Measurement of changes in fluorescence resonance energy transfer between gonadotropin-releasing hormone receptors in response to agonists.
Oligomerization of membrane-bound G-protein-coupled receptors has recently emerged as an important step in cellular signaling. Fluorescence resonance energy transfer (FRET) has undergone a revival as the method of choice for demonstrating in vivo protein-protein interactions and receptor dimerization. We have used chimeras of gonadotropin-releasing harmone (GnRH) receptors and various fluorescent proteins to investigate receptor dimerization in relation to receptor activation. Two pairs of FRET-compatible fluorescent proteins were used: sapphire with topaz, and enhanced green fluorescent protein (eGFP) with dsRed. Changes in the ratio between acceptor and donor fluorescence were measured after addition of buserelin, a GnRH agonist, and antide, a GnRH antagonist. For both pairs of fluorescent proteins, an increase in the ratio of acceptor to donor intensities was observed immediately after addition of buserelin as would be predicted if FRET occurred due to the microaggregation of receptors conjugated with different fluorescent proteins. No change in FRET was observed in time for cells in medium or after addition of antide. The increase in FRET signal was not uniform throughout a cell.
Cornea
Anda
A
Oregon Regional Primate Research Center and Oregon Health and Science University, Beaverton 97006, USA.
Conn
P Michael
PM
D000818:Animals; D037421:Anthozoa; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology; D002064:Buserelin / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D006727:Hormone Antagonists / Q000494:pharmacology; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009842:Oligopeptides / Q000494:pharmacology; D051381:Rats; D011966:Receptors, LHRH / Q000819:agonists* / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000737:chemistry / Q000378:metabolism
D018931:Antineoplastic Agents, Hormonal; D006727:Hormone Antagonists; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009842:Oligopeptides; D011966:Receptors, LHRH; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C057022:iturelix; D002064:Buserelin