| Abstrakt: |
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. |
