PMID: 11595658

 

    Legend: Sugar

Title : N-glycosylation of CRF receptor type 1 is important for its ligand-specific interaction

Abstract :

1. The corticotropin-releasing factor (CRF) receptor type 1 ( CRFR1 ) contains five potential N-glycosylation sites : N38, N45, N78, N90, and N98
2. Cells expressing CRFR1 were treated with tunicamycin to block receptor glycosylation
3. The nonglycosylated receptor did not bind the radioligand and had a decreased cAMP stimulation potency in response to CRF
4. To determine which of the polysaccharide chain(s) is/are involved in ligand interaction, the polysaccharide chains were deleted using site-directed mutagenesis of the glycosylation consensus, N-X-S/T
5. Two sets of mutations were performed for each glycosylation site : N to Q and S/T to A, respectively
6. The single mutants Q38, Q45, Q78, Q90, Q98, A40, A47, A80, A92, and A100 and the double mutants A40/A47 and A80/A100 were well expressed, bound CRF , sauvagine (SVG), and urotensin-I (UTS-I) with a normal affinity, and increased cAMP accumulation with a high efficiency
7. In contrast, the combined mutations A80/A92/A100, A40/A80/A92/A100, and A40/A47/A80/A92/A100 had low levels of expression, did not bind the radioligand, and had a decreased cAMP stimulation
8. These data indicate the requirement for three or more polysaccharide chains for normal CRFR1 function