Calcitonin and Amylin Receptor Peptide Interaction Mechanisms: INSIGHTS INTO PEPTIDE-BINDING MODES AND ALLOSTERIC MODULATION OF THE CALCITONIN RECEPTOR BY RECEPTOR ACTIVITY-MODIFYING PROTEINS

Abstract

Receptor activity-modifying proteins (RAMP1-3) determine the selectivity of the class B G protein-coupled calcitonin receptor (CTR) and the CTR-like receptor (CLR) for calcitonin (CT), amylin (Amy), calcitonin gene-related peptide (CGRP), and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 Trp-84 or RAMP2 Glu-101 contacting the distinct CGRP/AM C-terminal residues. It is unclear whether RAMPs use a similar mechanism to modulate CTR affinity for CT and Amy, analogs of which are therapeutics for bone disorders and diabetes, respectively. Here, we reproduced the peptide selectivity of intact CTR, AMY1 (CTR·RAMP1), and AMY2 (CTR·RAMP2) receptors using purified CTR extracellular domain (ECD) and tethered RAMP1- and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound salmon calcitonin (sCT). Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scanning mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TN(T/V)G motif prior to the C terminus. After this motif, the peptides diverged; the sCT C-terminal Pro was crucial for receptor binding, whereas the AC413/rAmy C-terminal Tyr had little or no influence on binding. Accordingly, mutant RAMP1 W84A- and RAMP2 E101A-CTR ECD retained AC413/rAmy binding. ECD binding and cell-based signaling assays with antagonist sCT/AC413/rAmy variants with C-terminal residue swaps indicated that the C-terminal sCT/rAmy residue identity affects affinity more than selectivity. rAmy(8-37) Y37P exhibited enhanced antagonism of AMY1 while retaining selectivity. These results reveal unexpected differences in how RAMPs determine CTR and CLR peptide selectivity and support the hypothesis that RAMPs allosterically modulate CTR peptide affinity.

Keywords: G protein-coupled receptor (GPCR); RAMP; allosteric regulation; cell surface receptor; glycoprotein; mutagenesis; peptide hormone; structure-function.

FIGURE 1.

Expression and purification of CTR ECD and RAMP1/2-CTR ECD fusion proteins in HEK293T cells. A, protein constructs designed for this study. Five pairs of (Gly-Ser) were used as a linker between RAMP1/2 and CTR ECDs. Amino acid numbers used for the ECDs are indicated above the diagram. The schematic shown is not scaled with real protein size. B, gel filtration elution profiles of MBP-CTR ECD, MBP-RAMP1-CTR ECD, and MBP-RAMP2-CTR ECD fusion proteins. C, left image is non-reducing SDS-PAGE analysis. Molecular mass markers are shown in the 1st lane and are labeled in kDa. Tris-glycine native gel analysis is shown in the right panel where a slight upward shift was observed for the E101A mutant protein due to the loss of a negative charge on Glu-101. The gels were stained with Coomassie Brilliant Blue.

FIGURE 2.

Sequence alignment of peptide ligands for calcitonin and amylin receptors. Sequences were aligned with ClusterX2.1, and similar residues are shown in black bold characters and yellow boxes determined with ESPript 3.0. sCT and AC413 sequences with green background were used for alanine-scanning experiments.

FIGURE 3.

Peptide binding profiles with CTR ECD and the fusion proteins of RAMP1/2-CTR ECD. A, competition binding with CTR ECD. B and C, competition binding with RAMP1/2-CTR ECD fusion proteins. MBP-CTR ECD (150 nm) with B-sCT (150 nm), MBP-RAMP1-CTR (200 nm) with B-AC413 (100 nm), and MBP-RAMP2-CTR (120 nm) with B-AC413 (100 nm) were used to obtain IC₅₀ of non-biotinylated competitive peptides for A–C, respectively. Representative binding curves of at least three independent experiments are shown except hαCGRP(8–37) with CTR and AM(22–52) with CTR, RAMP1-CTR, and RAMP2-CTR (n = 2). The means of duplicate samples with S.E. are shown in the binding curves.

FIGURE 4.

Binding of alanine-substituted sCT(22–32) peptides to CTR ECD and RAMP1/2-CTR ECD fusion proteins and homology model of sCT-bound CTR ECD. sCT(22–32) peptides with an alanine mutation were used at 10 μm concentration for CTR ECD (A) and RAMP1/2-CTR ECD (B and C) binding. The concentrations of MBP-ECD proteins and biotinylated peptides used for the binding assay were the same as shown in Fig. 3. The averages of two independent experiments with duplicate samples are shown with S.E. in the bar graphs. The mutational effects on peptide binding are indicated with dark gray color, and stronger effects are indicated with additional slash lines. D, homology model of sCT(22–32) bound to the CTR ECD. The hydrophobic patch and binding pocket are labeled with A and B, respectively. The hydrogen bond of sCT(22–32) Thr-25 and CTR Asp-101 is shown as a red dotted line.

FIGURE 5.

Binding of alanine-substituted AC413(6–25) peptides to CTR ECD and RAMP1/2-CTR ECD fusion proteins and homology model of the AC413-bound RAMP1·CTR ECD complex. AC413(6–25) peptides with an alanine mutation were used at 100 and 10 μm concentrations for CTR ECD (A) and RAMP1/2-CTR ECD fusion proteins (B and C) binding, respectively. R11A and P16A mutant peptides were excluded for all ECD binding, and G21A mutant peptide was excluded only for CTR binding due to peptide insolubility. The concentrations of MBP-ECD proteins and biotinylated peptides used for the binding assay were the same as shown in Fig. 3. The averages of two independent experiments with duplicate samples are shown with S.E. in the bar graphs. The mutational effects on peptide binding are indicated with dark gray color, and stronger effects are indicated with additional slash lines. D, homology model of AC413(15–25) bound to the RAMP1·CTR ECD complex. The hydrophobic patch and binding pocket are labeled with A and B, respectively. The hydrogen bond of AC413(15–25) Thr-18 and CTR Asp-101 is shown as a red dotted line.

FIGURE 6.

Contribution of C-terminal residues of AC413(6–25) and rAmy(8–37) to RAMP1/2-CTR ECD fusion protein binding. A, full competition binding curves with AC413(6–25) peptide with Y25A mutation. For RAMP1-CTR ECD, Y25F mutant peptide was used to examine the molecular interaction mediated by the aromatic ring of Tyr-25. B, mutational effects of Y37A on rAmy(8–37) binding to RAMP1/2-CTR ECD. The concentrations of MBP-ECD proteins and biotinylated peptides used for the binding assay were the same as shown in Fig. 3. Representative binding curves of three independent experiments are shown except the binding experiments of rAmy(8–37) Y37A with CTR (n = 2). The means of duplicated samples and S.E. are shown in the binding curves.

FIGURE 7.

Effects of RAMP1 W84A and RAMP2 E101A on peptide binding to RAMP1/2-CTR ECD fusion proteins. A, superposition of crystal structures of CGRP analog-bound RAMP1-CLR ECD (Protein Data Bank code 4RWG) and AM-bound RAMP2-CLR ECD (Protein Data Bank code 4RWF) highlighting the pocket occupied by the C-terminal residue of the peptides. The hydrogen bonds between AM Tyr-52 and RAMP2 Glu-101 and between C-terminal amides of AM and CGRP analog and Thr-122 backbone of CLR ECD are shown as red dotted lines. B and C, saturation binding of B-AC413 with RAMP1 W84A-CTR ECD fusion protein and RAMP2 E101A-CTR ECD fusion protein on the left and their competitive binding on the right. 100 nm mutant ECD fusion proteins were used for saturation binding assay. Competitive binding assay was performed under the same concentrations as shown in Fig. 3. Representative saturation and competitive binding curves of three independent experiments are shown. The means of the duplicate samples and S.E. are shown in the binding curves.

FIGURE 8.

Effects of C-terminal swap mutations of sCT(22–32) and AC413(6–25)/rAmy(8–37) on CTR ECD and RAMP1/2-CTR ECD fusion protein binding. A, sCT(22–32)P32Y/AC413(6–25)Y25P; B, rAmy(8–37)Y37P binding to CTR ECD and RAMP1/2-CTR ECD fusion proteins. The concentrations of MBP-ECD proteins and biotinylated peptides used for the binding assay were the same as shown in Fig. 3. Representative competitive binding curves of three independent experiments are shown. The means of duplicate samples and S.E. are shown in the binding curves.

FIGURE 9.

Antagonism of AC413(6–25) Y25P/Y25A and rAmy(8–37) Y37P/Y37A at the CTR and AMY₁ receptors in COS-7 cells. A, AC413(6–25) antagonism against hCT-mediated cAMP production at CTR. B, AC413(6–25) antagonism against rAmy-mediated cAMP production at AMY₁. C, AC413(6–25) antagonism against hCT-mediated cAMP production at AMY₁. D, rAmy(8–37) antagonism against hCT-mediated cAMP production at CTR. E, rAmy(8–37) antagonism against rAmy-mediated cAMP production at AMY₁. F, rAmy(8–37) antagonism against hCT-mediated cAMP production at AMY₁. Concentration-response curves of cAMP production were generated by nonlinear regression using the log(agonist) versus response equation of Prism 5.0. Representatives of at least three independent experiments are shown. The means of duplicate or triplicate samples and S.E. are shown in the cAMP production curves. See Table 3 for pA₂ values for the antagonists.