Abstract
Luteinizing hormone receptors (LHR) are G protein-coupled receptors (GPCR) found primarily in female and male reproductive organs where they play a critical role in ovulation and sperm maturation, respectively, as well as maintenance of sex hormone production in both sexes. The role of oligomerization in LHR function is of considerable interest and not well understood. The oligomerization state of LHR has been suggested to have a significant role in signaling, desensitization and internalization of this receptor after activation by either luteinizing hormone (LH) or human chorionic gonadotropin (hCG) [2-8]. Overexpression of membrane proteins such as LHR may result in molecular crowding and may lead to increased protein oligomerization [10]. We hypothesize that LHR are present in the plasma membrane as constitutive small clusters or, alternatively, exist as dimers or mixture of monomers and dimers in the absence of hormone and then undergo varying degrees of aggregation after binding hCG. These differences in LHR organization depend on expression levels of LHR which may, in turn, affects LHR activity. In this project, we examined the effect of LHR expression levels on receptor oligomerization using polarized homo-transfer fluorescence resonance energy transfer (homo-FRET) methods to evaluate receptor interactions in cell lines stably expressing averages of 10,000 receptors per cell, 32,000 receptors per cell, 123,000 receptors per cell or 560,000 receptors per cell. In addition, we measured levels of cyclic adenosine monophosphate (cAMP), a second messenger involved in signal transduction which is produced in response to activation of LHR. This study demonstrated that the oligomerization state of LHR depends on the expression level of LHR, i.e. the number of receptors per cell or the concentration of LHR per unit area. Although LHR appear as in dimers or oligomers in the plasma membrane when receptor expression levels are low, it is clear that, with increased expression levels, LHR are found in larger structures exhibiting lower values for initial anisotropy. The effect of hCG binding on LHR was dependent on the expression level of receptors in the absence of bound hormone. The greatest effect of hCG occurred in cells expressing low numbers of LHR per cell where receptors were able to undergo further aggregation in response to hormone binding. The effect of hCG on highly expressed LHR was negligible; LHR, when highly expressed, were already extensively aggregated and did not undergo measurable changes in their aggregation state. Deglycosylated human chorionic gonadotropin (DG-hCG) had modest effects on cells expressing comparatively few LHR per cell since these receptors were already present in small clusters. Depletion of plasma membrane cholesterol using MβCD caused a decrease in intracellular cAMP level accompanied by decrease in cluster size of LHR as expression level of LHR increased. Together these results are important to our understanding of the roles that the expression levels of LHR, the oligomerization state of LHR and the plasma membrane play in LHR function. The organization of lipids in the bulk membrane and in membrane microdomains affects the ability of LHR to signal as does protein density, particularly when receptor crowding has occurred. These studies also suggest, more generally, that protein organization in the plasma membrane may function as an important pharmacological target that merits further exploration.