Glycosylation of bombesin receptors: characterization, effect on binding, and G-protein coupling.
In the present study, we investigated the nature and the importance of glycosylation of two mammalian bombesin receptors, the neuromedin B receptor (NMB-R) and the gastrin-releasing peptide receptor (GRP-R), using chemical cross-linking and enzymatic deglycosylation. [125I]-(D-Tyr0)NMB cross-linked to native NMB-R on rat C-6 glioblastoma cells or rat NMB-R transfected into BALB 3T3 cells revealed a single broad band, M(r) = 63,000, on both cell types that was not altered by DTT. NMB inhibited cross-linking specifically and saturably with an IC50 of 4.8 and 6.1 nM for C-6 and NMB-R transfected cells, respectively, and there was a close correlation between its ability to inhibit binding and its ability to inhibit cross-linking. A single broad band of M(r) = 82,000 was cross-linked with [125I]GRP on mouse GRP-R transfected BALB 3T3 cells. Peptide-N4-(N-acetyl-beta- glucosaminyl)asparagine amidase F (PNGase F) digestion increased the mobility of the original band in C-6, NMB-R, and GRP-R transfected cell membranes. Endoglycosidase H (Endo-H) and endoglycosidase F2 (Endo-F2) digestion had no effect on both transfected cells. Neuraminidase digestion slightly increased the mobility of the original band in NMB-R transfected cell membranes; however, it had no effect on GRP-R transfected cell membranes. Endo-alpha-N-acetylglucosaminidase (O-glycanase) digestion subsequent to neuraminidase treatment showed no additional effect on either receptor. Serial partial deglycosylation of cross-linked NMB-Rs with PNGase F treatment for different incubation periods revealed one band of partially glycosylated receptor (53 kDa) besides the fully glycosylated and fully deglycosylated ones, showing that NMB-R has two oligosaccharide chains. Similarly, three partially deglycosylated species (72, 62, and 52 kDa) are seen with the GRP-R, indicating that the GRP-R has four oligosaccharide chains. Treatment of unlabeled membranes with PNGase F followed by affinity labeling resulted in fully deglycosylated NMB-R or 75% deglycosylated GRP-R. Deglycosylation of the NMB-R did not alter its affinity for NMB or alter G-protein coupling; however, 75% deglycosylation of the GRP-R both decreased its affinity for GRP and altered its ability to couple to G-proteins. The present results demonstrate that NMB-R on native and transfected cells is an N-linked sialoglycoprotein with two triantenary and/or tetraantenary complex oligosaccharide chains. The apparent M(r) of this sialoglycoprotein is 63,000, and this protein does not contain disulfide-linked subunits or O-linked carbohydrates.(ABSTRACT TRUNCATED AT 400 WORDS)
Kusui
T
T
Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Benya
R V
RV
Battey
J F
JF
Jensen
R T
RT
D016475:3T3 Cells / Q000187:drug effects / Q000378:metabolism; D000581:Amidohydrolases / Q000494:pharmacology; D000818:Animals; D003432:Cross-Linking Reagents; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005909:Glioblastoma / Q000473:pathology; D006031:Glycosylation; D006596:Hexosaminidases / Q000494:pharmacology; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008565:Membrane Proteins / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D051381:Rats; D018004:Receptors, Bombesin / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D018004:Receptors, Bombesin; D006596:Hexosaminidases; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019204:GTP-Binding Proteins