Glycosylation of the gastrin-releasing peptide receptor and its effect on expression, G protein coupling, and receptor modulatory processes.
Many gastrointestinal G protein-coupled receptors are glycosylated; however, which potential glycosylation sites are actually glycosylated and their role in receptor transduction or receptor modulation (internalization, down-regulation, desensitization) is largely unknown. We used site-directed mutagenesis to address these issues with the gastrin-releasing peptide receptor (GRP-R). Each of the four potential glycosylation sites was mutated by converting the Asn (N) to Gln (Q). Transient expression in CHOP cells demonstrated that changing Asn(24) or Asn(191) inhibited GRP-R cell surface expression, whereas elimination of Asn(5) and Asn(20) had no effect. Using ligand cross-linking studies in stable mutants expressed in Balb 3T3 cells, all four potential extracellular sites were glycosylated with carbohydrate residues of approximately 13 kDa on Asn(5), 10 kDa on Asn(20), 5 kDa on Asn(24), and 9 kDa on Asn(191). Removal of three glycosylation sites (N5,20,24,Q mutant) did not alter receptor affinity or G protein coupling; therefore, it could be speculated that deglycosylation at Asn(191) might be responsible for the altered G protein coupling seen with complete enzymatic deglycosylation of the native receptor previously reported. Removal of any single glycosylation site did not interfere with GRP-R induced chronic desensitization or down-regulation. However, elimination of all three NH(2)-terminal sites (N5,20,24) markedly attenuated both processes, with no effect on acute homologous desensitization and with only a minimal alteration of GRP-R internalization, supporting the findings of other studies that suggest that chronic desensitization and down-regulation are functionally coupled, distinct from acute desensitization and distinct from internalization. These data show that separate and specific glycosylation sites are important for GRP-R trafficking to the cell surface, ligand binding, G protein coupling, chronic desensitization, and down-regulation.
Benya
R V
RV
Department of Medicine, University of Illinois School of Medicine, Chicago, Illinois, USA.
Kusui
T
T
Katsuno
T
T
Tsuda
T
T
Mantey
S A
SA
Battey
J F
JF
Jensen
R T
RT
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D016384:Consensus Sequence; D015536:Down-Regulation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006031:Glycosylation; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D018004:Receptors, Bombesin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018004:Receptors, Bombesin; D019204:GTP-Binding Proteins