Glycosylation of bombesin receptors: characterization, effect on binding, and G-protein coupling

Biochemistry. 1994 Nov 8;33(44):12968-80. doi: 10.1021/bi00248a005.


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)

MeSH terms

  • 3T3 Cells / drug effects
  • 3T3 Cells / metabolism
  • Amidohydrolases / pharmacology
  • Animals
  • Cross-Linking Reagents
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • GTP-Binding Proteins / metabolism*
  • Glioblastoma / pathology
  • Glycosylation
  • Hexosaminidases / pharmacology
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
  • Membrane Proteins / drug effects
  • Mice
  • Mice, Inbred BALB C
  • Molecular Weight
  • Neuraminidase / pharmacology
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Protein Binding / drug effects
  • Protein Binding / physiology
  • Rats
  • Receptors, Bombesin / chemistry*
  • Receptors, Bombesin / metabolism
  • Transfection
  • Tumor Cells, Cultured


  • Cross-Linking Reagents
  • Membrane Proteins
  • Receptors, Bombesin
  • Hexosaminidases
  • Neuraminidase
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
  • glycopeptide alpha-N-acetylgalactosaminidase
  • Amidohydrolases
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • GTP-Binding Proteins