Beschreibung:
<jats:p>The calcitonin receptor (CTR) has a large extracellular domain (ECD) with multiple <jats:italic>N</jats:italic>‐glycosylation sites. An asparagine (Asn)‐linked <jats:italic>N</jats:italic>‐acetylglucosamine (GlcNAc) of CTR ECD N130 was previously reported to enhance peptide hormone binding affinity for CTR ECD. CTR forms a complex with an accessory protein RAMP, and the RAMP:CTR complex gains affinity for peptide hormone amylin as the amylin receptor (AMY). Although <jats:italic>N</jats:italic>‐glycosylation of AMY ECD was reported to enhance peptide hormone affinity, it remains underexplored which <jats:italic>N</jats:italic>‐glycosites of AMY ECD are responsible for peptide affinity enhancement and it is unclear whether an Asn‐linked GlcNAc of the <jats:italic>N</jats:italic>‐glycosites plays a critical role. Here, I investigated the role of the Asn‐linked GlcNAc of CTR N130 in the affinity of an antagonistic amylin analog (AC413) for AMY<jats:sub>2</jats:sub> ECD (the RAMP2 ECD:CTR ECD complex). I used Endo H‐treated CTR ECD in which <jats:italic>N</jats:italic>‐glycans were trimmed to an Asn‐linked GlcNAc on each of the <jats:italic>N</jats:italic>‐glycosites. I incubated Endo H‐treated CTR ECD with excess of glycan‐free RAMP2 ECD to produce the RAMP2 ECD:CTR ECD complex. Using this coincubation system, I found that the RAMP2 ECD complex with Endo H‐treated CTR ECD with N130D mutation showed a fourfold decrease in AC413 affinity compared with the RAMP2 ECD complex with Endo H‐treated CTR ECD WT. In contrast, RAMP2 ECD <jats:italic>N</jats:italic>‐glycosylation did not affect peptide binding affinity. These results indicate that the Asn‐linked GlcNAc of CTR N130 is an important peptide affinity enhancer for AMY<jats:sub>2</jats:sub> ECD and reveals a significant role of the Asn‐linked GlcNAc in AMY<jats:sub>2</jats:sub> function.</jats:p>