This project will investigate structure-activity relations in the human glycoprotein hormones from two structural standpoints: primary amino acid sequence and post-translational modifications. To define the role of subunit sequence regions in target-organ action (Aim 1) we will: (a) prepare chemically modified subunits of human choriogonadotropin (hCG) for testing of recombination, receptor binding, adenylate cyclase activation and steroidogenesis. The results are expected to provide information on the importance of several amino acid sequence positions unique to the human hormones. (b) Synthesize several peptide fragments for use in preparing sequence-specific antisera for neutralization studies and evaluation of conformational changes. Two peptides with potential for amphiphilic helix formation will also be tested for direct interaction with ovarian membrane receptors. A post-translational modification clearly important in the action of hCG is the asparagine-linked side-chain complex carbohydrate. Extending our previous work characterizing the biological effects of deglycosylated hCG, we will explore the biochemical basis for the interaction of carbohydrate with cell membrane components to influence """"""""coupling"""""""" of receptor to postreceptor events (Aim 2). We will determine requirements for carbohydrate specificity by preparing and testing hCG with modified, otherwise intact oligosaccharides, and will attempt to remove the chains intact from the subunit for assay of binding and cyclase activity. We will use carbohydrate coupling procedures to add oligosaccharide chains to specific locations on deglycosylated or intact hCG subunits. These will be tested for their ability to recombine and elicit hCG responses in ovarian cells. If bioactive products are obtained, we will vary the configuration of added carbohydrate for define specificity. Finally, evidence for a subunit conformational change after deglycosylation will be studied by use of the region-specific antisera as probes for alterations involving different regions of the molecule. Through understanding the way in which these structural features mediate hormone action we hope to suggest new directions for design of antagonists, or potent agonists, for a range of potential clinical applications in reproductive endocrinology.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD012851-07A1
Application #
3312009
Study Section
Endocrinology Study Section (END)
Project Start
1979-09-30
Project End
1990-07-31
Budget Start
1986-08-01
Budget End
1987-07-31
Support Year
7
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199
Keutmann, H T; Rubin, D A (1993) A subunit interaction site in human luteinizing hormone: identification by photoaffinity cross-linking. Endocrinology 132:1305-12
Keutmann, H T; Hua, Q X; Weiss, M A (1992) Structure of a receptor-binding fragment from human luteinizing hormone beta-subunit determined by [1H]- and [15N]nuclear magnetic resonance spectroscopy. Mol Endocrinol 6:904-13
Milius, R P; Keutmann, H T; Ryan, R J (1990) Molecular modeling of residues 38-57 of the beta-subunit of human lutropin. Mol Endocrinol 4:859-68
Keutmann, H T; Mason, K A; Kitzmann, K et al. (1989) Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin. Mol Endocrinol 3:526-31
Keutmann, H T; Charlesworth, M C; Kitzmann, K et al. (1988) Primary and secondary structural determinants in the receptor binding sequence beta-(38-57) from human luteinizing hormone. Biochemistry 27:8939-44
Ryan, R J; Charlesworth, M C; McCormick, D J et al. (1988) The glycoprotein hormones: recent studies of structure-function relationships. FASEB J 2:2661-9
Keutmann, H T; Charlesworth, M C; Mason, K A et al. (1987) A receptor-binding region in human choriogonadotropin/lutropin beta subunit. Proc Natl Acad Sci U S A 84:2038-42
Amir, S M; Kubota, K; Tramontano, D et al. (1987) The carbohydrate moiety of bovine thyrotropin is essential for full bioactivity but not for receptor recognition. Endocrinology 120:345-52
Ryan, R J; Keutmann, H T; Charlesworth, M C et al. (1987) Structure-function relationships of gonadotropins. Recent Prog Horm Res 43:383-429
Calvo, F O; Keutmann, H T; Bergert, E R et al. (1986) Deglycosylated human follitropin: characterization and effects on adenosine cyclic 3',5'-phosphate production in porcine granulosa cells. Biochemistry 25:3938-43

Showing the most recent 10 out of 11 publications