Asn‐linked N–acetylglucosamine of the amylin receptor 2 extracellular domain enhances peptide ligand affinity

The calcitonin receptor (CTR) has a large extracellular domain (ECD) with multiple N‐glycosylation sites. An asparagine (Asn)‐linked N‐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 N‐glycosylation of AMY ECD was reported to enhance peptide hormone affinity, it remains underexplored which N‐glycosites of AMY ECD are responsible for peptide affinity enhancement and it is unclear whether an Asn‐linked GlcNAc of the N‐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(2) ECD (the RAMP2 ECD:CTR ECD complex). I used Endo H‐treated CTR ECD in which N‐glycans were trimmed to an Asn‐linked GlcNAc on each of the N‐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 N‐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(2) ECD and reveals a significant role of the Asn‐linked GlcNAc in AMY(2) function.