Abstract
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 Asn24 or Asn191 inhibited GRP-R cell surface expression, whereas elimination of Asn5 and Asn20 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 Asn5, 10 kDa on Asn20, 5 kDa on Asn24, and 9 kDa on Asn191. 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 Asn191 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 NH2-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.
Footnotes
- Received April 28, 2000.
- Accepted August 17, 2000.
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Send reprint requests to: Dr. Robert T. Jensen, NIH/NIDDK/DDB, Bldg. 10/9C-103, 10 Center Dr. MSC 1804, Bethesda, MD 20892-1804. E-mail: robertj{at}bdg10.niddk.nih.gov
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This work was supported partially by National Institutes of Health Grant DK51168 and a Veterans Affairs Merit Review (to R.V.B.).
- The American Society for Pharmacology and Experimental Therapeutics
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