Glycosylation of human CRLR at Asn123 is required for ligand binding and signaling.
Calcitonin receptor-like receptor (CRLR) constitutes either a CGRP receptor when complexed with receptor activity-modifying protein 1 (RAMP1) or an adrenomedullin receptor when complexed with RAMP2 or RAMP3. RAMP proteins modify the glycosylation status of CRLR and determine their receptor specificity; when treated with tunicamycin, a glycosylation inhibitor, CHO-K1 cells constitutively expressing both RAMP2 and CRLR lost the capacity to bind adrenomedullin. Similarly, in HEK293 EBNA cells constitutively expressing RAMP1/CRLR receptor complex CGRP binding was remarkably inhibited. Whichever RAMP protein was co-expressing with CRLR, the ligand binding was sensitive to tunicamycin. There are three putative Asn-linked glycosylation sites in the extracellular, amino terminal domain of CRLR at positions 66, 118 and 123. Analysis of CRLR mutants in which Gln was substituted for selected Asn residues showed that glycosylation of Asn123 is required for both the binding of adrenomedullin and the transduction of its signal. Substituting Asn66 or Asn118 had no effect. FACS analysis of cells expressing FLAG-tagged CRLRs showed that disrupting Asn-linked glycosylation severely affected the transport of the CRLR protein to the cell surface on N66/118/123Q mutant, and slightly reduced the level of the cell surface expression of N123Q mutant compared with wild-type CRLR. But other single mutants (N66Q, N118Q) had no effect for other single mutants. Our data shows that glycosylation of Asn66 and Asn118 is not essential for ligand binding, signal transduction and cell surface expression, and Asn123 is important for ligand binding and signal transduction rather than cell surface expression. It thus appears that glycosylation of Asn123 is required for CRLR to assume the appropriate conformation on the cell surface through its interaction with RAMPs.
Kamitani
S
S
Shionogi Research Laboratories, Shionogi and Co. Ltd., 2-5-1 Mishima, Settsu-shi, 566-0022, Osaka, Japan.
Sakata
T
T
D053607:Adrenomedullin; D019943:Amino Acid Substitution; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D058286:Calcitonin Receptor-Like Protein; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins / Q000235:genetics / Q000494:pharmacology; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; C545502:RAMP1 protein, human; C545503:RAMP2 protein, human; C545504:RAMP3 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin; D018000:Receptors, Peptide; D014415:Tunicamycin; D053607:Adrenomedullin; D000242:Cyclic AMP; D015740:Calcitonin Gene-Related Peptide