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]-α-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.
Footnotes
-
DOI: 10.1124/jpet.103.055525.
-
ABBREVIATIONS: MSH, melanocyte-stimulating hormone; MCR, melanocortin receptor; GPCR, G protein-coupled receptor; GFP, green fluorescent protein; HS014, Ac-Cys-Glu-His-(d-Nal)-Arg-Trp-Gly-Cys-Pro-Pro-Lys-Asp-NH2; MTII, [Ac-Nle-c[Asp-His-d-Phe-Arg-Trp-Lys]-NH2]; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide; SHU9119, [Ac-Nle-c[Asp-His-d-Nal(2′)-Arg-Trp-Lys]-NH2]; NDP-MSH, [Nle4-d-Phe7]-α-melanocyte-stimulating hormone; PBS, phosphate-buffered saline; PKA, protein kinase A; HA-hMC4R, hemagglutinin-tagged human MC4R.
- Received June 9, 2003.
- Accepted August 21, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
JPET articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|