Functional and molecular properties of the human recombinant Y4 receptor: resistance to agonist-promoted desensitization
| Autor: | T, Voisin, M, Goumain, A M, Lorinet, J J, Maoret, M, Laburthe |
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| Rok vydání: | 2000 |
| Předmět: |
Time Factors
Cloning Organism Down-Regulation Fluorescent Antibody Technique CHO Cells Pancreatic Polypeptide Transfection Cricetinae Cyclic AMP Animals Humans Drug Interactions Peptide YY Virulence Factors Bordetella Cells Cultured Binding Sites Dose-Response Relationship Drug Cell Membrane Colforsin Neuropeptides Recombinant Proteins Rats Receptors Neuropeptide Y Cross-Linking Reagents Pertussis Toxin Protein Binding |
| Zdroj: | The Journal of pharmacology and experimental therapeutics. 292(2) |
| ISSN: | 0022-3565 |
| Popis: | 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, and1 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. |
| Databáze: | OpenAIRE |
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