No description available.

No description available.

No description available.

No description available.

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.

The regulation of gene expression at the transcriptional level is one of the paramount mechanisms for maintaining control of growth, development and metabolic homeostasis. The Liver X Receptor (LXRa) is a novel member of the nuclear hormone receptor superfamily of transcription factors, which was originally isolated in our laboratory. Subsequent studies have revealed that LXRa is an essential transcriptional regulator of cholesterol homeostasis and a number of potent LXRa activators, including the oxysterol 22(R)-hydroxycholesterol have also been identified. As other members of the superfamily, LXRa exerts its regulatory control of target genes directly by binding to LXRa-responsive enhancer elements (LXREs), located upstream of the target gene promoter. Our laboratory initially demonstrated that LXRa heterodimerizes with the Retinoid X Receptor (RXRa) and cooperatively binds to a synthetic LXRE (DR4- LXRE), which consists of direct repeats of the hexad core consensus sequence spaced by four nucleotides. Tc date, two naturally occurring LXREs have been identified, including the LXRE--L\MTV element, located in the promoter region of the mouse mammary tumor vims long terminal repeat and the CYP7 A-LXRE element, located in the proximal promoter region of the rat cholesterol a-hydroxylase gene. In order to delineate the mechanism by which LXRa mediates the transcriptional regulation of target genes, a series of highly integrated characterization studies were initiated. Our initial interest was identifying the transactivation properties ofLXRa. Thus, a series of tramient transfection studies were performed, which investigated the effect of various LXREs, ligands/activators and cell lines on LXRa.-mediated transactivation. Ultimately, these studies revealed that the LXRa.-mediated transcriptional response was highly varied and specifically dependent upon the response element, ligand and cell line employed. Thus, these investigations indicate the specificity and great diversity in the nuclear hormone receptor-mediated transcriptional regulation of target genes. Furthermore, these studies resulted in the establishment of a viable and efficient transient transfection assay for further LXRa. in vivo investigations. Nuclear hormone receptors, including LXRa., are comprised of several modular domains termed AlB, C, D and E. A number of recent studies have implicated the highly divergent AlB domain of variety of nuclear receptors, and their isoforms, as a participant in transactivation. Specifically, these nuclear receptors have been shown to posses, within their respective AlB domains, an autonomous ligand-independent transactivation function termed the AF-1 domain, which can either function independently or can synergize with the E domain of the same receptor. Thus, determination of whether or not the 97 amino acid AI B domain of LXRa. participated in LXRa.-mediated transactivation became a main focus; in our investigation of LXRa.. In vitro EMSA analysis revealed that deletion of up to 63 amino acids of the N-terminal region of the LXRa. AlB domain did not effect either LXRa./ RXR.a. heterodimerization nor cooperative binding to LXREs. In vivo transient transfection assays further illustrated that theN-terminal 63 amino acids of the LXR.a. AlB domain were dispensable for LXR.a./RXR.a.-mediated transactivation. Therefore, as determined by the limitations of these assays, theNIV terminal63 amino acids of the LXRa AlB domain do not participate in neither transactivation nor heterodimerization and subsequent binding to LXR.Es. Transcriptional regulation, mediated by members of the nuclear hormone receptor superfamily, has been shown to involve multiple auxiliary co-factors, which modulate receptor-mediated tnmsactivation. These co-factors can either serve to repress (corepressors) or activate (co-activators) transcription not only through blocking or facilitating interactio r1s, respectively, between receptors and the basal transcription machinery but also through chromatin remodeling. Thus, the identification of LXRainteracting co-facton and the subsequent investigation of their ability to modulate LXRamediated transactiva1ion, were of particular interest. We demonstrated, via utilization of in vitro GST-binding assays, that LXRa interacts with RIP 140, SRC-1a and SMRT cofactors in a ligand-independent manner. Furthermore, these studies illustrate that the LXRa AF-2 core domain is necessary for efficient RIP 140 and SRC-1a binding. Surprisingly, this domain appears to impede, although not absolutely, the SMRTILXRa interaction, which has also been observed for the Retinoic Acid Receptor (RAR)/SMRT interaction. Functional studies ofLXRa, RXRa and RIP 140 indicate that RIP 140 antagonizes LXRa/RXR.a-mediated transactivation, which suggests that RIP 140 may serve to attenuate the transcriptional response of nuclear receptors modulated by other, more potent co-activators, as previously suggested in Peroxisome Proliferator-activated receptor a (PPARa);RIP 140 studies. As well, it is apparent that neither'the RIP 140/LXRa interaction nor the RIP 140-mediated repression of LXRa activity is effected upon deletion of the N-terminal 63 amino acids of the LXR.a. AlB domain. Interestingly, functional studies of LXR.a., RXR.a. and the partial SRC-1a clone, which lacks the Nterminal PAS-bHLH domain, indicate that this SRC-1a clone antagonized LXR.a.IRXR.a.mediated transactivation. While this result may simply demonstrate the necessity for a full length SRC-1a clone it may also indicate SRC-1 isoform-specific differences as previously illustrated in Estrogen Receptor (ER)/SRC-1 studies. Lastly, preliminary functional studies of LXR.a., RXR.a. and S:MR.T indicate that S:MR.T has no significant effect on LXR.a./RXR-mediated transactivation. These tentative results indicate that while LXR.a. and SMRT interaction in solution, S:MR.T may not be able to interact with LXR.a. when bound to DNA, and is thus unable to modulate LXR.a.-mediated transcriptional activation as previously demonstrated for the PP ARy and the orphan receptor Rev Erb. Taken together, the investigations presented in this study of LXR.a., further our understanding of not only the mechanism by which LXR.a. mediates its transcriptional response, but also hew nuclear receptors achieve specificity and diversity in the activation of target gene expression. / Thesis / Master of Science (MS)

WW-domain binding protein-2 (WBP-2) was cloned as an E6-associated protein (E6-AP) interacting protein and its role in steroid hormone receptor (SHR) function was investigated. We show that WBP-2 differs from other SHR coactivators, as it specifically enhanced the transactivation functions of progesterone receptor (PR) and estrogen receptor (ER alpha), whereas it had no significant effect on the androgen receptor, glucocorticoid receptor or the activation functions of p53 or VP-16. We also demonstrated that, like other well characterized coactivators, WBP-2 contains an intrinsic activation domain. Depletion of endogenous WBP-2 with small interfering RNAs indicated that normal physiological protein level of WBP-2 was required for the proper functioning of ER alpha and PR. Moreover, chromatin immunoprecipitation (ChIP) assays demonstrate the hormone-dependent recruitment of WBP-2 onto an estrogen-responsive promoter. As we initially identified WBP-2 as an E6-AP interacting protein, we investigated whether WBP-2 and E6-AP function in concert. Our data shows that WBP-2 and E6-AP each enhance PR function and when co-expressed they additively enhance the transactivation functions of PR. However, WBP-2 was also able to enhance the transactivation functions of ER alpha and PR in mouse embryonic fibroblast cells generated from E6-AP knockout mice lines, suggesting that the coactivation functions of WBP-2 was not dependent on E6-AP. The further elucidate the molecular mechanism of action of WBP-2; we dissected the functional importance of the polyproline (PY) motifs contained within the WBP-2 protein. Mutational analysis suggests that one of three PY motifs, PY3 of WBP-2 was essential for its coactivation and intrinsic activation functions. In this study, we also demonstrate that the WBP-2 binding protein, Yes-kinase associated protein 1 (YAP1) acts as a secondary coactivator of ER alpha and PR. However, the coactivation function of YAP1 is revealed only in the presence of wild-type WBP-2 and not with the PY motif 3 mutant WBP-2. This is consistent with our observations that, unlike the wild-type WBP-2, the PY motif 3 mutant WBP-2 does not interact with YAP1. Our quantitative reChIP assays demonstrates an estrogen-dependent recruitment and association of ER alpha with both WBP-2 and YAP1. The hormone-dependent recruitment of YAP1 to ER alpha responsive promoter is dependent on the physiological expression levels of WBP-2. This is consistent with, our observation that the coactivation functions of YAP1 is dependent on WBP-2, and is also in agreement with other known secondary coactivators that get recruited to SHR responsive promoter via their interaction with primary coactivators. Surprisingly, the association of WBP-2 with ER alpha and its recruitment to the ER alpha target promoter was abrogated by YAP1 knock-down, suggesting that WBP-2 and YAP1 may stabilize each other at the promoter, and consequently, are functionally interdependent. Taken together our data establish the role of WBP-2 and YAP1 as selective coactivators for ER alpha and PR transactivation pathways.

The glycoprotein hormones, Luteinizing Hormone (LH), human Chorionic Gonadotropin (hCG), Follicle Stimulating Hormone (FSH) and Thyroid Stimulating Hormone (TSH) are heterodimeric proteins with an identical α-subunit associated noncovalently with the hormone specific β-subunit and play important roles in reproduction and overall physiology of the organism (Pierce & Parsons, 1981). The receptors of these hormones belong to the family of G-protein coupled receptors (GPCR) and have a large extracellular domain (ECD)comprising of 9-10 leucine rich repeats (LRR) followed by a flexible hinge region, a seven helical transmembrane domain (TMD) and a C terminal cytoplasmic tail (Vassart et al, 2004). Despite significant sequence and structural homologies observed between the ECDs of the receptors and the specific β-subunits of the hormones, the hormone-receptor pairs exhibit exquisite specificity with very low cross-reactivity with other members of the family. 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, 2005b; Fox et al, 2001; Wu et al, 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. Binding of FSH to FSHR occurs in the large extracellular NH2-terminal domain where the participation of the LRRs (amino acids 18-259) is essential to determine the ligand selectivity (Dias & Van Roey, 2001; Fan & Hendrickson, 2005a; Szkudlinski et al, 2002). In fact, mutations in these regions lead to reduction in binding of the agonist to the receptor. It is not known how the signal from the large extracellular domain liganded complex is transmitted to the TMD (amino acids 367-695). It is envisioned that hormone binding to the LRRs leads to series of conformational changes leading activation of the TMD resulting in signal transduction. The recently reported crystal structure of the single chain form of FSH in complex with the leucine rich repeats of the FSHR (amino acids 1-268) (Fan & Hendrickson, 2005b), although provides detailed understanding of the molecular interactions of the LRRs with the hormone, fails to provide any insights into mechanism of receptor activation as the information regarding critical interaction of the hormone with TMD. This structure also did not provide any information on the role of the hinge region (amino acids 259-366) that connects the LRRs to the TMD in hormone binding and activation of the receptor. In the present study an attempt has been made to understand the role of the hinge region in hormone binding and signal transduction. The overall objective of the study is to elucidate the molecular details of the hormone receptor interactions, particularly FSH-FSHR interaction. Antibodies to glycoprotein hormones and their receptors have often provided insights into the mechanism of hormone-receptor interactions and signal transduction. While the TSH receptor antibodies and their effects on the overall physiology have been well documented (Khoo & Bahn, 2007; Rapoport & McLachlan, 2007), reports of such antibodies against FSHR or LHR and their possible effects on the reproductive functions are not available. In the present study, effects of FSHR antibodies with different specificities on FSH-FSHR interactions have been investigated. Antibodies to different regions of rat FSHR, were raised and extensively characterized and their effects of FSH-FSHR interactions and signaling were investigated. It was found that a polyclonal antibody against the hinge of the receptor (RF2 antiserum, amino acids 218-336), while having no significant effect on hormone binding and response could stimulate the receptor by itself bypassing the hormone. This stimulation of FSHR was very specific as this antiserum could not stimulate LHR or TSHR and could be blocked by preincubating the antibody with the antigen. Through competition experiments with different synthetic peptides of human FSHR, a stretch of hinge region corresponding to amino acids 296-331 was identified as the site recognized by the stimulatory antibody. This antibody did not interfere in hormone binding and could also bind to the pre-formed hormone-receptor complex suggesting that the binding site of the antibody may not participate directly in hormone binding. Subsequently the antibody was extensively characterized for its effect of hormone receptor interactions (Chapter 2). Previous studies considered the hinge region to be an inert linker connecting the LRRs to the TMD, a structural entity without any known functional significance (Vlaeminck-Guillem et al, 2002). However, the data with RF2 antibody suggested a direct role of the hinge region in signal transduction. Therefore, a systematic study to dissect the role to hinge region in hormone binding and signal transduction was conducted. Several truncations, deletions, activating and inactivating point mutations in the FSHR were generated to understand the mechanism of receptor activation. Firstly, these mutant receptors were characterized for their ability to translocate to the cell surface when transfected in the cultured mammalian cells. Secondly, affinity of all the mutant receptors for the hormone was determined in order to understand the effect of mutations on hormone binding. Finally, the cAMP response of these mutant receptors to the hormone and the stimulatory antibody was investigated to understand the effects of mutations on signal transduction. The results are described in Chapter 3. The hormone binding analysis and the affinity measurement of the mutant receptors showed that the LRRs are involved in high affinity hormone binding while the hinge region may not contribute to the process. This is in agreement with the crystal structure data which showed that the hormone was bound to the truncated receptor fragment representing only the LRRs (Fan & Hendrickson, 2005b). These binding data also corroborated the earlier data indicating that the antibodies against the hinge region do not interfere in hormone-receptor interactions. Further, the analysis of different N-terminally truncated receptor mutants provided strong evidence indicating that the constraining intramolecular interactions between the extracellular and the transmembrane domains are required to maintain the FSHR in an inactive conformation in the absence of an agonist. The analysis of the constitutive basal activity of the mutant receptors in absence of hormone suggested that certain regions of the extracellular domain had an attenuating effect over the TMDs that prevented constitutive activation of the receptor. This was demonstrated by a marked increase in the basal constitutive activity of the receptor upon the complete removal of its extracellular domain. Detailed analysis of the mutants suggested that LRR portion does not contribute to this attenuating effect, but it is the hinge region that perhaps interacts with the TMDs and dampens its basal constitutive activity. This attenuating effect was further narrowed down to a small stretch of 35 amino acids (296-331) within the hinge region. It was striking that the similar stretch was identified as the binding site of the stimulatory receptor antibody. In pharmacology, an ‘inverse agonist’ is an agent which binds to the receptor and reverses the constitutive activity of receptors. Thus the hinge region of the receptor could be termed as a ‘tethered inverse agonist’ of the TMD, since it is covalently associated with the TMD and their interactions dampen the basal constitutive activity of the receptor. However, careful comparison of the activities of the mutants (receptors harboring deletions and gain-of-function mutations) with maximally stimulated wild-type FSHR indicated that these mutations of the receptor resulted only in partial activation of the serpentine domain suggesting that only the ECD in complex with the hormone is the full agonist of the receptor. Moreover, the hinge region stabilizes the TMD in an inactive conformation and the activating mutations disengage the inhibitory ECD–TMD interactions bringing about partial activation of the receptor. Most interestingly, the deletion of amino acids 296-331 from hFSHR resulted in no further response to the hormone indicating that this part of the receptor is also critical for hormonal activation, perhaps playing a dual role in the attenuation of the basal activity and a direct involvement in the hormonal activation of the receptor. Progressive sequential deletions of ten amino acids from 290 to 329 yielded similar results (high basal cAMP production with concomitant loss of hormone and antibody response) clearly demonstrating that the integrity of this region is absolutely essential for hormonal activation. In conclusion, the study provides a conclusive evidence to show that the hinge region of FSHR, although not involved in primary high affinity hormone binding, plays a critical role in the modulation of the receptor activity in absence, as well as, presence of the hormone. A large array of reproductive abnormalities is associated with malfunctioning of FSHR. To explore the possibility of using the stimulatory antibodies for therapeutic purpose, three inactivating mutations of hFSHR were analyzed. In corroboration with the earlier reports (Doherty et al, 2002; Touraine et al, 1999), the mutants A419T and L601V are incapable of transducing the signal, despite having adequate cell surface expression and wild type affinities for the hormone, mainly because of defective TMD. The RF2 antibody failed to elicit any response from these mutants suggesting that its ability to activate the receptor depends on the status of the TMD. Interestingly, the activating mutant D576G, which showed very high basal cAMP production, could be stimulated by both antibody and the hormone to the nearly wild type levels suggesting that in this mutant the interactions between the hinge region and TMD are similar to that of wild type and higher basal cAMP production could be due to different interactions of the TMD with the G-Proteins. Structure-function studies of glycoprotein hormones and their receptors have been hampered due to low levels of expression of the properly folded proteins in heterologous systems (Chazenbalk & Rapoport, 1995; Hong et al, 1999b; Peterson et al, 2000; Sharma & Catterall, 1995; Thomas & Segaloff, 1994). Previous studies from the laboratory have shown that the Pichiapastoris,which blends the advantages of both bacterial and mammalian expression systems, can be used to hyper-express biologically active hormones (Blanchard et al, 2008; Gadkari et al, 2003; Samaddar et al, 1997). In addition, the same expression system has been used to produce single chain hormone analogs (Roy et al, 2007; Setlur & Dighe, 2007). Further, methodologies for Pichiafermentation and purification of recombinant hormones from the fermentation media have been wellestablished in the laboratory. Chapter 4 describes the work carried out to express, purify and characterize a fully functional hFSHR extracellular domain. Thus a stage is now set to attempt structural studies with the receptor. The results are discussed at the end of each of these chapters and future directions have been discussed at the end of this thesis.

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.

E6-associated protein (E6-AP), which was originally identified as an ubiquitin-protein ligase, also functions as a co-activator that enhances the hormone-dependent transactivation of estrogen (ER) and progesterone (PR) receptors. To investigate the in vivo role of E6-AP in mammary gland development, we generated transgenic mouse lines that specifically overexpress either wild-type human E6-AP (E6-APWT) or the ubiquitin-protein ligase defective mutant E6-AP (E6-APC833S) in the mammary gland. Here we show that overexpression of E6-APWT results in impaired mammary gland development. In contrast, overexpression of E6-APC833S or loss of E6-AP (E6-APKO) increases lateral branching and alveolus-like protuberances in the mammary gland. We also show that the mammary phenotypes observed in the E6-AP transgenic and knockout mice are in large part due to the alteration of PR-B protein levels. RNAi-mediated knockdown of E6-AP in T47D breast cancer cells increased PR-B protein levels and stability. In vitro ubiquitination assay using purified E6-AP and PR-B reinforce these conclusions and demonstrate that E6-AP promotes PR-B turnover in an ubiquitin-dependent manner. Furthermore, we also show that E6-AP regulates progesterone-induced Wnt-4 expression by modulating the steady state level of PR-B in both mice and in human breast cancer cells. This novel mechanism appears to regulate normal physiology of the mammary gland and its dysregulation may prove to contribute importantly to mammary cancer development and progression. To test this hypothesis, we examined the E6-AP transgenic mice for tumor formation over a period of 6, 9, 12, 18 and 24 months. Our data shows that, unlike the E6-APWT mice that show normal phenotype, the E6-APC833S mice develop mammary hyperplasia at high penetrance (80%); with a median latency of 18 months. Our findings indicate that the inactivation of the E3-ligase function of E6-AP is sufficient to initiate the process of mammary tumor development. These findings strongly suggest that E6-AP may act as a tumor suppressor by down regulating the ER-alpha, PR-B and thereby their signaling pathways.