The objective of this project since its inception has been to establish the structural requirements for biological activity of the glycoprotein hormones related to reproduction. The complex nature of these molecules has hindered efforts to determine the sequence regions important for receptor binding, post-receptor activation and subunit association. In work to date we have used synthetic peptides and analogs to identify and characterize at least two receptor-binding regions in the beta subunit of human luteinizing hormone (LH) and choriogonadotropin (hCG). Under this renewal we will continue to develop a functional model for LH/hCG beta subunit through Specific Aims that explore structure-activity relations from four perspectives.
Aim 1 will focus ont he continued characterization of the binding sequence (38-57) by use of nuclear role for secondary structure in gonadotropin action. Analogs will be prepared to determine biological effects of substitutions predicted by NMR to alter primary or secondary structure.
In Aim 2 we will use additional peptides to map the binding regions throughout the subunit, and determine whether their location on the subunit is important for specificity. Their interrelationships in constituting the binding domains in the whole hormone will be assessed through synthesis of fragments containing more than one site.
Aim 3 will correlate the binding of subunit sequences to distinct regions on the LH/hCG receptor by use of fluorescence perturbation and anti-receptor peptide antibodies. Transferred nuclear Overhauser (TRNOE) NMR spectroscopy will be used to detect conformational changes in subunit peptides upon binding to receptor.
In Aim 4 we will determine sequences in the beta subunit that are responsible for interaction with alpha subunit, using J-correlated (COSY) NMR spectroscopy of alpha in presence of spin- labeled beta subunit peptides, and chemical identification at alpha sequences after crosslinking by photoaffinity labeled beta fragments. These approaches should offer unique information regarding the structural behavior of specific subunit sequences in solution, complementing anticipated studies by others using crystallographic and recombinant DNA methodology. The results can be expected to provide new insights into the chemical basis for defects in gonadotropin-receptor action and impaired reproductive function, and to facilitate future design of hormonal analogues for use in regulation of fertility.
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