The plasma membrane Ca2+-pump (PMCA) is a Ca2+-Mg2+- ATPase that expels Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+. Four genes (PMCAl to PMCA4) encode various isoforms ofthis pump. Caloxin is a novel peptide that was selected for binding to the second putative extracellular domain of PMCAl. Caloxin inhibits the Ca2+- Mg2+-ATPase in human erythrocyte leaky ghosts which primarily contain PMCA4. The objectives of this thesis are to delineate the mechanism of this inhibition and to determine its PMCA isoform selectivity. Caloxin inhibition of the PMCA pump in erythrocyte ghosts is non-competitive with respect to the substrates Ca2+ and ATP and the activator calmodulin. This was expected because the high affinity binding site for Ca2+ and sites for ATP and calmodulin are intracellular whereas caloxin is a peptide selected for binding to the second extracellular domain of the pump. In the reaction cycle, PMCA forms a 140 kDa acylphosphate enzyme intermediate from ATP (forward reaction) or orthophosphate (reverse reaction). Caloxin inhibits the acylphosphate formation in the forward but not in the reverse reaction. These results suggest that caloxin inhibits conformational changes required during the reaction cycle of the pump. In COS-M6 cells overexpressing PMCA4, caloxin inhibited the Ca2+ -Mg2+ATPase but with a marginally lower affinity than in the erythrocyte ghosts. Caloxin inhibition was also observed in insect cells overexpressing PMCA2. Several unsuccessful attempts were made to overexpress functional PMCAl. The work on isoform selectivity would require high level of expression of various PMCA isoforms and problems related to this requirement are discussed. Caloxin appears to inhibit the PMCA pumps by binding to the second putative extracellular domain and thus affecting the conformation of the protein. This is the first identified role of an extracellular domain of a PMCA pump. / Thesis / Master of Science (MSc)

-- / The nicotinic acetylcholine receptors (nAChRs) are transmembrane proteins, composed of five subunits and belong to the superfamily of ligand gated ion channels The nAChRs are distinguished according to their topological and pharmacological characteristics in muscle and nervous type. Both the muscle and the nervous type are involved in the execution of many physiological functions (eg, nerve impulse transmission) but respectively in the pathogenesis of many diseases (eg Myasthenia Gravis,Parkinson's,Alzheimer's).This makes imperative the need to design drugs that target specific to each type of receptor. A prerequisite for achieving this objective is to study the structure of the extracellular regions of the receptor, as it is known that the specific areas are recognised by the cholinergic ligands and the abnormal antibodies. The α7 subunit of the human nicotinic acetylcholine receptor, can be used as a model for this study as It is expressed as a homopentamer. Wanting therefore to avoid the large and hydrophobic transmembrane regions of the receptor that would hinder the achievement of the objective, we focused on the extracellular domain (ECD) of the receptor .So, according to the above, a recombinant form of the extracellular region of the receptor was constructed and expressed previously in our laboratory (Zouridakis et al., 2009). The recombinant protein was (α7-mut10-myc-His), expressed in soluble form, in sufficient concentration and showed about three times greater affinity for I125-a-bgtx compared to the wild type (α7-ΔCDwt). Furthermore, studies of dynamic light scattering and electron microscopy confirmed the formation of homopentamer molecules. Moreover, the deglycosylated form of the protein displayed all these enhanced features, allowing the entry of crystallization experiments with both the glycosylated and the deglycosylated form. In order to further improve the specific mutant, new recombinant forms of the extracellular region of the α7 subunit of the nAChR were constructed. The recombinant forms were expressed with different expression tags in their N-or C-terminal in order to improve the folding of the molecule. The FLAG-α7-mut10-myc-His was produced in greater quantity and Ηts deglycosylated form differs significantly, indicating probably a more homogeneous protein population. Also, analysis of the molecule bygel filtration showed the predominant formation of a homopentamer molecule and the absence of high molecular weight aggregates. This protein, has enhanced features compared to the α7-mut10-myc-His and thus can proceed to crystallization trials. The second part of the study refers to the construction concateremers of the α7ECD. Σwo peptide linkers varying in their length were used. The mutant which carried the smaller linker (AGS)8, showed greater solubility compared to the more extended one (AGS)11.

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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.

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