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.
Flahaut
Marjorie
M
Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland.
Pfister
Corinne
C
Rossier
Bernard C
BC
Firsov
Dmitri
D
D053607:Adrenomedullin; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D015740:Calcitonin Gene-Related Peptide / Q000378:metabolism; D058286:Calcitonin Receptor-Like Protein; D016384:Consensus Sequence; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D006031:Glycosylation; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin / Q000737:chemistry / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014981:Xenopus
D058286:Calcitonin Receptor-Like Protein; D004220:Disulfides; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D011134:Polysaccharides; D058261:Receptor Activity-Modifying Proteins; D058265:Receptors, Adrenomedullin; D018003:Receptors, Calcitonin; D018000:Receptors, Peptide; D011994:Recombinant Proteins; D053607:Adrenomedullin; D015740:Calcitonin Gene-Related Peptide; D003545:Cysteine