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. 2019 Mar 18;2(3):183-197.
doi: 10.1021/acsptsci.9b00010. eCollection 2019 Jun 14.

Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation Through Extracellular Loops of the Calcitonin Receptor

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Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation Through Extracellular Loops of the Calcitonin Receptor

Vi Pham et al. ACS Pharmacol Transl Sci. .
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Abstract

Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY3 receptors (AMY3R) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMY3R is thus important for peptide drug targeting of this receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by calcitonin (CT) and related peptides (Dal Maso, E., et al. (2019) The molecular control of calcitonin receptor signaling. ACS Pharmacol. Transl. Sci. 2, 31-51). That work revealed ligand- and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMY3Rs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMY3R, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the receptor.

Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Effect of alanine mutation on the cell surface expression of the AMY3R and CTR. (A–C) AMY3R; (D–F) CTR (extracted from Dal Maso et al, 2018). (A, D) Top view of the active, sCT-bound, AMY3R (A) or CTR (6NIY) (D) model with the extracellular surface subject to alanine scanning depicted in gray (A) or off-white (D) (combined surface/cpk representation). The rest of the protein complex is shown in ribbon representation. CTR (blue, AMY3R; dark red, CTR), sCT peptide (dark red, AMY3R; aquamarine, CTR), RAMP3 (green). The receptor ECD is omitted for clarity. (B, E) Map of the effect of mutation on cell surface receptor expression colored according to the legend in panel F. (C, F) Effect of alanine mutation of ECL2 and ECL3 on CTR expression monitored by FACS of anti-c-Myc antibody binding to the N-terminal c-Myc epitope on the receptor. Data are normalized to the expression of the wild-type (WT) receptor (100%). Significant differences in the level of cell surface expression were determined by one-way ANOVA followed by Dunnett’s post-test comparison to the WT. P < 0.05 was used to denote significance, and colored according to the magnitude of change. Individual values (separate experiments) are shown within the bars.
Figure 2
Figure 2
Identification of key amino acids of AMY3R ECL2 and ECL3 for peptide binding affinity (log Ki). (A) sCT; (B) hCT; (C) pCT; (D) rAmy; (E) hCGRP. Mutations that significantly decreased peptide affinity in radioligand competition assay are colored dark orange (≤10-fold effect) or red (>10-fold effect), with mutated amino acids without significant alteration to log Ki colored gray. Amino acid mutations where there was an insufficiently robust functional effect to quantify by radioligand competition binding are depicted in black. The receptor ECD is not shown for clarity, with the CTR TM bundle in blue ribbon and RAMP3 in green ribbon. Quantitative data are reported in Table 1.
Figure 3
Figure 3
Alanine mutation of ECL2 and ECL3 of AMY3R alters cAMP functional affinity (log KA) in a peptide-specific manner. Functional affinities derived from operational fitting of concentration–response curves in cAMP accumulation for alanine mutation of ECL2 (A-E) and ECL3 (F-J) are displayed as log KA. Significance of changes was established by comparison of the WT to the other receptor mutants following one-way ANOVA and Dunnett’s post-test with P < 0.05 accepted as significant. Mutants that gave significant reductions between 3- and 10-fold are colored orange, and those with reductions greater than 10-fold are colored red. Mutants giving significant increases in log KA are colored green. Where data were insufficiently robust to derive a reliable value for log KA no symbol is shown (ND). Quantitative data are reported in Table 2.
Figure 4
Figure 4
Alanine mutation of ECL2 and ECL3 of AMY3R has limited effect on pERK functional affinity (log KA). Functional affinities derived from operational fitting of concentration–response curves in ERK phosphorylation for alanine mutation of ECL2 (A–E) and ECL3 (F–J) are displayed as log KA. No significant changes in log KA from WT were seen for receptor mutants following one-way ANOVA and Dunnett’s post-test with P < 0.05 accepted as significant. Where data were insufficiently robust to derive a reliable value for log KA no symbol is shown (ND). Quantitative data are reported in Table 3.
Figure 5
Figure 5
Alanine mutation of ECL2 and ECL3 of AMY3R alters functional affinity (log KA) in a peptide- and pathway-specific manner. Functional affinities derived from operational fitting of concentration–response curves in cAMP accumulation (A–E) and pERK (F–J) are displayed as Δlog KA from wild-type. Illustrated is a top view of the AMY3R model with the extracellular surface subject to alanine scanning depicted (combined surface/cpk representation). Mutations that significantly alter peptide functional log KA are colored according to the magnitude of effect, with mutated amino acids without significant alteration to log KA colored gray. Amino acid mutations for which there was an insufficiently robust functional effect to quantify by operational modeling are depicted in black. The receptor ECD and peptide are not shown for clarity, with the CTR TM bundle in blue ribbon and RAMP3 in green ribbon. Red arrows in panels I and J indicate residues at the apex of ECL3 that are affected for Amy and CGRP but not CT peptides.
Figure 6
Figure 6
Alanine mutation of ECL2 and ECL3 has distinct effects on AMY3R and CTR cAMP functional affinity (log KA). Functional affinities derived from operational fitting of concentration–response curves in cAMP accumulation are displayed as Δlog KA from wild-type for AMY3R (A–E) and CTR (F–J). Illustrated are top views of the receptors with the extracellular surface subject to alanine scanning depicted (combined surface/cpk representation). Mutations that significantly alter peptide functional log KA are colored according to the magnitude of effect, with mutated amino acids without significant alteration to log KA colored gray. Amino acid mutations where there was an insufficiently robust functional effect to quantify by operational modeling are depicted in black. The receptor ECD and peptide are not shown for clarity, with the CTR TM bundle in blue ribbon and RAMP3 in green ribbon in panels A–E, and CTR TM bunding in red ribbon in panels F–J. Data for CTR functional affinity are from Dal Maso et al., 2018.
Figure 7
Figure 7
Alanine mutation of ECL2 and ECL3 has distinct effects on AMY3R and CTR pERK functional affinity (log KA). Functional affinities derived from operational fitting of concentration–response curves in pERK are displayed as Δlog KA from wild-type for AMY3R (A–E) and CTR (F–J). Illustrated are top views of the receptors with the extracellular surface subject to alanine scanning depicted (combined surface/cpk representation). Mutations that significantly alter peptide functional log KA are colored according to the magnitude of effect, with mutated amino acids without significant alteration to log KA colored gray. Amino acid mutations where there was an insufficiently robust functional effect to quantify by operational modeling are depicted in black. The receptor ECD and peptide are not shown for clarity, with the CTR TM bundle in blue ribbon and RAMP3 in green ribbon in A-E, and CTR TM bunding in red ribbon in F-J. Data for CTR functional affinity are from Dal Maso et al., 2018.
Figure 8
Figure 8
Alanine mutation of ECL2 and ECL3 of AMY3R alters cAMP efficacy (log τc) in a peptide-specific manner. Peptide efficacy (log τ) was derived from operational fitting of concentration–response curves in cAMP accumulation for alanine mutation of ECL2 (A–E) and ECL3 (F–J), and corrected for cell surface receptor expression to yield Log tauc. Significance of mutation effect was established by comparison of the WT to the other receptor mutants following one-way ANOVA and Dunnett’s post-test with P < 0.05 accepted as significant. Mutants that significantly reduced log τc are colored orange (≤10-fold change), or red (>10-fold change). Mutants that significantly increased log τc are colored green (≤10-fold change, light green; >10-fold, dark green). Where data were insufficiently robust to derive a reliable value for log τc no symbol is shown (ND). Quantitative data are reported in Table 4.
Figure 9
Figure 9
Alanine mutation of ECL2 and ECL3 of AMY3R alters pERK efficacy (log τc) in a peptide-specific manner. Peptide efficacy (log τ) was derived from operational fitting of concentration–response curves in pERK for alanine mutation of ECL2 (A–E) and ECL3 (F–J), and corrected for cell surface receptor expression to yield log τc. Significance of mutation effect was established by comparison of the wild-type to the other receptor mutants following one-way ANOVA and Dunnett’s post-test with P < 0.05 accepted as significant. Mutants that significantly reduced log τc are colored orange (≤10-fold change), or red (>10-fold change). Where data were insufficiently robust to derive a reliable value for log τc no symbol is shown (ND). Quantitative data are reported in Table 5.
Figure 10
Figure 10
Alanine mutation of ECL2 and ECL3 of AMY3R alters peptide efficacy (log τc) in a pathway-specific manner. Efficacy values derived from operational fitting of concentration–response curves in cAMP accumulation (A–E) and pERK (F–J) are displayed as Δlog τc from wild-type. Illustrated is a top view of the AMY3R model with the extracellular surface subject to alanine scanning depicted (combined surface/cpk representation). Mutations that significantly alter peptide log τc are colored according to the magnitude of effect, with mutated amino acids without significant alteration to log τc colored gray. Amino acid mutations for which there was an insufficiently robust functional effect to quantify by operational modeling are depicted in black. The receptor ECD and peptide are not shown for clarity, with the CTR TM bundle in blue ribbon and RAMP3 in green ribbon.
Figure 11
Figure 11
Alanine mutation of ECL2 and ECL3 has distinct effects on cAMP peptide efficacy for AMY3R and CTR. Peptide efficacy, derived from operational fitting of concentration–response curves in cAMP accumulation, are displayed as Δlog τc from wild-type for AMY3R (A–E) and CTR (F–J). Illustrated are top views of the receptors with the extracellular surface subject to alanine scanning depicted (combined surface/cpk representation). Mutations that significantly alter peptide functional log τc are colored according to the magnitude of effect, with mutated amino acids without significant alteration to log τc colored gray. Amino acid mutations for which there was an insufficiently robust functional effect to quantify by operational modeling are depicted in black. The receptor ECD and peptide are not shown for clarity, with the CTR TM bundle in blue ribbon and RAMP3 in green ribbon in A–E, and CTR TM bunding in red ribbon in F–J. Data for CTR peptide efficacy are from Dal Maso et al., 2018.
Figure 12
Figure 12
Alanine mutation of ECL2 and ECL3 has distinct effects on pERK peptide efficacy for AMY3R and CTR. Peptide efficacy, derived from operational fitting of concentration–response curves in pERK, are displayed as Δlog τc from wild-type for AMY3R (A–E) and CTR (F–J). Illustrated are top views of the receptors with the extracellular surface subject to alanine scanning depicted (combined surface/cpk representation). Mutations that significantly alter peptide functional log τc are colored according to the magnitude of effect, with mutated amino acids without significant alteration to log τc colored gray. Amino acid mutations for which there was an insufficiently robust functional effect to quantify by operational modeling are depicted in black. The receptor ECD and peptide are not shown for clarity, with the CTR TM bundle in blue ribbon and RAMP3 in green ribbon in A–E, and CTR TM bundling in red ribbon in F–J. Data for CTR peptide efficacy are from Dal Maso et al., 2018.

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