The LH receptor (LH-R) is a member of the G-protein-coupled receptor family and comprises an extracellular N-terminal half and a membrane- associated C-terminal half of a similar size The long-term goal of this project is to define the structural basis of hormone-dependent LH receptor-modulation and subsequent G-protein-activation. Hormonal responses are observed only after the hormone interacts with the receptor. Understanding how the receptor-modulation induces signal transduction necessitates the elucidation of hormone-receptor interactions and the determination of contact sites between the hormone and the receptor. The immediate objective is to determine these contact sites and to understand hCG-receptor interactions. The results of our studies suggest the presence of one or more hormone- receptor contact sites in each of hCGalpha, hCGbeta, the N-terminal half of LH-R and the C-terminal half of LH-R. In the HCGALPHA subunit, the C- terminal 4AAs (-Tyr-His-Lys-Ser) are essential for receptor-binding. AlphaPeptide (-Tyr-His-Lys-Ser) binds to the C-terminal half and stimulates CAMP synthesis and, particularly, AlphaLys91 is important for CAMP inducibility. This 4 amino acid peptide appears to have two sites, one for a receptor-contacting and the other for stimulation of CAMP synthesis. These and other contacts sites between HCG and LH-R will be determined in this study. To facilitate this study, we have prepared the wild type and various hCGs, LH-Rs, the C-terminal half of LH-R and the N-terminal half of LH-R (some naturally occurring soluble receptors). These hormones and receptors are expressed in stably transfected cell lines. For large quantity preparations, these CDNAS have been constructed in Vaculovirus and will be expressed in new more efficient SF21 cells. In addition, a number of HCG alpha peptides and LH-R peptides are available and antibodies against these peptides are being prepared. By using hormone binding and CAMP assays as well as affinity-labeling, we are at a breakthrough point to determine hormone-receptor contact sites and to identify specific amino acid residues in these sites. In addition, we hope to define the relationship of the high affinity HCG site in the N- terminal half and the low affinity HCG site in the C-terminal half of LH-R as well as the molecular mechanisms of HCG-dependent receptor- modulation to activate G-proteins. These results will serve as a model for not only other glycoproteins and their receptors but also bioactive peptides such as substance P and their receptors.
Showing the most recent 10 out of 58 publications
