Cloning and functional expression of the hNPY Y5 receptor in human endometrial cancer (HEC-1B) cells

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Abstract
Aiming to develop a functional assay for the human NPY Y5 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-Y5-FLAG and the pcDEF3-Y5 vectors. After optimization of the transfection procedure, the binding of [3H]propionyl-NPY to transiently and stably expressed Y5 receptors was determined. The affinities of NPY, NPY derivatives, and rPP (pNPY >= p(Leu31Pro34)NPY = p(2-36)NPY >= p(D-Trp32)NPY > p(13-36)NPY > rPP) were in accordance with the NPY Y5 receptor subtype. For [3H]propionyl-pNPY approximately 1.7 × 105 and 1 × 106 binding sites per transiently and stably transfected cell, respectively, were determined. The KD 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 Y5 receptor. In these cells, 10 nM pNPY inhibited the forskolin-stimulated cAMP synthesis by 75%. This effect was partially antagonized by the Y5 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 Y5 receptor remained unchanged for more than 30 passages.Key words: human NPY Y5 receptor, HEC-1B cells, stable expression, radioligand binding, cAMP assay.