Related Experiment Video
Updated: Oct 17, 2026
Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods
Published on: May 11, 2018
Cloning and functional expression of the hNPY Y5 receptor in human endometrial cancer (HEC-1B) cells
C Moser1, G Bernhardt, J Michel
1Institute of Pharmacy, University of Regensburg, Germany.
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 > or = p(Leu31Pro34)NPY = p(2-36)NPY > or = 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 x 10(5) and 1 x 10(6) 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.
Insights
Researchers developed a functional assay for the human Neuropeptide Y Y5 receptor (NPY Y5 receptor) using HEC-1B cells. Stable expression allowed for reliable radioligand binding and functional studies of the NPY Y5 receptor.
Area of Science:
- Pharmacology
- Molecular Biology
- Cell Biology
Background:
- The Neuropeptide Y Y5 receptor (NPY Y5 receptor) is a G protein-coupled receptor implicated in various physiological processes.
- Developing reliable assays for studying receptor function is crucial for drug discovery and understanding receptor pharmacology.
Purpose of the Study:
- To establish a functional assay for the human NPY Y5 receptor utilizing adenylyl cyclase activity.
- To characterize ligand binding and functional responses of the NPY Y5 receptor in transfected HEC-1B cells.
Main Methods:
- HEC-1B cells were transfected with vectors expressing the human NPY Y5 receptor.
- Radioligand binding assays using [3H]propionyl-NPY were performed on transiently and stably transfected cells.
- Functional assays measured inhibition of forskolin-stimulated cAMP synthesis.
Main Results:
- Both transient and stable transfections yielded high expression of functional NPY Y5 receptors.
- Ligand affinities for NPY and its derivatives were consistent with the NPY Y5 receptor subtype.
- Stably transfected cells demonstrated inhibition of cAMP synthesis by NPY, partially antagonized by a Y5-specific antagonist.
- Receptor characteristics remained stable for over 30 passages in stably transfected cells.
Conclusions:
- HEC-1B cells stably expressing the human NPY Y5 receptor provide a robust system for both radioligand binding and functional assays.
- This assay system is suitable for routine studies and characterization of NPY Y5 receptor ligands and antagonists.
