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 green fluorescent protein (GFP) fused to the C-terminus of the rat LH receptor (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 response to 10 nM LH or hCG. In fluorescence photobleaching recovery studies at 37 degrees C, 54 +/- 13% of unoccupied rLHR-GFP were laterally mobile with a diffusion coefficient D of 16 +/- 3.5 x 10(-10)cm2sec-1. Introduction of 10 nM LH for 1 h slowed receptor lateral diffusion to 6.6 +/- 1.3 x 10(-10)cm2sec-1 and reduced fluorescence recovery after photobleaching 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. Interaction of receptors with LH or hCG caused clustering of rLHR-GFP receptors, significantly restricting lateral diffusion.
Department of Cell and Molecular Biology, Colorado State University, Fort Collins 80523, USA.
R D
RD
Horvat
S
S
Nelson
C M
CM
Clay
B G
BG
Barisas
D A
DA
Roess
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011940:Receptor Aggregation; D011974:Receptors, LH / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*
D006063:Chorionic Gonadotropin; D008164:Luminescent Proteins; D011974:Receptors, LH; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP