Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors

We have used spot fluorescence photobleaching recovery methods to measure t he lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to gr een fluorescent protein (GFP) after binding of either GnRH agonists or anta gonist. Before ligand binding, GnRHR-GFP exhibited fast rates of lateral di ffusion (D = 18 +/- 2.8 x 10(-10)cm(2)sec(-1)) and high values for fraction al fluorescence recovery (%R) after photobleaching (73 +/- 1%). Increasing concentrations of agonists, GnRH or D-Ala(6)-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mo bile receptors. Increasing concentrations of the GnRH antagonist Antide slo wed the rate of receptor diffusion but had no effect on the fraction of mob ile receptors, which remained high. To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-assoc iation, we measured the fluorescence resonance energy transfer efficiency b etween GnRHR-GFP and yellow fluorescent protein-GnRHR. There was no energy transfer between GnRHR on untreated cells. Treatment of cells with GnRH ago nists led to a concentration-dependent increase in the energy transfer betw een GnRH receptors to a maximum value of 16 +/- 1%. There was no significan t energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nm. These data provide direct evidence that, bef ore binding of ligand, GnRHR exists as an isolated receptor and that bindin g of GnRH agonists, but not antagonist, leads to formation of large complex es that exhibit slow diffusion and contain receptors that are self-associat ed.