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Role of homotropic association of luteinizing hormone receptors in hormone mediated signaling

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dc.contributor.authorCrenshaw, Shirley Ann, author
dc.contributor.authorBarisas, B. George, advisor
dc.contributor.authorvan Orden, Alan, committee member
dc.contributor.authorRickey, Dawn, committee member
dc.contributor.authorLevinger, Nancy, committee member
dc.contributor.authorRoess, Deborah, committee member
dc.date.accessioned2007-01-03T08:09:45Z
dc.date.available2007-01-03T08:09:45Z
dc.date.issued2012
dc.description.abstractG 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.
dc.format.mediumborn digital
dc.format.mediumdoctoral dissertations
dc.identifierCrenshaw_colostate_0053A_11021.pdf
dc.identifierETDF2012400229CHEM
dc.identifier.urihttp://hdl.handle.net/10217/67425
dc.languageEnglish
dc.language.isoeng
dc.publisherColorado State University. Libraries
dc.relation.ispartof2000-2019
dc.rightsCopyright 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.
dc.subjectBRET
dc.subjectoligomerization
dc.subjectluteinizing hormone receptor
dc.subjectFRET
dc.titleRole of homotropic association of luteinizing hormone receptors in hormone mediated signaling
dc.typeText
dcterms.rights.dplaThis Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).
thesis.degree.disciplineChemistry
thesis.degree.grantorColorado State University
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (Ph.D.)

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