Restricted Diffusion of Luteinizing Hormone Receptors

Single particle tracking was used to evaluate lateral motions of individual FLAGtagged human luteinizing hormone (LH) receptors expressed on Chinese hamster ovary (CHO) cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared to LH receptors on untreated cells, LH receptors on cells treated with 100 nM hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Like LH receptors labeled with Au-hCG, LH receptors labeled with Audeglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nM hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nM hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homo-transfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell-surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.