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Functional and molecular properties of the human recombinant Y4 receptor: resistance to agonist-promoted
T Voisin1, M Goumain, A M Lorinet
1Unité de Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale U410, Faculté de Médecine Xavier Bichat, Paris, France. tvoisin@bichat.inserm.fr
Abstract:
After stable transfection of Chinese hamster ovary cells with the human Y4 receptor, clone 29 was isolated and studied for receptor properties. The following data were obtained: 1) one class of binding site was identified by analysis of (125)I-human pancreatic polypeptide (hPP) binding to cell membranes with a K(d) value of 0. 26 nM and a B(max) value of 1.44 pmol/mg protein; 2) the K(i) values for inhibition of (125)I-hPP binding by hPP, human peptide YY (hPYY), human neuropeptide Y (hNPY), and analogs were hPP (0.7 nM) < rat PP (47 nM) < hPYY (94 nM) < h[Leu(31)-Pro(34)]NPY (124 nM) << hNPY = porcine NPY(13-36) = rat D-[Trp(32)]NPY (>1 microM); 3) cross-linking experiments using (125)I-hPP identified a single M(r) 60,000 glycosylated Y4 receptor; and 4) the natural peptides hPP, hPYY, and hNPY inhibited forskolin-stimulated cAMP production in clone 29 cells with EC(50) values of 0.56 nM, 218 nM, and >1 microM, respectively. The inhibitory effect of hPP was abolished when cells were incubated with pertussis toxin, indicating a pertussis toxin-sensitive G(i) protein-mediated event. 5) Exposure of cells to 10 nM hPP for 24 h resulted in the absence of modification of binding capacity (1.38 versus 1.44 pmol/mg protein in control cells) or affinity (0.31 versus 0.26 nM in control cells); there also was no modification in the potency and efficacy of hPP in inhibiting forskolin-stimulated cAMP. Immunofluorescence indicated that the Y4 receptor was not internalized within the cells after 24-h treatment with 10 nM hPP. These data support that Y4 receptors are resistant to agonist-promoted desensitization and internalization. Clone 29 cells provide a valuable tool to further characterize the pharmacological aspects of human Y4 receptor.
Insights
This study found that human Y4 receptors in clone 29 cells are resistant to desensitization and internalization by agonists. These cells offer a valuable tool for further pharmacological characterization of the human Y4 receptor.
Area of Science:
- Pharmacology
- Cell Biology
- Receptor Biology
Background:
- The human Y4 receptor plays a role in various physiological processes.
- Understanding its pharmacological properties is crucial for potential therapeutic applications.
Purpose of the Study:
- To characterize the binding and signaling properties of the human Y4 receptor in a stably transfected cell line.
- To investigate the receptor's response to agonist stimulation, including desensitization and internalization.
Main Methods:
- Stable transfection of Chinese hamster ovary (CHO) cells with the human Y4 receptor.
- Radioligand binding assays using (125)I-human pancreatic polypeptide (hPP).
- Inhibition studies with various neuropeptides.
- Cross-linking experiments to identify receptor size.
- Forskolin-stimulated cAMP production assays.
- Pertussis toxin treatment.
- Immunofluorescence microscopy.
Main Results:
- Characterization of a single class of high-affinity binding sites for hPP on clone 29 cell membranes (K(d) = 0.26 nM).
- Differential binding affinities for various related peptides, with hPP showing the highest affinity.
- Identification of a 60,000 M(r) glycosylated Y4 receptor.
- Agonists (hPP, hPYY, hNPY) inhibited forskolin-stimulated cAMP production in a dose-dependent manner, mediated by a pertussis toxin-sensitive G(i) protein.
- No significant desensitization or internalization of Y4 receptors was observed after prolonged agonist exposure.
Conclusions:
- Human Y4 receptors expressed in clone 29 cells exhibit high affinity for hPP and mediate G(i) protein-coupled signaling.
- The Y4 receptor is resistant to agonist-induced desensitization and internalization.
- Clone 29 cells represent a valuable model system for detailed pharmacological studies of the human Y4 receptor.