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Signaling complexes formed by luteinizing hormone receptor trans-activation




Shanta, Hanan Ali A., author
Barisas, B. George, advisor
Roess, Deborah A., advisor
Crans, Debbie C., committee member
Miller, Charles W., committee member

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Signal transduction by luteinizing hormone (LH) receptors depends on hormone activation of these receptors, a process important for mammalian reproduction. The LH receptor, a member of the G protein-coupled receptor (GPCR) family, undergoes hormone-induced LH receptor dimerization and/or oligomerization and translocation into small membrane compartments where receptors are confined and exhibit slow lateral diffusion. However, the organization of the signaling complex confined within these structures is not clear. In this project, we used single particle tracking methods to evaluate the lateral motions of wild type receptor FLAG-LHR-YFP and mutant receptors defective in hormone binding (LHR-hCG,+cAMP) or defective in signal transduction (LHR+hCG,-cAMP) after exposure to human chorionic gonadotropin (hCG). These studies showed that, when wild type LH receptors and mutant receptors are coexpressed and treated with 100 nM hCG, there are decreases in receptor lateral diffusion, the number of receptor-occupied membrane microdomains and the size of receptor-containing membrane microdomains. These results suggest that wild type LH receptors are capable of both cis-activation of nearby wild type LH receptors and transactivation of LHR-hCG,+cAMP , a receptor that is not able to bind hCG. We then investigated interactions between wild type LH receptors and mutant receptors using homo-transfer fluorescence resonance energy transfer (FRET) methods. We showed that LH receptors associate with one another and that the extent of self-association increases in response to increasing hCG concentrations. Using homo-transfer FRET methods, we showed that mutant LH receptors are trans-activated by wild type receptors and undergo aggregation in response to 100 nM hCG despite being unable to bind hCG directly. Finally, we evaluated cAMP levels in cis-activated and trans-activated LH receptors using ICUE3, an EPAC-based reporter molecule for cAMP. We determined that increases in intracellular cAMP occur in cells expressing wild type receptors and exposed to increasing concentrations of hCG. Similarly, cells co-expressing mutant receptors exhibit increased cAMP when there is a 1:10 transfection ratio of LHR+hCG,-cAMP to LHR-hCG,+cAMP indicating that trans-activation is occurring. Disruption of membrane microdomains by pre-treatment of cells with 10 nM methyl-β-cyclodextrin for an hour has a negative effect on cAMP levels which indicates the importance of cholesterol-containing microdomains in signal transduction by LH receptors. Together these results demonstrate that trans-activated LH receptors can undergo receptor aggregation in response to hormone binding and can signal effectively despite the absence of a signal-transduction sequence in the mutant receptor.


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