Molecular mimicry by an antiidiotypic monoclonal antibody to gonadotropin releasing hormone : Binding and signalling properties

Abstract

This thesis deals with the establishment and characterization of monoclonal antibodies (mAbs) to gonadotropin-releasing hormone (GnRH) receptor, with particular emphasis on the investigations into the abilities of the mAbs to transduce signals upon binding to the GnRH receptor using in vitro cultures of organs or cell lines expressing GnRH receptors.

GnRH receptor belongs to the family of G-protein coupled receptors (GPCRs) exhibiting the typical feature of the presence of seven transmembrane domains with three extracellular and three intracellular loops. GnRH, produced by the hypothalamic neurons, binds to GnRH receptors on the anterior pituitary gonadotrophs, thereby inducing the release of luteinizing and follicle-stimulating hormones (LH and FSH), which in turn orchestrate the activity of the gonads. Although the pituitary is the major site of action of GnRH, binding sites for GnRH have also been located on various extra-pituitary tissues, including the placenta, brain, breast, ovary, testis, lymphocytes, and certain neoplastic tissues. Despite the unprecedented explosion in studies in the area of the GnRH receptor, there is a paucity of information on several aspects, including the sites on the receptor involved in ligand binding, signal transduction, and the mechanism of desensitization of the receptor. Studies on the signaling events initiated upon binding of GnRH to its receptor reveal the participation of dual pathways, namely Ca²?-Protein Kinase C (PKC) and cyclic AMP (cAMP)-Protein Kinase A (PKA) cascades in GnRH-mediated gonadotropin synthesis/release. Although the role of the Ca²?-PKC pathway has been well established in GnRH-mediated signaling, the involvement of the cAMP-PKA pathway has been a subject of debate.

Studies on biological receptors for several ligands, including LH, FSH, thyroid-stimulating hormone (TSH), growth hormone (GH), prolactin, insulin, and ?-adrenergic ligands, have been greatly facilitated by the use of antibodies. Several of these antibodies have been found to mimic their respective ligands in terms of transducing signals upon binding to the receptor. Owing to the ease with which they can be purified in a stable form, anti-receptor antibodies with signal-transducing abilities serve as tools in obtaining greater details regarding the ligand-binding sites on the receptor and the cascade of events in the ligand-induced signal transduction pathway. It was, therefore, proposed to raise antibodies to the GnRH receptor and investigate their abilities to transduce signals upon binding to the receptor. Two approaches have been widely used to raise antibodies to receptors. These include the anti-idiotypic approach, in which antibodies are raised to the idiotype of an antibody recognizing the ligand, and the anti-peptide approach, which involves raising antibodies to synthetic peptides corresponding to different segments of the receptor. The latter is thus possible only when the sequence of the receptor is known. Before the cloning of the GnRH receptor, antibodies to the receptor were raised earlier in our laboratory through the anti-idiotypic approach. The cloning and sequencing of the GnRH receptor from several mammalian species, including human, mouse, rat, sheep, and cow, in 1992-1993, prompted the use of the anti-peptide approach for the establishment of mAbs to the GnRH receptor.

The main objectives of the present investigation include:

Establishment of mAbs to a synthetic peptide corresponding to the extracellular domain (ECD) of the human GnRH receptor.

Characterization of the anti-peptide mAbs as well as the anti-idiotypic mAb raised earlier in terms of their ability to recognize the native GnRH receptor on pituitary and extra-pituitary sites.

Investigation of the abilities of the anti-peptide and anti-idiotypic mAbs to stimulate the release of gonadotropins in cultures of placental villi and rat pituitaries.

Investigation of the abilities of the mAbs to mediate calcium signaling in a mouse gonadotroph cell line, namely ?T3-1.

Investigation of the abilities of GnRH and the mAbs to induce cAMP levels and the correlation between elevated cAMP levels and the release of gonadotropins in cultures.

Delineation of the role of the cAMP-PKA cascade in GnRH-mediated signaling in the absence of Ca²?-PKC series of signaling events under conditions of blockade of calcium channels or inhibition of PKC.

Chapter 1 of this thesis provides a general introduction summarizing the currently available information on GPCRs, GnRH, and its receptor, as well as the various approaches to raise antibodies to receptors. Chapter 2 includes details of the materials used and the experimental procedures adopted. Chapter 3 deals with studies on the establishment and characterization of mAbs to the GnRH receptor obtained through two different approaches, namely the anti-idiotypic and anti-peptide routes. In the former approach, an anti-idiotypic mAb, namely 4D10C1, was raised earlier in the laboratory to the idiotype of a GnRH-specific mAb. In the latter approach, anti-peptide mAbs were obtained to a synthetic peptide corresponding to the N-terminal 1-29 amino acids of the ECD of the human GnRH receptor. The ECDs in some GPCRs play a crucial role in ligand binding, while in a few other GPCRs, the extracellular loops together form the ligand-binding pocket. Since the ECD of the GnRH receptor has 35 amino acids and consists of stretches with high antigenic indices, the N-terminal 1-29 amino acids of the ECD of the human GnRH receptor were used as the immunogen. mAbs were obtained by fusion of myeloma cells with splenocytes from mice immunized with the ECD peptide conjugated to a carrier protein, namely bovine serum albumin (BSA). Two representative mAbs, namely A9E4 and F1G4, were characterized further. Both mAbs showed binding to the ECD peptide as demonstrated by enzyme-linked immunosorbent assay (ELISA) and dot-blotting. A major problem encountered with the use of peptide immunogens is that the antibodies capable of binding the peptide may not always necessarily recognize the parent protein. Therefore, the binding of the mAbs to the native GnRH receptor protein on pituitary and extra-pituitary tissues/cell lines was determined by immunohistochemistry/immunocytochemistry and Western blotting. The anti-peptide mAb F1G4 showed strong binding to human pituitary and human placenta but poor binding to rat testis, suggesting that mAb F1G4 may have low affinity for the rat GnRH receptor. Furthermore, the mAb recognized GnRH receptors on a human breast carcinoma cell-line, namely T47D, and a human hepatocarcinoma cell-line, namely HepG2, as demonstrated by immunocytochemistry. The binding of the anti-idiotypic mAb 4D10C1 was then tested. mAb 4D10C1 showed binding to human, rat, and sheep pituitaries, human placenta, and rat testis, as well as T47D and HepG2 cells.

Chapter 4 describes the results from investigations on the signal-transducing abilities of the anti-peptide and anti-idiotypic mAbs using in vitro cultures of human term placental villi, rat pituitaries, and a mouse gonadotroph cell-line, namely ?T3-1. The anti-idiotypic mAb 4D10C1 exerted a dose-dependent increase in the levels of human chorionic gonadotropin (hCG) in cultures of human term placental villi. Likewise, increased levels of LH and FSH were seen on stimulation by mAb 4D10C1 in cultures of rat pituitaries. In both cultures, namely those of placental villi and rat pituitaries, the molar concentration of the mAb required to exert maximal response was about fivefold lower than that of GnRH, probably due to the greater stability of the mAb compared to GnRH or due to the higher affinity of the mAb to bind to the GnRH receptor in comparison to GnRH. That the signaling actions of mAb 4D10C1 are a consequence of binding to the GnRH receptor was demonstrated by using a GnRH antagonist. For this, rat pituitaries were incubated with the antagonist prior to induction with GnRH or mAb 4D10C1, followed by monitoring the levels of LH and FSH. The GnRH antagonist blocked mAb-mediated increase in LH and FSH levels, thereby suggesting that the mAb exerts its stimulatory actions on binding to the GnRH receptor. The anti-peptide mAb F1G4 neither stimulated nor altered GnRH-induced gonadotropin release, indicating that the site on the receptor recognized by mAb F1G4 may not be sufficient to bring about signal transduction. The other anti-peptide mAb, namely A9E4, also neither stimulated nor altered GnRH-induced gonadotropin release. Having tested the ability of the mAb to stimulate gonadotropin levels, it was proposed to further test its signaling property in terms of stimulation of the levels of the second messenger, namely calcium, in ?T3-1 cells. mAb 4D10C1 caused an increase in the levels of [Ca²?] in the cells. The response was found to be similar to that seen on induction with GnRH, in that it was biphasic, with a rapid transient peak followed by a slower sustained rise. mAb-mediated increase in [Ca²?] could be blocked by prior treatment of the cells with GnRH antagonist.

Chapter 5 details the observations from studies on the effect of GnRH/mAb 4D10C1 on cAMP levels in cultures. Since the role of cAMP in GnRH-mediated signaling has been a subject of debate, it was proposed to first test the ability of GnRH to stimulate cAMP levels in cells prior to studying the antibody-mediated effects. A dose-dependent increase in cAMP levels was exerted by GnRH in cultures of human term placental villi and rat pituitaries. Likewise, mAb 4D10C1 also stimulated cAMP levels in both cultures. The correlation between cAMP and gonadotropin release was tested by using an activator of adenylate cyclase, namely forskolin, wherein the levels of gonadotropins were monitored on stimulation with forskolin. In both cultures, namely placental villi and rat pituitaries, forskolin exerted a dose-dependent increase in the levels of gonadotropins. It was of interest to further demonstrate the role of cAMP in GnRH-mediated signaling by monitoring the GnRH-induced levels of gonadotropins in the absence of the Ca²?-PKC pathway. Towards this objective, pituitaries were incubated with calcium channel blockers, namely verapamil and nifedipine, or a PKC inhibitor, namely staurosporine, prior to induction with GnRH or mAb 4D10C1. It was surprising to see decreased levels of GnRH/mAb-induced LH and FSH in tissues treated with the inhibitors compared to those that were not treated. It was of interest to further determine the GnRH/mAb-induced cAMP levels in the treated tissues. Against expectation, the levels of cAMP were also found to be decreased in the treated tissues. A decrease in cAMP levels upon blockade of calcium channels or inhibition of PKC suggests the involvement of Ca²?-PKC-dependent adenylate cyclase in GnRH-mediated signaling and the possibility of cross-talk between the Ca²?-PKC and cAMP-PKA pathways. From the results detailed in Chapters 4 and 5, it becomes evident that the anti-idiotypic mAb to GnRH mimics GnRH in all the signaling events tested, including stimulation of the second messengers, namely calcium and cAMP, as well as the gonadotropin levels. The agonistic property of the mAb prompts its application in the better understanding of the GnRH receptor.