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Title: The LH receptor : structure/activity relationships using anti-peptide antibodies
Author: Pallikaros, Zakos
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1996
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Site-directed antibodies were used to investigate the structure/activity relationships of the LH/CG receptor. Polyclonal antibodies were raised against synthetic peptides corresponding to regions within the extracellular N-terminal domain (antibody 1: Arg48-Glu65; antibody 2: Tyr187-Asp206) and the cytoplasmic C-terminal domain (antibody 3: Cys622-Ala636) of the receptor. Affinity purified antibodies proved to be peptide-specific by both ELISA and dot-blotting assays. On Western blots of membrane proteins prepared from superovulated rat ovaries, mouse Leydig tumour (MA10) cells and rat testes, all three antibodies recognised a single broad band (95- 100 kDa) corresponding to the putative LH/CG receptor. The 95-100 kDa protein also bound 125I-hCG on ligand blots and this binding was inhibited by the two N-terminal antibodies. Antibody 1 significantly inhibited 125I-hCG binding to intact MA10 cells to a greater extent than did antibody 2, whereas antibody 3 and pre-immune IgG were without effect. Similarly both N-terminal antibodies significantly inhibited the cAMP and progesterone response of MA10 cells to LH (antibody 1 being more potent than antibody 2), whereas antibody 3 and pre-immune IgG had no significant effect. Both the cAMP and progesterone responses of MA10 cells to dbcAMP, cholera-toxin and forskolin were unaffected by any of the antibodies, as was the stimulation of membrane adenylyl cyclase activity by NaF and a GTP analogue. These results indicate that the antibodies inhibit LH action at the level of the LH/CG receptor and that residues Arg48-Glu65 (and to a lesser extent Tyr187-Asp206) are close to the binding site for LH/CG. Data obtained with antibody 3 suggest that antibody binding to the C-terminal residues Cys622-Ala636 does not interfere with hormone binding or actions. The differential effects of these antibodies on LH-induced signal transduction provide important information on the structure / activity relationships of the LH receptor.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available