Functional and molecular properties of the human recombinant Y4 receptor: resistance to agonist-promoted desensitization.
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.
Voisin
T
T
Unité 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
Goumain
M
M
Lorinet
A M
AM
Maoret
J J
JJ
Laburthe
M
M
D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019976:Cloning, Organism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000502:physiology*; D004347:Drug Interactions; D005455:Fluorescent Antibody Technique; D006801:Humans; D009479:Neuropeptides / Q000494:pharmacology; D010191:Pancreatic Polypeptide / Q000378:metabolism*; D019894:Peptide YY / Q000494:pharmacology*; D037342:Pertussis Toxin; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D017476:Receptors, Neuropeptide Y / Q000737:chemistry* / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D003432:Cross-Linking Reagents; D009479:Neuropeptides; D017476:Receptors, Neuropeptide Y; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D019894:Peptide YY; D005576:Colforsin; C109491:neuropeptide Y4 receptor; D010191:Pancreatic Polypeptide; D000242:Cyclic AMP; D037342:Pertussis Toxin