Article: article from journal or magazin.
N-Glycosylation and conserved cysteine residues in RAMP3 play a critical role for the functional expression of CRLR/RAMP3 adrenomedullin receptor.
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.
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, 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, Xenopus
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