Peptide binding to CTR ECD and RAMP-CTR ECD complexes 1 Calcitonin and amylin receptor peptide interaction mechanisms: Insights into peptide-binding modes and allosteric modulation of the calcitonin receptor by receptor activity-modifying proteins

Receptor activity-modifying proteins (RAMP13) determine the selectivity of the class B GPCRs calcitonin receptor (CTR) and the CTR-like receptor (CLR), for calcitonin (CT), amylin (Amy), CGRP, and adrenomedullin (AM) peptides. RAMP1/2 alter CLR selectivity for CGRP/AM in part by RAMP1 W84 or RAMP2 E101 contacting the distinct CGRP/AM C-terminal residues. It is unclear if 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 RAMP1and RAMP2-CTR ECD fusion proteins and antagonist peptides. All three proteins bound sCT. Tethering RAMPs to CTR enhanced binding of rAmy, CGRP, and the AMY antagonist AC413. Peptide alanine-scan mutagenesis and modeling of receptor-bound sCT and AC413 supported a shared non-helical CGRP-like conformation for their TNT/VG 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 W84Aand 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. Receptor activity-modifying proteins are single-pass transmembrane proteins (RAMP1-3 in humans) that form heteromeric complexes with several G protein-coupled receptors (GPCRs) and thereby regulate their cell-surface expression and pharmacology (1,2). RAMPs are best characterized for their effects on two class B GPCRs, the calcitonin receptor (CTR) and CTRlike receptor (CLR) (3). CTR/CLR and their complexes with RAMPs give rise to at least seven pharmacologically distinct receptors (not including splice variants) in humans that exhibit unique selectivity profiles for six related calcitonin (CT) family peptide agonists: CT, amylin (Amy), http://www.jbc.org/cgi/doi/10.1074/jbc.M115.713628 The latest version is at JBC Papers in Press. Published on February 19, 2016 as Manuscript M115.713628 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Peptide binding to CTR ECD and RAMP-CTR ECD complexes 2 calcitonin gene-related peptides alpha and beta (αCGRP, βCGRP), adrenomedullin (AM) and adrenomedullin2/intermedin (AM2) (2-4). CLR:RAMP1 is the CGRP receptor at which CGRP has greater potency than AM. CLR:RAMP2 and CLR:RAMP3 are the AM1 and AM2 receptors, respectively, at which AM is more potent than CGRP (1). AM2 activates the CGRP and AM receptors, but is most potent at AM2 (5). CTR alone is the CT receptor at which CT is more potent than Amy. CTR complexes with RAMP1, 2, or -3 form distinct AMY1, AMY2, and AMY3 receptor subtypes, respectively (6-9), at which CT and Amy are roughly equipotent; the AMY1 and AMY3 receptors also respond to CGRP (9-11). This complex system provides a paradigm for understanding modulation of GPCRs by accessory proteins, but much remains unknown regarding the mechanisms of RAMP-altered peptide recognition. Several of the diverse biological actions of CT family peptides are clinically relevant thus highlighting the need to understand peptide recognition mechanisms to facilitate drug development. CGRP and AM are potent vasodilators (12). Antagonism of the neurogenic inflammatory effects of CGRP is actively pursued as migraine therapy (13) and the cardioprotective effects of AM suggest that agonists of its receptor(s) may be of value for various cardiovascular disorders (14). The potent CT receptor agonist salmon calcitonin (sCT) has long been used to treat bone disorders taking advantage of its essentially irreversible binding to the human receptor (15) and the ability of exogenous CT to affect bone turnover (16). Amy controls plasma glucose levels via AMY receptor activation that causes slowed gastric emptying, inhibition of glucagon secretion, and reduction of food intake (17). The Amy analog pramlintide is available for types I and II diabetes and AMY receptor activation is under consideration as an obesity treatment (17,18). Recently, amylin-mediated metabolic alteration was also shown to induce regression of p53-deficient tumors (19). Despite these drug development successes and potential, relatively little is known about how CT and Amy bind their receptors and next-generation analogs with more favorable properties are actively pursued (20,21). Peptide agonist binding to class B GPCRs follows a “two-domain” model (22). Binding of the peptide C-terminal region to the N-terminal extracellular domain (ECD) of the receptor contributes to affinity and selectivity and facilitates binding of the N-terminal peptide region to the receptor seven transmembrane domain (7TM), which activates the receptor. N-terminally truncated agonist peptides thus act as antagonists. The two-domain model applies to the CT family peptides and their receptors, but with the additional complication of the RAMP subunits (23). Our understanding of CT family peptide receptor binding is most advanced for CGRP and AM (24). Recombinant soluble ECDs of RAMP1 and RAMP2 formed complexes with the CLR ECD that bound their respective peptides, albeit with lower affinity than the full-length receptors (25-28). Although these complexes were stable enough to co-purify, we devised a fusion protein approach in which the CGRP and AM1 receptor ECD complexes were engineered as tethered RAMP1-CLR ECD and RAMP2-CLR ECD fusions to further stabilize the 1:1 heterodimers (29). Crystal structures of the fusion proteins bound with C-terminal antagonist fragments of a CGRP analog or AM revealed that the peptides occupy a shared binding site on CLR with largely unstructured conformations containing a β-turn near their C-terminus (24). This β-turn structure is distinct from the α-helical conformation observed for several other class B GPCR ligands (30-34). RAMP1 and RAMP2 each make a single important contact to the peptides. The C-terminal CGRP analog F37 and AM Y52 occupy a pocket on CLR adjacent to the RAMP subunits. RAMP1 W84 or RAMP2 E101 augments the pocket and contacts CGRP analog F37 or AM Y52, respectively. These RAMP-mediated contacts are important for peptide affinity and contribute to selectivity. RAMPs thus alter CLR selectivity in part by a direct peptide contact mechanism. Nonetheless, allosteric modulation of CLR by the RAMPs may also contribute to selectivity because swapping of the C-terminal residues of CGRP and AM was insufficient to exchange their receptor preferences and subtle differences in CLR conformation were evident in the two structures (24). It is unclear to what extent the recent findings for the CGRP and AM1 receptors translate to CTR and AMY receptors. Chimeric receptor studies by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Peptide binding to CTR ECD and RAMP-CTR ECD complexes 3 indicated that the RAMP ECDs dictated ligand selectivity at CTR (11,35), but there are no reports of purification and characterization of the CTR ECD or its complexes with RAMP ECDs to confirm these findings. The stoichiometry of CTR:RAMP complexes has not been studied. There are well-characterized differences between RAMP complexes with CTR and CLR. CLR is an obligate heterodimer; without RAMP subunits it cannot reach the cell surface (1). In contrast, CTR can traffic to the cell surface and function independent of RAMPs (3). The ligand selectivity patterns conferred by RAMPs at CLR and CTR are different. Whereas RAMP1 and RAMP2 swap the selectivity of CLR for CGRP and AM, their association with CTR has only minor effects on CT affinity and potency (9,11). Moreover, RAMP1 and RAMP2 both confer enhanced affinity for the same ligand, Amy, at CTR despite their favoring binding of different peptides, CGRP or AM, to CLR (9). These properties hint that RAMPs may use different mechanisms to alter peptide selectivity at CLR and CTR. Here, we purified CTR ECD and tethered RAMP1-CTR and RAMP2-CTR ECD fusion proteins and investigated their peptide interactions. Mutagenesis of peptide ligands and RAMP1/2 ECDs and computational modeling were employed to probe the conformations of receptor ECD-bound CT and Amy or Amy analogs and selected results were extended to intact receptors via cell-based signaling assays. We also swapped the C-terminal Pro of CT for the C-terminal Tyr of Amy and vice versa to investigate the role(s) of peptide C-terminal residues in affinity and selectivity at CTR. Our results reveal significant differences in how RAMPs function at CTR and CLR and are consistent with the hypothesis that RAMPs modulate CTR peptide affinity by an allosteric mechanism. EXPERIMENTAL PROCEDURES Plasmid construction and purification Human CTRA isoform, RAMP1, and RAMP2 cDNAs were obtained from the cDNA resource center. Gene fragments encoding E. coli maltose binding protein (MBP) ending with an NAAAEF linker sequence, CTR ECD, RAMP1 ECD, or RAMP2 ECD were assembled into the pHLsec vector designed for secreted expression from mammalian cells (36) using PCR/restriction enzyme/DNA ligase-based cloning methods or the Gibson Assembly method using Gibson Assembly Master Mix (New England Biolabs). Primer sequences are available upon request. The CTR fusion protein designs were similar to those previously reported for CLR (29). The RAMP and CTR ECDs were tethered with a flexible (GlySer)5 linker sequence. The following three plasmids were constructed by inserting DNA encoding the desired MBP fusion constructs between the AgeI and KpnI sites of pHLsec: pHLsec/MBP-hCTR.36-151-(His)6; pHLsec/MBPhRAMP1.24-111-(Gly-Ser)5-hCTR.36-151-(His)6; and pHLsec/MBP-hRAMP2.55-140-(Gly-Ser)5hCTR.36-151-(His)6 (amino acid numbers indicated). Amino acid substitutions were introduced into RAMP1 (W84A) or RAMP2 (E101A) using the Gibson Assembly method. All plasmids were confirmed by automated DNA sequencing of the coding regions performed by the OUHSC Laboratory for Molecular Biology and Cytometry Research core facility. Purification of the plasmids for use in transient transfections used the Macherey-Nagel Midi kit or Qiagen Giga kit according to the manufacturer’s directions. Protein expression and purification Human embryonic kidney 293T (HEK293T) cells were grown in 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) with 50 U/mL of penicillin and 50 μg/mL of streptomycin supplemented with 10% fetal bovine serum. For expression of MBP-CTR ECD, MBPRAMP1/2-CTR ECD, and MBP-RAMP2 [E101A]-CTR ECD, HEK293T cells were transiently transfected with the expression construct in five T175 cm 2 flasks using 30 ml culture volume and 50 μg of plasmid DNA per flask with polyethyleneimine transfection reagent according to standard methods (36). All postexpression processing and purification steps were carried out at 4°C. Cell culture media were collected 72 h after transfection, centrifuged to remove remaining cells and the supernatant (~ 150 mL) was filtered (0.22 m, Corning) and dialyzed overnight against 4 L of 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM Imidazole using 6-8 kDa molecular weight cut off dialysis membrane. Due to poor expression, the MBP-RAMP1 [W84A]CTR ECD protein required scale-up into six by gest on N ovem er 9, 2017 hp://w w w .jb.org/ D ow nladed from Peptide binding to CTR ECD and RAMP-CTR ECD complexes 4 expanded surface area roller bottles (with 1700 cm 2 surface area for each, Corning) with 350 ml culture volume and 500 μg of plasmid DNA per bottle. In addition, after transfection the temperature was lowered to 30°C and the culture media were harvested after 4 days. The media (~ 2.1 L) were centrifuged to remove cells, and the supernatant was filtered (0.22 m, Millipore), concentrated to ~ 150 mL by tangential flow filtration using three Minimate TM TFF capsules (molecular weight cut off 10 kDa) connected in parallel and the Pall Minimate TM TFF system, and finally dialyzed as above. After dialysis, the proteins were purified by immobilized-metal affinity and size exclusion chromatography using an AKTA purifier system (GE Healthcare). A 5 ml pre-packed Ni-chelating sepharose column (GE Healthcare) was equilibrated in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 25 mM Imidazole, 10% (v/v) glycerol, followed by sample loading and extensive washing in equilibration buffer before step elution with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 275 mM Imidazole, 10% (v/v) glycerol. Peak fractions were pooled and spin concentrated using Pierce  Concentrators with 9 kDa molecular weight cut off. The concentrated sample was loaded on a 320 ml bed volume Superdex 200 HR column (GE Healthcare) equilibrated in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol. Peak fractions were pooled and dialyzed overnight against 1 L of storage buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50% (v/v) glycerol) and stored as aliquots at -80°C. The courses of the purifications were monitored by SDS-PAGE and native-gel electrophoresis performed as previously described (29). Protein concentrations were determined by UV absorbance at 280 nm using extinction coefficients calculated based on Tyr, Trp, and Cystine residues, and are stated in terms of the monomers. Final yields were ~ 1 mg of protein from five T175 flasks for MBP-CTR ECD, MBP-RAMP1/2-CTR ECD, and MBP-RAMP2 [E101A]-CTR ECD and from six expanded surface area roller bottles for MBP-RAMP1 [W84A]-CTR ECD.