Gonadotropin-releasing hormone receptor microaggregation - Rate monitored by fluorescence resonance energy transfer

Gonadotropin-releasing hormone (GmRH) regulates pituitary gonadotropin rele ase and is a therapeutic target for human and animal reproductive diseases, In the present study we have utilized the technique of fluorescence resona nce energy transfer to monitor the rate of GnRH receptor-receptor interacti ons. This technique relies on the observation that the degree of physical i ntimacy of molecules can be assessed by the tendency of proximal fluorophor es to exchange energy. Our data indicate that GnRH agonist, but not antagon ist, occupancy of the GnRH receptor promotes physical intimacy (microaggreg ation) between receptors. The time course indicates that this occurs prompt ly (<1 min) after occupancy and persists for at least 80 min and within the physiologically relevant range of the releasing hormone. The process measu red is not inhibited by 0.1 mM vinblastin, 2 <mu>M cytochalasin D, or 3 mM EGTA, an observation that distinguishes it from macroaggregation (patching, capping, and internalization). These observations, along with reports from other laboratories, are consonant with a growing body of evidence that ind icates that microaggregation is an early event following agonist occupancy of the receptor and part of the mechanism by which effector regulation occu rs.