AN ABSTRACT OF THE DISSERTATION OF LAN HAI, for the Doctor of Philosophy degree in Molecular Cellular and Systemic Physiology, presented on 11th December, 2015 at Southern Illinois University Carbondale. TITLE: EFFECT OF CONSTITUTIVELY ACTIVATED LUTEINIZING HORMONE RECEPTOR ON THE MOUSE REPRODUCTIVE SYSTEM MAJOR PROFESSOR: Dr. Prema Narayan The luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is crucial for fertility, and genetic mutations in LHCGR cause adverse effects in reproductive development. Among the activating mutations identified in LHCGR, replacement of aspartic acid 578 by glycine (D578G) is the most common inherited mutation. Boys with this mutation undergo puberty by 2-4 years, caused by elevated testosterone in the context of prepubertal luteinizing hormone levels and present with Leydig cell hyperplasia. Clinically, these symptoms are associated with familial male-limited precocious puberty (FMPP). Our lab has published a mouse model for FMPP (KiLHRD582G) with D582G mutation equivalent to D578G in human LHCGR. We have previously demonstrated that KiLHRD582G male mice exhibited precocious puberty, Leydig cell hyperplasia and elevated testosterone and was a good model for FMPP. However, unlike women with the D578G mutation who show no abnormal phenotype, our studies revealed that female KiLHRD582G mice were infertile. KiLHRD582G female mice exhibit precocious puberty and irregular estrous cyclicity. A temporal study from 2-24 weeks of age indicated elevated steroid levels and upregulation of steroidogenic acute regulatory protein as well as several steroidogenic enzyme genes. Ovaries of KiLHRD582G mice exhibited significant pathology with the development of large hemorrhagic cysts as early as 3 weeks of age, extensive stromal cell hyperplasia with luteinization, numerous atretic follicles and granulosa cell tumors. Anovulation could not be rescued by exogenous gonadotropins. The body weights of the KiLHRD582G mice was higher that wild type counterparts, but there were no differences in the body fat composition. Hyperandrogenism and polycystic ovary phenotype was not accompanied by impaired glucose tolerance. Blocking the androgen action and estrogen synthesis indicated that reproductive phenotype was primarily due to excess estradiol. These studies demonstrate that activating LHCGR mutations do not produce the same phenotype in humans and mice and clearly illustrates species differences in the expression and regulation of LHCGR in the ovary. As we use male KiLHRD582G mice as breeders, we observed that the KiLHRD582G mice became progressive infertile, and only 8% of KiLHRD582G were fertile at 6 months of age despite normal sperm production. The infertile KiLHRD582G males were not able to form copulatory plugs in WT females, and mating studies suggested that the KiLHRD582G males were not capable of mating and/or ejaculating. Sexual behavioral testing revealed that the infertile KiLHRD582G males were capable of mounting the receptive WT females but were unable to achieve ejaculation indicating a problem with erectile and/or ejaculatory function. To address the reason for the ejaculatory dysfunction, we performed histopathological analysis of the accessory glands and penis. Hematoxylin and eosin staining showed that the normal columnar epithelium was replaced by pseudostratified columnar epithelium in the ampulla and several aggregates of chondrocyte metaplasia were apparent in the penile body of KiLHRD582G male mice. A temporal study indicated the histopathological changes in ampulla and penile body initiated at 7-8 and 12 weeks of age, respectively. Immunohistochemistry indicated that the chondrocytes stained positive for collagen type II, SOX9 and androgen receptor in the nucleus and for LHCGR in the cytoplasm. Penile fibrosis is a major cause of erectile dysfunction and is characterized by an increased collagen/smooth muscle ratio. However, our Image J analysis, hydroxyproline assay and western blot showed that KiLHRD582G penile body exhibited reduced levels of smooth muscle actin but similar total collagen content compared to WT mice. Thus, penile fibrosis was not responsible for the progressive infertility of adult KiLHRD582G mice. We also observed Leydig cell adenoma and disruption of spermatogenesis at 1 year of age. Our results suggest FMPP patients may be susceptible to infertility and testicular tumors later in their life and a follow-up study of FMPP patients is recommended.

Abstract The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) that has a large N-terminal ligand binding ectodomain. The LHR ectodomain splice variant, expressed concomitantly with the full-length LHR in tissues, has an unknown biological function. GPCRs are a major pharmacological target, however, very little is known about the intracellular regulation of these receptors. In the present work, expression and maturation of the rat LHR and its variant were elucidated using both tissues and heterologous expression systems. A special effort was made to identify the role of developmental stage and tissue type on the LHR maturation and to find out about the molecular role of the ectodomain splice variant. We found two sites of localization for the receptor, namely the sensory system and urogenital tissues. This was demonstrated at mRNA and protein level and by rat LHR promoter-driven β-galactosidase (β-Gal) expression in the mice. In neurons, the β-Gal co-localized with the cytochrome P450 side chain cleavage enzyme, which may indicate a novel role in the neurosteroid synthesis. The neuronal LHR was expressed in the mature and immature protein forms in both developing and adult tissues, being able to bind hormone with similar high-affinity as gonadal receptors. In contrast, only immature receptors were detected in the fetal rat urogenital structures. A significant novel finding was substantial upregulation of the LHR in pregnant female rat adrenal glands and kidneys at a time that coincides with the differentiation of the fetal urogenital tissues. The mice overexpressing the ectodomain splice variant showed interference in pituitary-gonadal functions and morphological changes in the urogenital tissues. The studies showed that the variant was an endoplasmic reticulum (ER)-retained soluble protein. It accumulated in juxtanuclear regions of the ER together with ER folding chaperones and was a substrate for ER associated degradation (ERAD). The co-expression of the variant with the full-length receptor decreased the amount of receptors and misrouted them to the juxtanuclear ER subcompartment. Taken together, we suggest that the maturation of the LHR protein is developmentally and physiologically regulated at the post-translational level in tissues. The LHR ectodomain splice variant possibly modulates post-translationally the number of full-length receptors through physiological signals. Our observation of the chaperone and protein accumulation into a specific ER subcompartment may represent a protein quality control holding compartment for inefficiently/misfolded ERAD substrates.

Made available in DSpace on 2014-06-11T19:35:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T18:46:34Z : No. of bitstreams: 1 nogueira_mfg_dr_botfmvz.pdf: 892396 bytes, checksum: 471e1cec0bdbf86073d92bc94c87f460 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Em células da teca e da granulosa, de folículos bovinos, foram detectados quatro transcritos alternativos do receptor do hormônio luteinizante (LHR). Apenas dois deles são traduzidos em proteínas funcionais com afinidades distintas em relação aos ligantes. Em humanos e símios, a isoforma completa (full-length) tem afinidade pelo LH e hCG, enquanto que a isoforma que apresenta deleção do exon 10 tem afinidade somente pelo hCG. Além disso, isoformas com deleção do exon 3 foram observadas em ratos, embora nenhum outro estudo tenha investigado essa região do gene bovino. Objetivouse com este trabalho caracterizar o padrão da expressão do gene do LHR nas células da teca e da granulosa de folículos antrais bovinos. Ovários foram coletados em matadouro, os folículos (5-14 mm) foram dissecados e as células da teca e da granulosa separadas para extração de RNA total com Trizol. As concentrações de esteróides no fluido folicular foram determinadas por radioimunoensaio (RIE). A expressão gênica do LHR foi mensurada por RTPCR semiquantitativo com oligonucleotídeos iniciadores (primers) específicos para amplificar o fragmento entre o final da região extracelular e o final da intracelular (LHRBC; primers posicionados nos exons 9 e 11). A ocorrência de transcritos alternativos oriundos do início da região extracelular (LHRA; primers posicionados nos exons 2 e 9) foi investigada mediante amplificação por RT-PCR. Como controle interno no PCR, utilizou-se a expressão da GAPDH. Paralelamente, células da granulosa cultivadas in vitro foram tratadas com 1 ou 10 ng de FSH no meio de cultura. Mediante RT-PCR, foi investigada a expressão das isoformas do LHRBC nas células da granulosa cultivadas e tratadas com FSH... / Growth of dominant follicle in the absence of circulating FSH and the events following the LH surge that culminate in ovulation, are dependent on the interaction between LH and its receptor (LHR). Four LHR alternative transcripts were described in theca and granulosa cells from bovine follicles. Only two of them can be translated to functional proteins (receptors coupled with G protein) with different affinities to their ligands. In humans and marmosets, the full-length isoform has affinity to both LH and hCG molecules, whereas the isoform with deletion of only exon 10 has affinity to hCG exclusively. Additionally, isoforms with deletion of exon 3 were observed in rats, although no previous report have investigated this region of the bovine gene. The objective of this study was to characterize the pattern of gene expression of the LHR in theca and granulosa cells from bovine antral follicles. Additionally, LHR expression was determined in cultured granulosa cells under FSH treatment. From ovaries collected in abattoir, antral follicles were dissected (5 to 14mm of diameter), and samples of theca and granulosa cells were obtained to total RNA extraction (Trizol protocol). Steroids concentrations in the follicular fluid were determined by RIA. Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify part of extracellular region (LHRA; primers annealing on exons 2 and 9) and the fragment from the end of extracellular region, including the transmembrane domain and finishing near the end of intracellular region (LHRBC; primers annealing on exons 9 and 11). As internal control of the PCR, it was used GAPDH expression. Cultured granulosa cells were treated with 0, 1 or 10 ng of FSH (3 replicates each dose). As in vivo and positive control, theca cell sample was utilized to comparison... (Complete abstract, access undermentioned electronic address)

Orientador: Ciro Moraes Barros / Resumo: Em células da teca e da granulosa, de folículos bovinos, foram detectados quatro transcritos alternativos do receptor do hormônio luteinizante (LHR). Apenas dois deles são traduzidos em proteínas funcionais com afinidades distintas em relação aos ligantes. Em humanos e símios, a isoforma completa ("full-length") tem afinidade pelo LH e hCG, enquanto que a isoforma que apresenta deleção do exon 10 tem afinidade somente pelo hCG. Além disso, isoformas com deleção do exon 3 foram observadas em ratos, embora nenhum outro estudo tenha investigado essa região do gene bovino. Objetivouse com este trabalho caracterizar o padrão da expressão do gene do LHR nas células da teca e da granulosa de folículos antrais bovinos. Ovários foram coletados em matadouro, os folículos (5-14 mm) foram dissecados e as células da teca e da granulosa separadas para extração de RNA total com Trizol. As concentrações de esteróides no fluido folicular foram determinadas por radioimunoensaio (RIE). A expressão gênica do LHR foi mensurada por RTPCR semiquantitativo com oligonucleotídeos iniciadores ("primers") específicos para amplificar o fragmento entre o final da região extracelular e o final da intracelular (LHRBC; "primers" posicionados nos exons 9 e 11). A ocorrência de transcritos alternativos oriundos do início da região extracelular (LHRA; "primers" posicionados nos exons 2 e 9) foi investigada mediante amplificação por RT-PCR. Como controle interno no PCR, utilizou-se a expressão da GAPDH. Paralelamente, células da granulosa cultivadas in vitro foram tratadas com 1 ou 10 ng de FSH no meio de cultura. Mediante RT-PCR, foi investigada a expressão das isoformas do LHRBC nas células da granulosa cultivadas e tratadas com FSH... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Growth of dominant follicle in the absence of circulating FSH and the events following the LH surge that culminate in ovulation, are dependent on the interaction between LH and its receptor (LHR). Four LHR alternative transcripts were described in theca and granulosa cells from bovine follicles. Only two of them can be translated to functional proteins (receptors coupled with G protein) with different affinities to their ligands. In humans and marmosets, the full-length isoform has affinity to both LH and hCG molecules, whereas the isoform with deletion of only exon 10 has affinity to hCG exclusively. Additionally, isoforms with deletion of exon 3 were observed in rats, although no previous report have investigated this region of the bovine gene. The objective of this study was to characterize the pattern of gene expression of the LHR in theca and granulosa cells from bovine antral follicles. Additionally, LHR expression was determined in cultured granulosa cells under FSH treatment. From ovaries collected in abattoir, antral follicles were dissected (5 to 14mm of diameter), and samples of theca and granulosa cells were obtained to total RNA extraction (Trizol protocol). Steroids concentrations in the follicular fluid were determined by RIA. Gene expression of LHR was measured by semiquantitative RT-PCR with specific primers to amplify part of extracellular region (LHRA; primers annealing on exons 2 and 9) and the fragment from the end of extracellular region, including the transmembrane domain and finishing near the end of intracellular region (LHRBC; primers annealing on exons 9 and 11). As internal control of the PCR, it was used GAPDH expression. Cultured granulosa cells were treated with 0, 1 or 10 ng of FSH (3 replicates each dose). As in vivo and positive control, theca cell sample was utilized to comparison... (Complete abstract, access undermentioned electronic address) / Doutor

GPCRs, influencing myriads of cellular functions, are the members of the largest family of the membrane proteins. However, their structures and the signaling mechanisms still remain enigmatic. In case of the Glycoprotein Hormone Receptor (GpHR) family the structure-function relationship is less understood because of a large extra-cellular domain (ECD). This large ECD, consisting of Leucine Rich Repeats (LRRs) and membrane-proximal hinge region, is sufficient for specific binding to the hormone (Ascoli, Fanelli, & Segaloff, 2002), but for receptor activation, hormone binding is translated via a conformation wave starting at hinge region and relayed to the transmembrane domain. Several biochemical, immunological and molecular biological tools have been employed to elucidate the structure-function relationship of the hormones and their receptors. These studies also helped in deciphering some of the regions present in both the hormones and the receptors involved in maintaining the specificity of their interaction (Fan & Hendrickson, 2005; Fox, Dias, & Van Roey, 2001; Wu, Lustbader, Liu, Canfield, & Hendrickson, 1994). However, the complete understanding of the hormone‐receptor contact sites and mechanism of receptor activation are still an enigma. Understanding the molecular details of these phenomena can lead to the development of novel strategies of regulating hormone action or regulating receptor activation in a hormone independent manner. The crystal structure of FSHR ECD (amino acids 17-366) revealed that LRRs form a semicircular palm shaped structure with the C terminus region, designated as the hinge region, protruding out like a thumb. The hinge region, rather than being a separate functional unit, was found to be an integral part of the LRR domain, having two such repeats (LRR11 &12). LRR 11 is connected to LRR12 through a hairpin loop (amino acids 280-344) harboring the invariant sulfated tyrosine residue (sTyr) in YD/EY motif (X. Jiang et al., 2012). The heterodimeric hormones consisting of a common  subunit and a hormone specific  subunit, bind to the primary hormone binding site at LRR 4-6 as reported in the FSHR-FSH co crystal (Fan & Hendrickson, 2005). This primary binding of the hormone at LRR 4-6 creates a pocket (comprising of the residues P16α, L17α, F18α, F74α, L37β, Y39β, and P45β) in the hormone for secondary binding at sTyr residue. This interaction is proposed to initiate conformation change in the hinge region which further leads to FSHR activation (X. Jiang et al., 2012). Thus, the role of hinge region in GpHR activation got evolved from a linker to a switch, which decides the fate of the receptor activity (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). sTyr residue being conserved, presents itself as a potential player in activation mechanism of all the three receptors of the family (Bonomi, Busnelli, Persani, Vassart, & Costagliola, 2006; Kreuchwig, Kleinau, & Krause, 2013). Precise involvement of sTyr in GpHR activation is yet to be explored. The previous studies from the laboratory using the hinge region specific polyclonal and monoclonal antibodies established the unequivocal role of the hinge region in FSHR and TSHR activation (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). However, its function in LHR activation has not been conclusively established. Due to the unavailability of the structural information of LHR ECD/hinge, it is more difficult to study and explain the role of hinge region in LHR activation. The hormone independent signaling by point mutants of LHR also remains poorly understood. In the present study an attempt has been made to understand the role of the hinge region in LHR signaling and modulating role of LRRs in hinge mediated LHR activation. The present study was initiated with an overall objective of understanding the molecular details of LHR activation mechanism keeping hinge at the centre of the picture. To have clarity of this picture with a holistic view of the mechanism, multi-pronged approach was adopted. Initially, ScFvs against LHR hinge region were employed as tools to probe into the hormone‐receptor interactions. Antibodies against glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone‐receptor interactions and signal transduction (Agrawal & Dighe, 2009; Dighe & Moudgal, 1983; Gadkari, Sandhya, Sowdhamini, & Dighe, 2007; Gadkari et al., 2007; Kene, Nalavadi, Dighe, Iyer, & Mahale, 2004; Majumdar, Railkar, & Dighe, 2012a, 2012b). In this study, Single chain Fragment variables (ScFvs) against the hinge region of LH receptor have been employed to understand the mechanism of receptor activation. The effects of LHR ScFvs on hCG-LHR interactions have been investigated and three of the ScFvs, JE10, JE4 and JG1 could bypass the hormone and activate the receptor directly, with JE10 being the most potent one. The effect on the signaling was specific for LHR as no increase in cAMP response was observed for TSHR/FSHR in presence of these ScFvs. JE10 surprisingly was unique and could alter the hCG-LHR interaction by decreasing hormone affinity and simultaneously increasing the Bmax for the hormone. JE10 binding was decreased to the pre-formed hormone receptor complex suggesting that hCG and the stimulatory antibody show stearic hindrance at the binding sites on hinge or hormone binding induces conformational change in the epitope of JE10. The change in affinity and Bmax of the hormone by JE10 could be due to unmasking of new binding sites for hormones or an allosteric effect on the protomer interaction like explained in case of a small TMD specific allosteric modulator of FSHR (Xuliang Jiang et al., 2014). JE10 could also potentiate hCG signaling at sub-saturating concentrations of hCG, the precise mechanism of which is not clear. Through TSHR-LHR chimeric mutants, a stretch from amino acids 313-349, within the hinge region, was identified as the site recognized by JE10. In order to study structural features of the JE10 epitope, LHR ECD was modeled on the basis of FSHRED crystal structure. With most of the motifs being structurally conserved (CF3 and YPSHCCAFF); the major portion of the hinge region was found to be unstructured. This unstructured region harbored the JE10 epitope as well as the functionally important conserved sTyr residue. The CD spectra of LHR hinge in presence of ScFv JE10 suggested a ScFv induced helical conformation and stabilization of the hinge loop region, which was constrained in the homology model into helices. As loop was now constrained in the Mode 2, so was the interaction of sTyr, which was now in contact with positively charged residues, probably stabilizing its charge. The YEY motif mutants further confirmed the indirect essential role of Y331 in activation of LHR by JE10. Another approach followed to study hCG-LHR interactions was use of a series of LHR N-terminal truncation mutants and truncation mutants along with one of the LHR CAM (S277Q/D578Y). The effect of these truncations on hormone binding and receptor activation was investigated. The deletion of Cysteine box (Cb-1) of LHR (present at N-terminus of ECD) leads to abrogation of hCG binding, indicating importance of this region in maintaining ECD conformation required for hormone binding. This is the most unexplored region of the ECD. Though Cb-1 does not bind to the hormone directly (as is evident from the crystal structure) but it is indirectly essential for hormone binding. The basal activity of these truncated mutants was as low as that of the wild type LHR, reconfirming that no region of LHR ECD acts as an inverse agonist for the TMD (Karges, Gidenne, Aumas, Kelly, & Milgrom, 2005). Truncation mutants with CAM (double mutants) also showed low basal activity, suggesting that intact ECD is prerequisite for keeping LHR in a conformation, best suited for hormone binding and binding of G protein for activation. That best conformation still needs to be explored. Truncation mutants did not get stimulated by JE10 also. This observation is opposite to the previous studies in which FSHR/TSHR truncated mutants could be stimulated by hinge specific antibodies (Agrawal & Dighe, 2009; Majumdar & Dighe, 2012). This difference points out to the variations in which LHR hinge-TMD interactions prevail and lead to the receptor activation. This variation was also confirmed with a previous report in which the binding of TSHR-ECL specific antisera to wild type LHR and TSHR-LHR 6 chimeric mutant suggested that hinge of LHR does not seem to be constraining the TMD (Majumdar et al., 2012b). Thus the LHR TMD itself possesses all the inhibitory interactions, also indicated by the presence of most of the activating mutations in LHR TMD (Piersma, Verhoef-post, Berns, & Themmen, 2007). Protomer interaction is the newest aspect of GpHR activation mechanism and has not reached any conclusive, physiologically relevant explanations yet. By co-transfection of wild type LHR and ECD truncated mutants, this study suggests the LHR protomer interaction and proposes the involvement of allosteric effect of ECD on LHR protomer interaction. The effect of JE10 on activating and inactivating mutants of LHR were quite interesting. The ScFv could bind to the activating mutant D578Y (associated with precocious puberty). This mutant exhibited higher basal cAMP production, but was activated even further by the ScFv. The inactivating mutant A593P is a completely inactive receptor associated with (associated with pseudo-hermaphroditism. It does not respond to the hormone at all. The ScFv JE10 binds to this receptor and stimulates cAMP production. This observation is rather striking, as it is possible to activate a completely inactive mutant that could not be stimulated by the hormone by a binder specific for the hinge region. It is not clear how the binder that interacts with the hinge region affects the function of the inactive TMD thus providing an interesting tool to investigate the interactions between the hinge region and TMD that are probably key to understand the activation of GpHR. which has been shown to be central to the GpHR activation mechanism, (Agrawal & Dighe, 2009; Majumdar et al., 2012b; Schaarschmidt, Huth, Meier, Paschke, & Jaeschke, 2014). As per the recently suggested model by Deupi et. al., that each mutation and agonist can take a different pathway during activation (Kobilka & Deupi, 2007). The activated state induced by JE10 in D578Y and A593P seems to be different from the wild type LHR, with each activated receptor state having different capacity to bind to the G protein. The difference in G protein capacity in itself reflects the different receptor turnover or different Gs uncouplings or different Gs binding affinities, which needs to be further investigated, opening up another avenue for exploration. There is a lacuna in understanding the signal relay from the hinge to TMD. However, JE10 seems to be activating the wild type LHR and the mutants directly or indirectly by modulating the 6th helix of the TMD, known to be important for hormone independent activation of LHR (Fanelli, 2000; Latronico & Segaloff, 2007; Majumdar et al., 2012b). As evident from the absence of any hinge mediated constrain on LHR TMD and absence of uncharged residues present in LHR LRRD-TMD interface (LHR ECD Model 1), LHR hinge does not seem to be maintaining significant interactions with the TMD in absence of a ligand or in its basal state. Hormone/ agonist binding or activating mutations act as a positive regulator (inducing conformation change in hinge), required to bridge the interactions between LHR hinge and the TMD, which is supported by various studies in the past (Karges et al., 2005; Majumdar et al., 2012b; Nishi, Nakabayashi, Kobilka, & Hsueh, 2002; Osuga et al., 1997; Ryu, Gilchrist, Tung, Ji, & Ji, 1998; Zeng, Phang, Song, Ji, & Ji, 2001). This interaction bridged by the conformational change in the hinge region, seems to isomerize the closed state of LHR into an activated state. The present study supports the conformational induction model for receptor activation in which intramolecular interactions between the two domains (hinge-TMD) lead to the receptor activation. In conclusion, this study presents a possible mechanism of activation of LHR by a partial agonist ScFv, which induces the conformation change in the disordered loop region (a.a.313-349) of the hinge and stabilizes it into helical state. This conformation change is predicted to be important for relaying the activation signal to the TMD. The study also demonstrates the activation of a completely inactive mutant A593P by JE10, suggesting a distinct possibility of its use as a therapeutic tool in treating infertility caused by inactivating mutations in LHR. On a second note, the study extends the role of LRRs, apart from direct hormone binding, to an indirect allosteric role in hormone binding, LHR activation and functional stability. This functional stability does not seem to be restricted to a single LHR but also depends on its interaction with nearby protomers. Though there are evidences for and against each of the above discussed possibilities, as yet there is no accepted model that explains the precise steps of receptor activation, hence, the molecular details of these interactions needs to be investigated in future.

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