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 the lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to green fluorescent protein (GFP) after binding of either GnRH agonists or antagonist. Before ligand binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D = 18 +/- 2.8 x 10(-10)cm2 x sec(-1)) and high values for fractional fluorescence recovery (%R) after photobleaching (73 +/- 1%). Increasing concentrations of agonists, GnRH or D-Ala6-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mobile receptors. Increasing concentrations of the GnRH antagonist Antide slowed the rate of receptor diffusion but had no effect on the fraction of mobile receptors, which remained high. To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-association, we measured the fluorescence resonance energy transfer efficiency between GnRHR-GFP and yellow fluorescent protein-GNRHR: There was no energy transfer between GnRHR on untreated cells. Treatment of cells with GnRH agonists led to a concentration-dependent increase in the energy transfer between GnRH receptors to a maximum value of 16 +/- 1%. There was no significant energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nM. These data provide direct evidence that, before binding of ligand, GnRHR exists as an isolated receptor and that binding of GnRH agonists, but not antagonist, leads to formation of large complexes that exhibit slow diffusion and contain receptors that are self-associated.
Horvat
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RD
Cell and Molecular Biology Program, Colorado State University Fort Collins, Colorado 80523, USA.
Roess
D A
DA
Nelson
S E
SE
Barisas
B G
BG
Clay
C M
CM
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D016466:CHO Cells; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis / Q000378:metabolism; D004058:Diffusion; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D006727:Hormone Antagonists / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008164:Luminescent Proteins / Q000737:chemistry; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology*; D011966:Receptors, LHRH / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015398:Signal Transduction / Q000502:physiology; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D006727:Hormone Antagonists; D008164:Luminescent Proteins; D009842:Oligopeptides; D011966:Receptors, LHRH; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D007987:Gonadotropin-Releasing Hormone; C026031:LHRH, N-Et-AlaNH2(6)-; C057022:iturelix; D000242:Cyclic AMP