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 ¹²⁵I-human pancreatic polypeptide (hPP) binding to cell membranes with aK_d value of 0.26 nM and aB_max value of 1.44 pmol/mg protein; 2) theK_i values for inhibition of¹²⁵I-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[Leu³¹-Pro³⁴]NPY (124 nM) ≪ hNPY = porcine NPY(13-36) = rat d-[Trp³²]NPY (>1 μM); 3) cross-linking experiments using ¹²⁵I-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 EC₅₀ values of 0.56 nM, 218 nM, and >1 μM, 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.