N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor

Biochemistry. 2003 Sep 2;42(34):10333-41. doi: 10.1021/bi0347508.


The calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein-3 (RAMP3) can assemble into a CRLR/RAMP3 heterodimeric receptor that exhibits the characteristics of a high affinity adrenomedullin receptor. RAMP3 participates in adrenomedullin (AM) binding via its extracellular N-terminus characterized by the presence of six highly conserved cysteine residues and four N-glycosylation consensus sites. Here, we assessed the usage of these conserved residues in cotranslational modifications of RAMP3 and addressed their role in functional expression of the CRLR/RAMP3 receptor. Using a Xenopus oocyte expression system, we show that (i) RAMP3 is assembled with CRLR as a multiple N-glycosylated species in which two, three, or four consensus sites are used; (ii) elimination of all N-glycans in RAMP3 results in a significant inhibition of receptor [(125)I]AM binding and an increase in the EC(50) value for AM; (iii) several lines of indirect evidence indicate that each of the six cysteines is involved in disulfide bond formation; (iv) when all cysteines are mutated to serines, RAMP3 is N-glycosylated at all four consensus sites, suggesting that disulfide bond formation inhibits N-gylcosylation; and (v) elimination of all cysteines abolishes adrenomedullin binding and leads to a complete loss of receptor function. Our data demonstrate that cotranslational modifications of RAMP3 play a critical role in the function of the CRLR/RAMP3 adrenomedullin receptor.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adrenomedullin
  • Amino Acid Substitution
  • Animals
  • Binding Sites
  • Calcitonin Gene-Related Peptide / metabolism
  • Calcitonin Receptor-Like Protein
  • Consensus Sequence
  • Cysteine / genetics*
  • Cysteine / metabolism*
  • Disulfides / metabolism
  • Glycosylation
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Oocytes / metabolism
  • Peptides / metabolism
  • Polysaccharides / chemistry
  • Protein Binding
  • Protein Structure, Tertiary
  • Radioligand Assay
  • Receptor Activity-Modifying Proteins
  • Receptors, Adrenomedullin
  • Receptors, Calcitonin / chemistry
  • Receptors, Calcitonin / metabolism*
  • Receptors, Peptide / chemistry
  • Receptors, Peptide / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Xenopus


  • Calcitonin Receptor-Like Protein
  • Disulfides
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Peptides
  • Polysaccharides
  • Receptor Activity-Modifying Proteins
  • Receptors, Adrenomedullin
  • Receptors, Calcitonin
  • Receptors, Peptide
  • Recombinant Proteins
  • Adrenomedullin
  • Calcitonin Gene-Related Peptide
  • Cysteine