N-glycosylation of CRF receptor type 1 is important for its ligand-specific interaction.
The corticotropin-releasing factor (CRF) receptor type 1 (CRFR1) contains five potential N-glycosylation sites: N38, N45, N78, N90, and N98. Cells expressing CRFR1 were treated with tunicamycin to block receptor glycosylation. The nonglycosylated receptor did not bind the radioligand and had a decreased cAMP stimulation potency in response to CRF. 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. Two sets of mutations were performed for each glycosylation site: N to Q and S/T to A, respectively. 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. 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. These data indicate the requirement for three or more polysaccharide chains for normal CRFR1 function.
Assil
I Q
IQ
Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
Abou-Samra
A B
AB
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D029845:Amphibian Proteins; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D016384:Consensus Sequence; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008024:Ligands; D051379:Mice; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D036361:Peptide Hormones; D010455:Peptides / Q000378:metabolism; D011869:Radioligand Assay; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014415:Tunicamycin / Q000494:pharmacology; D014579:Urotensins / Q000378:metabolism
D029845:Amphibian Proteins; D008024:Ligands; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D014579:Urotensins; D014415:Tunicamycin; C025727:sauvagine; D003346:Corticotropin-Releasing Hormone; C029676:urotensin I; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine