Assistant Professor High Point University, Fred Wilson School of Pharmacy High Point, North Carolina, United States
Sangmin Lee (High Point University Fred Wilson School of Pharmacy)| Augen Pioszak (University of Oklahoma Health Sciences Center)
Amylin is a peptide hormone and its activation of amylin receptors controls blood glucose and food intake. Amylin receptor activation is a known drug target for diabetes and has potential for obesity treatment. The amylin receptor consists of the calcitonin receptor (CTR) and the accessary protein called receptor activity-modifying protein (RAMP). CTR is the receptor for peptide hormone calcitonin, whereas the heterodimer of CTR and RAMP gains affinity for peptide hormone amylin. Each of the CTR and RAMP has an extracellular domain (ECD), and CTR ECD is a known binding site for peptide hormones. However, whether RAMP ECD provides a binding site for peptide ligands remains elusive. RAMP has three types in humans and our structural model of the amylin receptor 2 ECD (RAMP2 ECD:CTR ECD complex) suggested the molecular interaction between RAMP2 ECD Glu101 and the C-terminal Tyr residue of an amylin analog AC413. We examined whether this molecular interaction contributes to AC413 binding for amylin receptor 2 ECD and enhances AC413 affinity. In addition, N-glycosylation of the amylin receptor ECD is known to increase the peptide ligand affinity. Despite, which N-glycosite is the most responsible for this effect and the role of the Asn-linked N-acetylglucosamine (GlcNAc) have been unexplored for amylin receptor 2 ECD. Based on our previous reports, we hypothesized that Asn-linked GlcNAc of CTR ECD Asn130 will enhance the peptide ligand affinity for amylin receptor 2 ECD. Here, we established the functional amylin receptor 2 ECD and applied mutagenesis and enzymatic deglycosylation to address the affinity enhancers. We used fluorescence polarization/anisotropy peptide binding assay to measure peptide ligand affinity for purified amylin receptor 2 ECD. Our results showed that the RAMP2 ECD Glu101 to alanine mutation significantly decreased the amylin analog affinity suggesting its potential interaction with the C-terminal Tyr residue of the amylin analog. Using glycan-trimmed CTR ECD, we showed that the Asn-linked GlcNAc residue of CTR ECD Asn130 is the most responsible among N-glycans for enhancing affinity for the amylin receptor 2 ECD. This study provides evidence that both RAMP2 ECD Glu101 and Asn-linked GlcNAc of CTR ECD Asn130 contribute to amylin analog binding as affinity enhancers.