Crenshaw, Shirley Ann, authorBarisas, B. George, advisorvan Orden, Alan, committee memberRickey, Dawn, committee memberLevinger, Nancy, committee memberRoess, Deborah, committee member2007-01-032007-01-032012http://hdl.handle.net/10217/67425G protein-coupled receptors (GPCR) are plasma membrane receptors involved in signal transduction and are an important target for drug discovery. Luteinizing hormone receptors (LHR) are GPCRs found on the reproductive organs of both males and females and promote spermatogenesis and ovulation. Understanding how these protein receptors function on the plasma membrane will lead to better understanding of the mammalian reproduction system and other GPCR systems. Studies in the past suggested that these receptors oligomerize after hormone binding, but recent studies performed with LHRs suggest that these receptors maybe constitutively oligomerized in the endoplasmic reticulum and on the plasma membrane. However, these experiments were performed on receptors expressed by transient transfection and using bioluminescence resonance energy transfer (BRET). These methods have potential weaknesses. Transient transfections typically yield a fraction of cells with very high receptor expression and BRET measurements are strongly weighted towards those cells. Hence, this overall approach may have yielded misleading results. Fluorescence energy transfer (FRET) is a similar technique to BRET but has advantages such as allowing imaging examination of single cells. Using FRET, LHR oligomerization was evaluated on cells treated with human chorionic gonadotropin (hCG) or deglycosylated-hCG, hormones which activate and inhibit the receptor function, respectively. FRET measurements demonstrated that, on the surfaces of transiently transfected cells, LHRs exhibit substantial intermolecular FRET which is very slightly increased by hCG treatment and very slightly reduced by exposure to DG-hCG. Closer examination of these data showed that all observed FRET depended linearly on receptor expression and approach zero at low expression levels. This suggests that FRET between LHR on these transiently-transfected cells may arise from inter-molecular proximity induced non-specifically by high receptor surface concentrations. To evaluate the receptor density on cells flow cytometry was used. Flow cytometry revealed that transiently-transfected LHRs are expressed over a broad range of surface densities, including very high expression levels. Using a mathematical model, the FRET efficiencies expected for various receptor surface densities were calculated. These calculations suggest that expression levels observed cytometrically could cause substantial amounts of FRET from molecular crowding and, particularly if the receptors are additionally concentrated in lipid rafts, most of the observed FRET signal could be attributed to non-specific concentration effects.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.BREToligomerizationluteinizing hormone receptorFRETText