The proposed studies relating to the structure and function relationships in human choriogonadotropin (hCG) and its receptor are a continuation of our ongoing studies. HCG and its subunits and the rat lutropin receptor have been expressed in an heterologous eukaryotic expression system, baculovirus-insect cell system. The hormone and its subunits were expressed in a high efficiency and could be purified by immunoaffinity chromatography. Since the recombinant hormones are similar to the native hormone and its subunits in immunological and biological properties, they can replace the native urinary hormones in all immunodiagnostic applications. More importantly, it has been possible to substitute selenomethionine for methionine in hCG. The recombinant selenomethionyl hCG has considerable potential in the determination of its three dimensional structure. The recombinant LH/hCG receptor expressed in baculovirus system occurs predominantly as a dimer.
The specific aims of the present studies are twofold. I. Studies on the structure and function relationships of gonadotropins will include the preparation and characterization of: (1) Recombinant selenomethionyl hCG on a large scale and to attempt to obtain x-ray diffraction grade crystals for three dimensional structural analysis. (2) Recombinant unglycosylated hCG and its subunits to resolve the question of the carbohydrate role in the function of hCG and selenomethionyl unglycosylated hCG. (3) The polypeptide and carbohydrate chains of recombinant hCG and its subunits. (4) Recombinant hCG, hCGalpha and hCGbeta on a large scale for further evaluation of their in vivo biological properties. (5) Carbohydrate mutants of hCGalpha and hCGbeta in baculovirus expression system to evaluate the contribution of individual N-linked carbohydrates to the function of hCG. II. Studies on the structure and function of rat LH/hCG receptor will include the preparation and characterization of: (1) Recombinant rat LH/hCG receptor. (2)Recombinant 1-294 amino acid residue extracellular polypeptide and its selenomethionyl analog. (3) Various deletion mutants of LH/hCG receptor in insect cells in order to delineate the ligand binding domain of the receptor and (4) Delineation of the hormone and Gs binding sites of the LH/hCG receptor and the possible involvement of extracellular and cytoplasmic loops in the ligand or Gs binding respectively. (5) Homologous reconstitution of LH receptor with GTP binding protein (Gs) and with Gs and adenylate cyclase or phospholipase C from rat ovarian membranes in liposomes to study the role of carbohydrate in the hormone and the receptor in the mechanism of signal transduction. All of the above studies should enhance our understanding of the regulation of human fertility and thus facilitate the development of safer methods of contraception.
Showing the most recent 10 out of 14 publications
