Functional role of N-linked glycosylation on the rat melanin-concentrating hormone receptor 1

• Published in: FEBS letters 2003-01, Vol.533 (1), p.29-34
• Main Authors: Saito, Yumiko, Tetsuka, Mitsue, Yue, Li, Kawamura, Yuuki, Maruyama, Kei
• Format: Article
• Language: English
• Subjects: Animals; Asparagine - chemistry; Binding Sites; Calcium influx; Calcium Signaling; Cell Line; DMEM, Dulbecco's modified Eagle's medium; Endo H, endoglycosidase H; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; Flag-MCHR1, amino-terminal Flag-tagged MCHR1; G-protein-coupled receptor; Glycosylation; GPCR, G-protein-coupled receptor; Humans; Ligand binding; Ligands; MCH, melanin-concentrating hormone; MCHR1, melanin-concentrating hormone receptor 1; Melanin-concentrating hormone; Mutagenesis, Site-Directed; N-glycosylation site; PBS, phosphate-buffered saline; PNGase F, peptide N-glycosidase F; Rats; Receptors, Pituitary Hormone - chemistry; Receptors, Pituitary Hormone - genetics; Receptors, Pituitary Hormone - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(02)03744-4

Melanin-concentrating hormone (MCH) is known to act through two G-protein-coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn 13, Asn 16 and Asn 23) for N-linked glycosylation in its extracellular amino-terminus which may modulate its reactivity. Site-directed mutagenesis of the rat MCHR1 cDNA at single or multiple combinations of the three potential glycosylation sites was used to examine the role of the putative carbohydrate chains on receptor activity. It was found that all three potential N-linked glycosylation sites in MCHR1 were glycosylated, and that N-linked glycosylation of Asn 23 was necessary for full activity. Furthermore, disruption of all three glycosylation sites impaired proper expression at the cell surface and receptor activity. These data outline the importance of the N-linked glycosylation of the MCHR1.