Dynamic interactions of luteinizing hormone receptors during signal transduction

There is increasing evidence that receptor-mediated signal transduction by G-protein coupled receptors can involve self-association and redistribution of plasma membrane receptors into specialized plasma microdomains or rafts. These membrane fragments are characterized by insolubility in cold Triton X-100 and localization to low density regions in sucrose gradients.;Here, we characterized the translocation of wild type rat and human luteinizing hormone (LH) receptors into rafts following the binding of human chorionic gonadotropin (hCG) via a sucrose gradient ultracentrifugation method. We have also examined constitutively active human LH receptors which, in contrast to wild type receptors, were self-associated in the absence of hormone with a significant fraction of the receptors localized in rafts.;Receptor localization in membrane rafts did not occur when cells were pretreated with 1% methyl-beta-cyclodextrin (MbetaCD), a cholesterol sequestering agent that reduces membrane cholesterol and disrupts membrane rafts. Pretreatment of cells with MbetaCD also significantly decreased levels of hormone-induced intracellular cAMP in wild type LHR, but not constitutively active receptors, and reduced the extent of receptor self-association as evaluated by fluorescence resonance energy transfer (FRET).;Translocation of LH receptors into rafts required a functional hormone-receptor complex. When CHO cells expressing FLAG-LHR-wt were treated with an hCG antagonist such as deglycosylated hCG, receptors remained in high density membrane fractions as did hCG-treated point mutated receptor LHR-K583R that has reduced responsiveness to hCG binding. On the other hand, extensive crosslinking of wild type LH receptors with anti-FLAG antibody and a second anti-mouse IgG, while elevating intracellular cAMP, did not drive LH receptors into the raft environment. Finally, we found that the LH receptor C-terminus and its ability to be palmitoylated might affect raft localization.;In conclusion, translocation of the LH receptor into specialized membrane microdomains may be important in hormone-mediated signaling and a characteristic of functional, hormone-occupied LH receptors. Nonetheless, under some conditions, the raft environment is not essential for cAMP-mediated signaling.