title: Agonist-dependent internalization of the human melanocortin-4 receptors in human embryonic kidney 293 cells.
abstract: A chimeric protein comprised of melanocortin-4 receptor (MC4R) and the green fluorescent protein (GFP) was created for studying receptor/ligand localization and trafficking. The ligand binding affinities and second messenger stimulation induced by MC4R-GFP closely resembled those of the wild-type receptor, suggesting functional integrity of the chimeric protein. As observed with a confocal microscope, in human embryonic kidney (HEK)-293 cells MC4R/GFP was distributed evenly along the cell membrane. Addition of [Nle4-d-Phe7]-alpha-melanocyte-stimulating hormone (NDP-MSH), a peptide MC4R agonist, induced receptor translocation into intracellular compartments in a time- and concentration-dependent manner. [Ac-Nle-c[Asp-His-d-Nal(2')-Arg-Trp-Lys]-NH2] (SHU9119), a potent MC4R antagonist, completely inhibited NDP-MSH-mediated internalization. MC4R-GFP internalization was unaffected by a protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89), but was impaired by pretreatment with inhibitors of endocytosis through clathrin-coated pits, hypertonic sucrose, or concanavalin A. Time-dependent colocalization of MC4R-GFP with rhodamine-transferrin, an early endosome marker, and with LysoTraker, a lysosome marker, was observed after short-term (45 min) and prolonged (20 h) agonist exposure, respectively. Rhodamine-[AcNle-c[Asp-His-d-Phe-Arg-Trp-Lys]-NH2] (MTII), a fluorescent derivative of an MC4R agonist, was found to cointernalize with MC4R-GFP into intracellular vesicles. No significant receptor recycling or segregation from the ligand was observed 60 min after removal of the agonist. In contrast, an antagonist rhodamine-Ac-Cys-Glu-His-(d-Nal)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH2 (HS014) bound to and colocalized with MC4R-GFP on the cell surface and did not stimulate receptor internalization. In sum, these results suggest that MC4R is subject to agonist-dependent endocytosis via clathrin-coated pits. Prolonged agonist exposure directs MC4R into lysosomes, possibly for degradation. Receptor and ligand recycling is not efficient for MC4R in HEK-293 cells.
authors.affiliation: Small Molecule Drug Discovery, Chiron Corporation, 4560 Horton St., Emeryville, CA 94608-2916, USA.
authors.forename: Zhenhai
authors.initials: Z
authors.lastname: Gao
authors.forename: Dachuan
authors.initials: D
authors.lastname: Lei
authors.forename: Julie
authors.initials: J
authors.lastname: Welch
authors.forename: Kathy
authors.initials: K
authors.lastname: Le
authors.forename: Jie
authors.initials: J
authors.lastname: Lin
authors.forename: Song
authors.initials: S
authors.lastname: Leng
authors.forename: David
authors.initials: D
authors.lastname: Duhl
mesh_terms: D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism*; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D009074:Melanocyte-Stimulating Hormones / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D011869:Radioligand Assay; D044105:Receptor, Melanocortin, Type 4 / Q000819:agonists* / Q000037:antagonists & inhibitors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013449:Sulfonamides
chemical_list: D004791:Enzyme Inhibitors; D007546:Isoquinolines; D008024:Ligands; D008164:Luminescent Proteins; D044105:Receptor, Melanocortin, Type 4; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D049452:Green Fluorescent Proteins; C103272:SHU 9119; D009074:Melanocyte-Stimulating Hormones; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide