Structural and functional analysis of the human vasoactive intestinal peptide receptor glycosylation. Alteration of receptor function by wheat germ agglutinin.
The vasoactive intestinal peptide (VIP) receptor from the human melanoma cell line IGR39 has been shown to be a 60-kDa glycoprotein. Using serial lectin affinity chromatography, as well as specific glycosidases, we demonstrate that VIP receptor-linked carbohydrates are predominantly tri- or tetraantennary sialylated N-linked oligosaccharides, 27% of which are fucosylated, and some may have terminal galactose residues. Treatment of 125I-VIP receptor complexes with peptide-N4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase revealed the presence of at least three N-linked carbohydrate chains/receptor polypeptide. To investigate the functional role of the carbohydrate moiety, 125I-VIP binding to IGR39 cell membranes was tested in the presence of soluble lectins. Among the lectins tested, only wheat germ agglutinin (WGA) was found to markedly inhibit VIP binding in a dose-dependent manner. Binding data indicated that the presence of the lectin led to a 3-fold increase in Kd value, from 0.15 to 0.44 nM, without any change in the number of available binding sites. The potent inhibitor of WGA binding, N,N',N"-triacetylchitotriose, completely reversed the effect of the lectin. On the other hand, VIP binding inhibition persisted even after neuraminidase treatment, suggesting that sialic acids were not directly involved. Furthermore, WGA inhibition was not abolished although most, if not all, VIP receptor oligosaccharides were converted to high mannose type structures by treating IGR39 cells with deoxymannojirimycin. Finally, whereas the pharmacological profile of VIP receptor was virtually identical, the presence of WGA greatly reduced the VIP-stimulated cAMP in IGR39 cells, indicating that the lectin alters the ability of the receptor to interact with the adenylate cyclase system.
Chochola
J
J
Centre National de la Recherche Scientifique URA 202, Université d'Aix-Marseille, France.
Fabre
C
C
Bellan
C
C
Luis
J
J
Bourgerie
S
S
Abadie
B
B
Champion
S
S
Marvaldi
J
J
el Battari
A
A
D001667:Binding, Competitive; D050260:Carbohydrate Metabolism; D002846:Chromatography, Affinity; D005576:Colforsin / Q000494:pharmacology; D003208:Concanavalin A / Q000737:chemistry; D000242:Cyclic AMP / Q000378:metabolism; D006031:Glycosylation; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D011499:Protein Processing, Post-Translational; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*; D014909:Wheat Germ Agglutinins / Q000737:chemistry
D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D014909:Wheat Germ Agglutinins; D003208:Concanavalin A; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP