Zusammenfassung
Aiming to develop a functional assay for the human NPY Y-5 receptor based on adenylyl cyclase activity, HEC-1B cells, in which cAMP synthesis can be efficiently stimulated with forskolin, were selected for the transfection with the pcDNA3-Y-5-FLAG and the pcDEF3-Y-5 vectors. After optimization of the transfection procedure, the binding of [H-3]propionyl-NPY to transiently and stably expressed Y-5 ...
Zusammenfassung
Aiming to develop a functional assay for the human NPY Y-5 receptor based on adenylyl cyclase activity, HEC-1B cells, in which cAMP synthesis can be efficiently stimulated with forskolin, were selected for the transfection with the pcDNA3-Y-5-FLAG and the pcDEF3-Y-5 vectors. After optimization of the transfection procedure, the binding of [H-3]propionyl-NPY to transiently and stably expressed Y-5 receptors was determined. The affinities of NPY, NPY derivatives, and rPP (pNPY greater than or equal to p(Leu(31)Pro(34))NPY = p(2-36)NPY greater than or equal to p(D-Trp(32))NPY > p(13-36)NPY > rPP) were in accordance with the NPY Y-5 receptor subtype. For [H-3]propionyl-pNPY approximately 1.7 + 10(5) and 1 + 10(6) binding sites per transiently and stably transfected cell, respectively, were determined. The K-D values were 2.4 +/- 0.4 and 1.7 +/- 0.2 nM, respectively. Due to the high expression of the receptor protein, both stably and transiently transfected cells can be conveniently used in routine radioligand binding studies. By contrast, functional assays were only feasible with HEC-1B cells stably expressing the Y-5 receptor. In these cells, 10 nM pNPY inhibited the forskolin-stimulated cAMP synthesis by 75%. This effect was partially antagonized by the Y-5 antagonist N-{trans-[4-(2-naphthylmethylamino)- methyl]cyclohexylmethyl}naphthalene-2-sulfonamide. Although the genetic variability of cancer cells is in principle incompatible with a stable phenotype, both ligand binding characteristics and functionality of the Y-5 receptor remained unchanged for more than 30 passages.
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