Mutations of the asparagine117 residue of a receptor activity-modifying protein 1-dependent human calcitonin gene-related peptide receptor result in selective loss of function.
The initially orphan human calcitonin (CT) receptor-like receptor (hCRLR) interacts with novel accessory receptor activity-modifying protein 1 (RAMP1) to reveal a functional CT gene-related peptide (CGRP) receptor. In mammalian cells, RAMP1 is required for mature N-glycosylation of the hCRLR predicted to occur at Asn(60), Asn(112), and/or Asn(117) in the amino-terminal extracellular domain. Here we have shown that the substitution of Asn(117) with Ala, Gln, Thr, or Pro abolished CGRP-evoked cAMP formation which was left unchanged when the Asn(117) was replaced with Asp. Moreover, the hCRLR and the Asn(117) mutants exhibited comparable N-glycosylation and cell surface expression, and the association with RAMP1 was only slightly impaired. In contrast, the hCRLR Asn(60,112) to Thr double mutant exhibited defective RAMP1-dependent N-glycosylation, and impaired cell surface expression and CGRP receptor function. Unlike Asn(60) and Asn(112), Asn(117) is normally not N-glycosylated, but essential for CGRP binding to the hCRLR-RAMP1 complex.
Gujer
R
R
Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery and Medicine, University of Zurich, Klinik Balgrist, 8008 Zurich, Switzerland.
Aldecoa
A
A
Bühlmann
N
N
Leuthäuser
K
K
Muff
R
R
Fischer
J A
JA
Born
W
W
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001216:Asparagine / Q000235:genetics*; D019556:COS Cells; D058286:Calcitonin Receptor-Like Protein; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000378:metabolism; D000242:Cyclic AMP / Q000096:biosynthesis; D005822:Genetic Vectors; D006031:Glycosylation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection
C545578:CALCRL protein, human; D058286:Calcitonin Receptor-Like Protein; D003432:Cross-Linking Reagents; D047908:Intracellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D016271:Proto-Oncogene Proteins c-myc; C545502:RAMP1 protein, human; D058262:Receptor Activity-Modifying Protein 1; D058261:Receptor Activity-Modifying Proteins; D018003:Receptors, Calcitonin; D018015:Receptors, Calcitonin Gene-Related Peptide; D011994:Recombinant Proteins; D001216:Asparagine; D000242:Cyclic AMP