Monks, Lara K.; (1998) The structure/function relationships of the murine Leydig tumour (MA10) cell, luteinising hormone receptor. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The LH/CG receptor is a member of the G-protein coupled receptor family. It is found expressed on testicular Leydig cells and ovarian granulosa-luteal and thecal cells. On binding its ligand LH, the activated LH/CG receptor initiates a cascade of intracellular signalling which results in the production of androgens and oestrogens. In this thesis the structural and functional relationships of the LH/CG receptor and in particular the process of LH/CG receptor desensitisation have been investigated. One of the aims of this work was to establish whether, when desensitised, the LH/CG receptor is phosphorylated. In order to achieve this it was first necessary to establish an immunoprecipitation protocol using antibodies which had previously been developed in this laboratory (Pallikaros et al. 1995). We used two different methods of immunoprecipitation and different LH/CG receptor antibodies, however, no immunoprecipitation of the LH/CG receptors from a solubilized membrane preparation occurred. Subsequently it was demonstrated that the LH/CG receptor antibodies did detect the LH/CG receptor in Leydig cells and ovarian preparation using immuncytochemical and confocal techniques and Western blotting. However, variations in the sensitivity of detection were observed depending on the method of fixation, nature of the tissue and the LH/CG receptor antibodies used. It was concluded, therefore, that the lack of immunoprecipitation may have been due to the inaccessibility of the antigenic sites of the LH/CG receptor to the antibodies (e.g. because of aggregation or degradation). Previous work from this laboratory had used an antisense strategy to produce C-terminally truncated LH/CG receptors in intact Leydig (MA10) tumour cells, some of which did not undergo ligand-stimulated desensitisation (West and Cooke 1991). We intended to use this antisense strategy to establish which regions of the LH/CG receptor underwent phosphorylation or were involved in coupling to Gs. However, because we could not reproduce the original results, we investigated the use of a cell free system. We successfully established that the rat LH/CG receptor could be synthesized in a coupled transcription/translation cell-free system. However, when the antisense oligonucleotides previously reported to cause truncated LH/CG receptors, were included in this cell-free system, they were found to have no effect on the size or level of LH/CG receptor synthesis. Preliminary studies indicated that antisense oligonucleotides targeted to the start site of translation may be more effective in preventing LH/CG receptor synthesis. In parallel with the antisense work, we amplified and cloned the LH/CG receptor from the MA10 cells so that we could generate truncated cDNAs by the use of restriction enzymes. These truncated cDNAs could then be expressed in a mammalian cell line and used to establish whether truncated LH/CG receptors could undergo ligand-induced desensitisation. The amplification and cloning of the whole LH/CG receptor proved difficult and so the amplification reaction was divided into two. The amplification and cloning of the C-terminal region of the MA10 LH/CG receptor was successfully performed. Sequence analysis confirmed that it was identical to that of the murine LH/CG receptor. However despite extensive optimisation procedures, it was not possible to amplify the first 1.1 kb of the receptor. Analysis of the predicted secondary structure formed by the mRNA indicated a considerable level of hairpin bend formation suggesting that this may have been responsible for preventing adequate primer hybridisation to the template. The putative kinases responsible for phosphorylation of the activated LH/CG receptor have not, as yet, been identified. In order to investigate the possible role of PKC, the effects of three PKC inhibitors (staurosporine, GF109203X and RO 31-8220) on LH-, cholera toxin- and forskolin-stimulated cAMP production in MA10 cells were investigated. It was found that staurosporine markedly increased, and GF109203X and RO 31-8220 decreased, the cAMP levels stimulated by these compounds. The effects of staurosporine were not via effects on LH binding, phosphodiesterase or cell viability. It is concluded that because the GF and RO compounds are more specific PKC inhibitors than staurosporine, that PKC-mediated phosphorylation may not be involved in LH-induced desensitisation. It is suggested that staurosporine may act via inhibition of CaM-kinase II.

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