N-Glycosylation of Asparagine 130 in the Extracellular Domain of the Human Calcitonin Receptor Significantly Increases Peptide Hormone Affinity

Abstract

The calcitonin receptor (CTR) is a class B G protein-coupled receptor that is activated by the peptide hormones calcitonin and amylin. Calcitonin regulates bone remodeling through CTR, whereas amylin regulates blood glucose and food intake by activating CTR in complex with receptor activity-modifying proteins (RAMPs). These receptors are targeted clinically for treatment of osteoporosis and diabetes. Here, we define the role of CTR N-glycosylation in hormone binding using purified calcitonin and amylin receptor extracellular domain (ECD) glycoforms and fluorescence polarization/anisotropy and isothermal titration calorimetry peptide-binding assays. N-glycan-free CTR ECD produced in Escherichia coli exhibited ~10-fold lower peptide affinity than CTR ECD produced in HEK293T cells, which yield complex N- glycans, or in HEK293S GnTI− cells, which yield core N-glycans (Man5GlcNAc2). PNGase F- catalyzed removal of N-glycans at N73, N125, and N130 in the CTR ECD decreased peptide affinity ~10-fold, whereas Endo H-catalyzed trimming of the N-glycans to single GlcNAc residues had no effect on peptide binding. Similar results were observed for an amylin receptor RAMP2-CTR ECD complex. Characterization of peptide-binding affinities of purified N→Q CTR ECD glycan site mutants combined with PNGase F and Endo H treatment strategies and mass spectrometry to define the glycan species indicated that a single GlcNAc residue at CTR N130 was responsible for the peptide affinity enhancement. Molecular modeling suggested that this GlcNAc functions through an allosteric mechanism rather than by directly contacting the peptide. These results reveal an important role for N-linked glycosylation in the peptide hormone binding of a clinically relevant class B GPCR.

Probing the Mechanism of Receptor Activity–Modifying Protein Modulation of GPCR Ligand Selectivity through Rational Design of Potent Adrenomedullin and Calcitonin Gene-Related Peptide Antagonists

“…The competition AlphaLISA assay is ill suited for determining accurate equilibrium constants, so we developed a fluorescence polarization/anisotropy peptide-binding assay similar to one we recently reported for the calcitonin receptor ECD (Lee et al, 2017) to enable a more thorough quantitative analysis of the binding of AM and CGRP variants to the purified tethered RAMP1/2-CLR ECD complexes (Supplemental Fig. S1A).…”

“…The orientation of the ECD complex with respect to the remainder of the receptor within the full-length complexes is unknown, and portions of the membraneembedded domains may also contact the C-terminal "ECDbinding" half of the peptides . In addition, the ECD complexes were expressed in E. coli so they are not N-glycosylated, which might affect peptide binding as for the related calcitonin receptor (Lee et al, 2017;Liang et al, 2017). Despite some disagreement in the degree of receptor selectivity, the relative activities of the AM/CGRP variants were reasonably consistent across the two assays.…”

“…Fluorescence Polarization/Anisotropy Peptide-Binding Assay. This assay was performed as previously described (Lee et al, 2017), except using FITC-Ahx-AM(37-52) S45W/Q50W as the probe, and Tween-20 was omitted from the reaction buffer. Saturation binding used 7 nM FITC-Ahx-AM(37-52) S45W/Q50W and the indicated concentrations of MBP-RAMP1/2 ECD-(GS) 5 -CLR ECD fusion proteins with a 1-hour incubation at room temperature.…”

“…Anisotropy values were corrected for the slight fluorescence intensity enhancement observed upon receptor binding (Supplemental Fig. S1E) as previously described (Lee et al, 2017). Competition binding assays used 7 nM FITC-peptide and 15 nM MBP-RAMP1 ECD-(GS) 5 -CLR ECD or 110 nM MBP-RAMP2 ECD-(GS) 5 -CLR ECD with the indicated concentrations of competitor peptides and a 2-hour incubation at room temperature.…”

“…Anisotropy values were not corrected for the slight decrease in total fluorescence intensity observed upon probe displacement because determination of the competitor K I value is insensitive to minor intensity changes (Roehrl et al, 2004). Equilibrium dissociation constants were determined for the labeled probe from the saturation experiments (log K D ) and for the unlabeled peptides from competition experiments (log K I ) by nonlinear regression fitting of the binding curves to the exact analytical equations of Roehrl et al (2004) using user-defined equations in GraphPad Prism version 5.0f as previously described (Lee et al, 2017). Roehrl et al (2004) eqs.…”