 1988; Elliott et al., 1997; Hoermann et al., lonate kinase, which appears to regulate LHCGR expression by has- 1992; Iles et al., 1990; Mock et al., 2000). Abnormalities in h- tening degradation of LHCGR mRNA (Menon et al., 2010). An hCG are also used to assess pregnancy-related risks—elevated inverse correlation was observed between LHCGR mRNA and mev- maternal blood levels during the first and second trimester are alonate kinase activity: when one was elevated, the other was de- associated with fetal aneuploidy risk such as Down syndrome creased (Nair et al., 2002). Moreover, inhibition of mevalonate while low h-hCG in the latter stages of pregnancy is emerging kinase prevented downregulation of LHCGR in response to hCG, as a putative marker of preeclampsia risk (Bahado-Singh et al., whereas overexpression of mevalonate kinase ameliorated induc- 2002; Cole, 2009). tion of LHCGR in the presence of FSH and estradiol (Ikeda et al., In the case of both LH and hCG, the multiple circulating forms 2008; Wang et al., 2007). complicate their measurement in blood and urine (Cole, 2009; Alternative splicing of LHCGR mRNA has been implicated in reg- O’Connor et al., 1998). For example, total hCG assays, which are ulation of the ovarian cycle and luteolysis (Dickinson et al., 2009; the preferred measurement tool used by clinical laboratories, are Minegishi et al., 2007). Splice variants differ in exon content and, effective for quantifying regular hCG, but are generally suboptimal consequently, in their ability to bind or respond to LH or hCG. for detecting h-hCG (Cole, 2009). Moreover, in addition to differ- Using human corpora lutea obtained at various stages of the ovar- ences in glycosylation and degradation rates, genetic variants have ian cycle, investigators have demonstrated quantitative, but not been found to impede detection of LH by immunoassay (O’Connor qualitative, differences in the profile of LHCGR splice variants, indi- et al., 1998; Pettersson and Soderholm, 1991). Detection limits must cating that the relative abundance of mRNA species changes also be taken into account when assessing gonadotropin presence or throughout the ovarian cycle but their identity did not (Dickinson absence in clinical testing. Extremes in gonadotropin concentrations et al., 2009; Madhra et al., 2004). 208 J. Choi, J. Smitz / Molecular and Cellular Endocrinology 383 (2014) 203–213

Fig. 1. Glycoprotein hormone receptor structure. (A) Illustration of the short intracellular, 7-pass transmembrane, and large extracellular domains characteristic of glycoprotein hormone receptors (e.g., the luteinizing hormone/choriogonadotropin receptor). (B) Three-dimensional schematic of the leucine-rich repeats (LRRs) within the extracellular domain. (C) Structure of an individual LRR, wherein X represents any amino acid. Reproduced with permission from Vassart et al. (2004).

In vitro evidence suggests that gonadotropin signaling through with infertility, including PCOS. A genome-wide association study LHCGR influences the expression of individual splice variants that detected a link between a polymorphic marker in the region (Dickinson et al., 2009). Previous experiments had shown that of LHCGR and PCOS in Han Chinese women (which was confirmed one of the identified LHCGR splice variants forms complexes with in a subsequent case-control cohort) spurred several similar investi- LHCGR or the FSH receptor, influencing receptor expression and gations in other ethnic groups (Chen et al., 2011; Shi et al., 2012). The preventing the latter from reaching the cell surface (Nakamura specific polymorphic marker associated with PCOS in the Han Chi- et al., 2004; Yamashita et al., 2005). More recently, a truncated nese population failed to correlate with PCOS in European popula- form of LHCGR lacking transmembrane or carboxyterminal do- tions (Eriksen et al., 2012; Mutharasan et al., 2013); however, mains negatively regulated the expression and activity of the linkage within the genomic region of LHCGR was maintained upon full-length protein (Dickinson et al., 2009). Further study is needed fine mapping in one study (Mutharasan et al., 2013). to elucidate the physiologic significance of LHCGR splice variants. Utilizing an actual LHCGR sequence variant rather than a neigh- Frank mutations in LHCGR are associated with developmental boring polymorphic marker, Capalbo and colleagues reported a cor- and reproductive abnormalities, including psuedohermaphrodism, relation between the 312N LHCGR allele and increased risk for PCOS micropenis, hypospadias, and infertility (Ascoli et al., 2002; Menon in women of Sardinian origin (Capalbo et al., 2012). A dose-depen- and Menon, 2012). Emerging data also suggest that polymorphisms dent effect was observed for this polymorphism, such that homozy- in the LHCGR sequence contribute to risk for conditions associated gosity of the 312N allele was associated with a further elevated risk

Table 3 Half-life in serum and differential downstream effects of r-hLH and r-hCG binding to LHCGR.

Cell type r-hLH r-hCG Half-life a b c t1/2a, range of mean, hrs 0.6–1.3 , 3.9–5.5 a b c t1/2b, range of mean, hrs 9–12 , 23–31 Response e ED50 (pM) COS-7/LHCGR 530.0 ± 51.2 107.1 ± 14.3 Time to maximal cAMP accumulationd COS-7/LHCGRe 10 min 1 h ERK1/2 activationf hGLC Strong Weak AKT activationf hGLC Strong Minimal Neuregulin 1 inhibition in presence of ERK1/2 and AKT pathway blockade hGLC Abrogated Unaffected CYP19A1 expression in presence of ERK1/2 pathway blockade hGLC Increased Unaffected

ERK1/2, extracellular signal-regulated protein kinases 1 and 2; r-hLH, recombinant human luteinizing hormone; r-hCG, recombinant human chorionic gonadotropin; LHCGR, luteinizing hormone/choriogonadotropin receptor; hGLC, human granulosa cells; t1/2a = distribution (initial) half-life; t1/2b = elimination (terminal) half-life a Le Cotonnec et al. (1998a) b Le Cotonnec et al. (1998b) c Trinchard-Lugan et al. (2002) d Doses used were consistent with ED50 for r-hLH and for r-hCG in COS-7/LHCGR cells. e COS-7/LHCGR cells constitutively express LHCGR. LHCGR expression in hGLC cells is under physiologic regulation. f 100 pM dose used for both r-hLH and r-hCG (Casarini et al., 2012). J. Choi, J. Smitz / Molecular and Cellular Endocrinology 383 (2014) 203–213 209

Fig. 2. Response of human granulosa cells (hGLC) to long-term stimulation with recombinant human luteinizing hormone (r-hLH) or recombinant human chorionic gonadotropin (r-hCG). Intracellular cyclic adenosine monophosphate (cAMP) (bars) and extracellular progesterone (lines) concentrations during chronic exposure to 500 pM r-hLH or 100 pM r-hCG in the presence of a non-selective phosphodiesterase inhibitor (IBMX 500 lM); control cells were not exposed to gonadotropins. Panels illustrate 3 time ranges: (A) 0–12 h; (B) 13–24 h; and (C) 25–36 h. Extracellular accumulation of cAMP continued to increase during the 36-h evaluation period (D). Reproduced with permission from Casarini et al. (2012). 210 J. Choi, J. Smitz / Molecular and Cellular Endocrinology 383 (2014) 203–213

The effects of gonadotropin binding appear to be regulated by both temporal and spatial control of cAMP-mediated signaling (Weedon-Fekjaer and Tasken, 2012). This cascade begins when LHCGR binds LH or hCG. This induces a conformational change in

LHCGR, leading to the activation of the stimulatory G-protein Gs (Puett et al., 2007). Gs, in turn, activates adenylate cyclase, which increases the intracellular pool of cAMP. Acting through an independent mechanism, LHCGR stimulation also activates the phospholipase C/inositol phosphate (PLC/IP) signaling pathway (Gilchrist et al., 1996). Given the high levels of LH or hCG required to activate PLC via LHCGR, it has been suggested that PLC-based signaling only occurs during the pre-ovulatory LH surge and during pregnancy (Gudermann et al., 1992). Consistent with this role, investigators have recently identified PLC as a medi- ator of final granulosa cell differentiation in response to LH (Dona- deu et al., 2011). Other signaling molecules/pathways including extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and AKT have been identified as major downstream players in LHCGR-mediated signaling, with putative roles in cell proliferation, differentiation, and survival (Ben-Ami et al., 2009; Noma et al., 2011; Palaniappan and Menon, 2010; Shiraishi and Ascoli, 2007). Together with cAMP/ PKA, the ERK1/2, and AKT pathways contribute to the regulation of oocyte and follicle maturation (Brown et al., 2010; Reizel et al., 2010). Although both hLH and hCG bind to LHCGR, emerging evidence suggests that the downstream effects differ. Much like LH and hCG were considered to be functionally equivalent, the result of receptor binding was traditionally presumed to be alike. New data, however, suggest that this is not true. Advances in gonadotropin purification and recombinant technology have allowed investigators to explore hormone-specific signaling cascades, with thought-provoking results. Casarini and colleagues performed a series of experiments examining changes in gene and protein expression/activation in response to recombinant hLH (r-hLH) or recombinant hCG (r-hCG) exposure in COS-7 cells constitutively expressing LHCGR Fig. 3. Theoretical pathways for the divergence in receptor-mediated signaling (COS-7/LHCGR) and in human granulosa cells (hGLC) (Casarini between luteinizing hormone (LH) and human chorionic gonadotropin (hCG). et al., 2012). Recombinant hCG was found to be five times more Although acting on the same receptor, accumulating evidence suggests that LH binding (A) has a greater impact (thicker arrow) on AKT and extracellular signal- potent than hLH with respect to cAMP production, with a com- regulated protein kinase (ERK1/2) phosphorylation than hCG does, whereas binding mensurate lower ED50 (Table 3). cAMP concentration reached its of hCG (B) generates a greater intracellular accumulation (thicker arrow) of cAMP maximum more quickly with r-hLH than with r-hCG in COS-7/ than does LH (Casarini et al., 2012; Gupta et al., 2012). Pathway components LHCGR cells (10 minutes and 1 hour, respectively). During ex- derived from Arey and Lopez (2011). AC, adenylate cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; Gs/Gq, G proteins; IP3, tended gonadotropin exposure in hGLC cells, in which LHCGR inositol triphosphate; LHCGR, luteinizing hormone/choriogonadotropin receptor; expression was physiologically regulated, cAMP levels cycled, with PKA, protein kinase A; PLC, phospholipase C; PKC, protein kinase C. a period of approximately 4–5 h (Fig. 2). Interestingly, extracellular cAMP levels were not cyclical. Peak intracellular cAMP levels of PCOS beyond that of a single copy. Interestingly, the 312N allele were generally higher with r-hCG than with r-hLH. Nonetheless, has been found to be less common among men with maldescended progesterone production was similar, with supernatant concentra- testes compared with unaffected controls (Simoni et al., 2008). tions continuing to increase throughout the 36-hour evaluation period. In addition, recombinant hLH stimulation of hGLC cells strongly activated ERK1/2 and AKT, but hCG only weakly affected 6.2. Signaling pathways these pathways (Table 3). Blockage of the ERK1/2 and/or AKT pathways influenced downstream gene expression in hGLC ex- LHCGR is capable of binding LH, hCG, and h-hCG, but not hCG posed to r-hLH but not those stimulated with r-hCG. To exclude free b-subunit (Cole, 2009, 2010). Nicked hCG binds LHCGR, but the possibility that these findings were specific to recombinant with a substantially lower affinity (Cole et al., 1991). Experiments gonadotropins that do not undergo post-translational modification using hCG and ovine LH indicate that binding of these hormones to of native proteins, the authors repeated the dose-response exper- LHCGR differentially influences receptor interaction with plasma iments using extractive hLH and hCG, with results comparable to membrane proteins (Roess et al., 1997), which may contribute to that of r-hLH and r-hCG. the subsequent divergence in signaling pathway activation. The Disparate effects on signaling between LH and hCG also have intricacies of LHCGR signaling are a subject of active investigation, been described in goat ovarian granulosa cells (Gupta et al., with many components and interfaces not yet defined. It is gener- 2012). In this model, sustained exposure in cell culture led to sig- ally understood that the cyclic adenosine monophosphate/protein nificantly greater cAMP accumulation with hCG vs. ovine LH kinase A (cAMP/PKA) pathway is a significant effector of down- (p < 0.05). Long-term culture in the presence of LH supported stream events that lead to ovulation and steroid biosynthesis. growth and proliferation of granulosa cells; hCG, on the other J. Choi, J. Smitz / Molecular and Cellular Endocrinology 383 (2014) 203–213 211 hand, had the opposite effect, slowing growth and increasing cell therapeutic tools to help manage clinical conditions such as doubling time. Consistent with the previously described experi- infertility. ments using human granulosa cells, phospho-ERK1/2 levels were greater after long-term exposure to LH compared with hCG. Using the aforementioned data in combination with known ele- Disclosure statement ments of LHCGR signaling, we have constructed a schematic illus- trating potential differences between LH- and hCG-mediated J.C. has nothing to disclose, J.S. has nothing to disclose. signaling pathways (Fig. 3). This diagram is an acknowledged sim- plification of the complex interactions between diverse cascades Acknowledgements that coordinately regulate gonadotropin response. Further exploration in different cell types and physiologic conditions is needed to Financial support for manuscript development was provided by fully characterize the unique aspects of signaling via LH or hCG. Ferring Pharmaceuticals, Inc. The authors thank Crystal Murcia, PhD, and Jill McCollam, 6.3. Extragonadal LHCGR PharmD, of The JB Ashtin Group, Inc., for assistance in preparing this manuscript for publication based on the authors’ initial draft Detection of LHCGR in regions other than ovarian cell types and and direction. 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