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Intrinsically Fluorescent Luteinizing Hormone Receptor Demonstrates Hormone-Driven Aggregation

1999; Elsevier BV; Volume: 255; Issue: 2 Linguagem: Inglês

10.1006/bbrc.1999.0185

1090-2104

Regina D. Horvat, Scott E. Nelson, Colin M. Clay, B. George Barisas, Deborah A. Roess,

Adipose Tissue and Metabolism

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°C, 54 ± 13% of unoccupied rLHR-GFP were laterally mobile with a diffusion coefficient D of 16 ± 3.5 × 10−10cm2sec−1. Introduction of 10 nM LH for 1 h slowed receptor lateral diffusion to 6.6 ± 1.3 × 10−10cm2sec−1and 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.