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 r ole in receptor transduction or receptor modulation (internalization, down- regulation, desensitization) is largely unknown. We used site-directed muta genesis to address these issues with the gastrin-releasing peptide receptor (GRP-R). Each of the four potential glycosylation sites was mutated by con verting the Asn (N) to Gln (Q). Transient expression in CHOP cells demonstr ated that changing Asn(24) or Asn(191) inhibited GRP-R cell surface express ion, whereas elimination of Asn(5) and Asn(20) had no effect. Using ligand crosslinking studies in stable mutants expressed in Balb 3T3 cells, all fou r potential extracellular sites were glycosylated with carbohydrate residue s of approximately 13 kDa on Asn(5), 10 kDa on Asn(20), 5 kDa on Asn(24), a nd 9 kDa on Asn(191). Removal of three glycosylation sites (N5,20,24, Q mut ant) did not alter receptor affinity or G protein coupling; therefore, it c ould be speculated that deglycosylation at Asn(191) might be responsible fo r the altered G protein coupling seen with complete enzymatic deglycosylati on of the native receptor previously reported. Removal of any single glycos ylation site did not interfere with GRP-R induced chronic desensitization o r down-regulation. However, elimination of all three NH2-terminal sites (N5 ,20,24) markedly attenuated both processes, with no effect on acute homolog ous desensitization and with only a minimal alteration of GRP-R internaliza tion, supporting the findings of other studies that suggest that chronic de sensitization and down-regulation are functionally coupled, distinct from a cute desensitization and distinct from internalization. These data show tha t separate and specific glycosylation sites are important for GRP-R traffic king to the cell surface, ligand binding, G protein coupling, chronic desen sitization, and down-regulation.