Role of N‐linked glycosylation on ligand binding and cellular expression of hMT1 and hMT2 melatonin receptors

• Published in: The FASEB journal 2006-03, Vol.20 (4), p.A250-A250
• Main Authors: Govorovska, Kristina A., Markowska, Magdalena, Kai, Li, Pazin, Marina, Gerdin, Matthew J., Masana, Monica I., Dubocovich, Margarita L.
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.20.4.A250-a

Melatonin (MLT) activates two pharmacologically distinct G‐protein‐coupled receptors, the hMT1 and hMT2. This study investigated the role of putative N‐linked glycosylation sites on ligand affinity and trafficking of hMT1 and hMT2 MLT receptors. Tunicamycin significantly decreased specific 2‐[125I]‐iodomelatonin binding to membranes from HEK293 cells transiently expressing either hMT1 or hMT2 receptors. The density of 2‐[125I]‐iodomelatonin binding was significantly decreased in mutants of all putative N‐linked glycosylation sites [hMT1N4A (61%), hMT1N10A (49%), hMT1N4N10A (40%), hMT2N4A (57%)] with no changes in binding affinity when compared to the wild types [hMT1: KD: 45 ± 5.7 pM; Bmax: 26.2 + 3.4 pmol/mg protein (n= 4); hMT2: KD: 57.1 ± 8.8 pM; Bmax: 4.2 + 9.1 pmol/mg protein (n= 4)]. Cellular distribution of Flag‐tagged wild type and mutant MLT receptors was determined by confocal microscopy. hMT1 WT and hMT1 N4A receptors localized primarily to cell membranes, while hMT1N10A and hMT1N4N10A localized to the cytoplasm. The pattern of cellular distribution of the hMT2N4A was similar to its parent wild type hMT2 receptor. Functional studies of point‐mutated receptors will be reported. These results suggest that N‐linked glycosylation of the hMT1 and hMT2 is important in the regulation of cellular expression and trafficking of melatonin receptors. Supported by MH 42922 (MLD), Polish Sci. Fellowship (MM), ASPET SURF (KG), F31 MH67320 (MJG).