Intrinsically fluorescent luteinizing hormone receptor demonstrates hormone-driven aggregation

The possibility that LH receptors exist as isolated molecules when unbound and aggregate upon binding gonadotropins has previously been untestable in viable cells for want of a suitable nonhormone probe. We have now expressed in CHO cells an intrinsically-fluorescent LH receptor involving enhanced g reen fluorescent protein (GFP) fused to the C-terminus of the rat LH recept or (rLHR-GFP), More than half of these receptors (54 +/- 4%) are located on the plasma membrane and are functional: cAMP levels increase 3-5 fold in r esponse to 10 nM LH or hCG, In fluorescence photobleaching recovery studies at 37 degrees C, 54 +/- 13% of unoccupied rLHR-GFP were laterally mobile w ith a diffusion coefficient D of 16 +/- 3.5 x 10(-10) cm(2)sec(-1). Introdu ction of 10 nM LH for 1 h slowed receptor lateral diffusion to 6.6 +/- 1.3 x 10(-10)cm(2)sec(-1) and reduced fluorescence recovery after photobleachin g to 27 +/- 1%. Following treatment with 1 nM hCG, rLHR-GFP were laterally immobile and were distributed into small fluorescent patches over the cell surface. Thus, unoccupied rLHR-GFP receptors apparently exist as dispersed plasma membrane proteins with comparatively fast lateral diffusion. Interac tion of receptors with LH or hCG caused clustering of rLHR-GFP receptors, s ignificantly restricting lateral diffusion. (C) 1999 Academic Press.