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 fo llowing data were obtained: 1) one class of binding site was identified by analysis of I-125-human pancreatic polypeptide (hPP) binding to cell membra nes 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 I-125-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 = porci ne NPY(13-36) = rat D-[Trp(32)]NPY (>1 mu M); 3) cross-linking experiments using I-125-hPP identified a single M-r 60,000 glycosylated Y4 receptor; an d 4) the natural peptides hPP, hPYY, and hNPY inhibited forskolin-stimulate d cAMP production in clone 29 cells with EC50 values of 0.56 nM, 218 nM, an d >1 mM, respectively. The inhibitory effect of hPP was abolished when cell s were incubated with pertussis toxin, indicating a pertussis toxin-sensiti ve 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 co ntrol 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 treat ment with 10 nM hPP. These data support that Y4 receptors are resistant to agonist-promoted desensitization and internalization. Clone 29 cells provid e a valuable tool to further characterize the pharmacological aspects of hu man Y4 receptor.