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

Biochemistry. 2001 May 8;40(18):5392-8. doi: 10.1021/bi002497v.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution / genetics
  • Animals
  • Asparagine / genetics*
  • COS Cells
  • Calcitonin Receptor-Like Protein
  • Cell Line, Transformed
  • Cell Membrane / genetics
  • Cell Membrane / metabolism
  • Cross-Linking Reagents / metabolism
  • Cyclic AMP / biosynthesis
  • Genetic Vectors
  • Glycosylation
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Iodine Radioisotopes
  • Membrane Proteins / biosynthesis
  • Membrane Proteins / genetics
  • Membrane Proteins / physiology*
  • Mutagenesis, Site-Directed*
  • Precipitin Tests
  • Protein Binding / genetics
  • Proto-Oncogene Proteins c-myc / genetics
  • Receptor Activity-Modifying Protein 1
  • Receptor Activity-Modifying Proteins
  • Receptors, Calcitonin / biosynthesis
  • Receptors, Calcitonin / genetics
  • Receptors, Calcitonin / metabolism
  • Receptors, Calcitonin Gene-Related Peptide / deficiency
  • Receptors, Calcitonin Gene-Related Peptide / genetics*
  • Receptors, Calcitonin Gene-Related Peptide / metabolism*
  • Recombinant Proteins / biosynthesis
  • Transfection

Substances

  • CALCRL protein, human
  • Calcitonin Receptor-Like Protein
  • Cross-Linking Reagents
  • Intracellular Signaling Peptides and Proteins
  • Iodine Radioisotopes
  • Membrane Proteins
  • Proto-Oncogene Proteins c-myc
  • RAMP1 protein, human
  • Receptor Activity-Modifying Protein 1
  • Receptor Activity-Modifying Proteins
  • Receptors, Calcitonin
  • Receptors, Calcitonin Gene-Related Peptide
  • Recombinant Proteins
  • Asparagine
  • Cyclic AMP