Binding of the vasodilator peptides adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) to the class B G protein-coupled receptor calcitonin receptor-like receptor (CLR) is modulated by receptor activity-modifying proteins (RAMPs). RAMP1 favors CGRP, whereas RAMP2 and RAMP3 favor AM. Crystal structures of peptide-bound RAMP1/2-CLR extracellular domain (ECD) heterodimers suggested RAMPs alter ligand preference through direct peptide contacts and allosteric modulation of CLR. Here, we probed this dual mechanism through rational structure-guided design of AM and CGRP antagonist variants. Variants were characterized for binding to purified RAMP1/2-CLR ECD and for antagonism of the full-length CGRP (RAMP1:CLR), AM (RAMP2:CLR), and AM (RAMP3:CLR) receptors. Short nanomolar affinity AM(37-52) and CGRP(27-37) variants were obtained through substitutions including AM S45W/Q50W and CGRP K35W/A36S designed to stabilize their -turn. K46L and Y52F substitutions designed to exploit RAMP allosteric effects and direct peptide contacts, respectively, yielded AM variants with selectivity for the CGRP receptor over the AM receptor. AM(37-52) S45W/K46L/Q50W/Y52F exhibited nanomolar potency at the CGRP receptor and micromolar potency at AM A 2.8-Å resolution crystal structure of this variant bound to the RAMP1-CLR ECD confirmed that it bound as designed. CGRP(27-37) N31D/S34P/K35W/A36S exhibited potency and selectivity comparable to the traditional antagonist CGRP(8-37). Giving this variant the ability to contact RAMP2 through the F37Y substitution increased affinity for AM, but it still preferred the CGRP receptor. These potent peptide antagonists with altered selectivity inform the development of AM/CGRP-based pharmacological tools and support the hypothesis that RAMPs alter CLR ligand selectivity through allosteric effects and direct peptide contacts.

The cardioprotective vasodilator peptide adrenomedullin 2/intermedin (AM2/IMD) and the related adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) signal through three heterodimeric receptors comprising the calcitonin receptor-like class B G protein-coupled receptor (CLR) and a variable receptor activity-modifying protein (RAMP1, -2, or -3) that determines ligand selectivity. The CGRP receptor (RAMP1:CLR) favors CGRP binding, whereas the AM (RAMP2:CLR) and AM (RAMP3:CLR) receptors favor AM binding. How AM2/IMD binds the receptors and how RAMPs modulate its binding is unknown. Here, we show that AM2/IMD binds the three purified RAMP-CLR extracellular domain (ECD) complexes with a selectivity profile that is distinct from those of CGRP and AM. AM2/IMD bound all three ECD complexes but preferred the CGRP and AM receptor complexes. A 2.05 Å resolution crystal structure of an AM2/IMD antagonist fragment-bound RAMP1-CLR ECD complex revealed that AM2/IMD binds the complex through a unique triple β-turn conformation that was confirmed by peptide and receptor mutagenesis. Comparisons of the receptor-bound conformations of AM2/IMD, AM, and a high-affinity CGRP analog revealed differences that may have implications for biased signaling. Guided by the structure, enhanced-affinity AM2/IMD antagonist variants were developed, including one that discriminates the AM and AM receptors with ∼40-fold difference in affinities and one stabilized by an intramolecular disulfide bond. These results reveal differences in how the three peptides engage the receptors, inform development of AM2/IMD-based pharmacological tools and therapeutics, and provide insights into RAMP modulation of receptor pharmacology.

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin 2/intermedin (AM2/IMD) have overlapping and unique functions in the nervous and circulatory systems including vasodilation, cardioprotection, and pain transmission. Their actions are mediated by the class B calcitonin-like G protein–coupled receptor (CLR), which heterodimerizes with three receptor activity–modifying proteins (RAMP1–3) that determine its peptide ligand selectivity. How the three agonists and RAMPs modulate CLR binding to transducer proteins remains poorly understood. Here, we biochemically characterized agonist-promoted G protein coupling to each CLR·RAMP complex. We adapted a native PAGE method to assess the formation and thermostabilities of detergent-solubilized fluorescent protein–tagged CLR·RAMP complexes expressed in mammalian cells. Addition of agonist and the purified Gs protein surrogate mini-Gs (mGs) yielded a mobility-shifted agonist·CLR·RAMP·mGs quaternary complex gel band that was sensitive to antagonists. Measuring the apparent affinities of the agonists for the mGs-coupled receptors and of mGs for the agonist-occupied receptors revealed that both ligand and RAMP control mGs coupling and defined how agonist engagement of the CLR extracellular and transmembrane domains affects transducer recruitment. Using mini-Gsq and -Gsi chimeras, we observed a coupling rank order of mGs > mGsq > mGsi for each receptor. Last, we demonstrated the physiological relevance of the native gel assays by showing that they can predict the cAMP-signaling potencies of AM and AM2/IMD chimeras. These results highlight the power of the native PAGE assay for membrane protein biochemistry and provide a biochemical foundation for understanding the molecular basis of shared and distinct signaling properties of CGRP, AM, and AM2/IMD.

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IntroductionMeningiomas are the most common primary central nervous system (CNS) tumor. They are most often benign, but a subset of these can behave aggressively. Current World Health Organization (WHO) guidelines classify meningiomas into three grades based on the histologic findings and presence or absence of brain invasion. These grades are intended to guide treatment, but meningiomas can behave inconsistently with regard to their assigned histopathological grade, influencing patient expectations and management. Advanced molecular profiling of meningiomas has led to the proposal of alternative molecular grading schemes that have shown superior predictive power. These include methylation patterns, copy number alterations, and mutually exclusive driver mutations affecting oncogenes, including BAP1, CDKN2A/B, and the TERT promoter, which are associated with particularly aggressive tumor biology. Despite the evident clinical value, advanced molecular profiling methods are not widely incorporated in routine clinical practice for meningiomas.ObjectiveTo assess the degree of concordance between the molecular profile of meningiomas and the histopathologic WHO classification, the current method of predicting meningioma behavior.MethodsIn a two-year single-institution experience, we used commercially available resources to determine molecular profiles of all resected meningiomas. Copy number aberrations and oncogenic driver mutations were identified and compared with the histopathologic grade.ResultsOne hundred fifty-one total meningioma cases were included for analysis (85.4% WHO grade 1, 13.3% WHO grade 2, and 1.3% grade 3). Chromosomal analysis of 124 of these samples showed that 29% of WHO grade 1 tumor featured copy number profiles consistent with higher grade meningioma, and 25% of WHO grade 2 meningiomas had copy number profiles consistent with less aggressive tumors. Furthermore, 8% harbored mutations in TERT, CDKN2A/B, or BAP1 of which 6% occurred in grade 1 meningiomas.ConclusionsRoutine advanced molecular profiling of all resected meningiomas using commercially available resources allowed for identification of a significant number of meningiomas whose molecular profiles were inconsistent with WHO grade. Our work shows the clinical value of integrating routine molecular profiling with histopathologic grading to guide clinical decision making.

The vasoactive peptides calcitonin gene‐related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2/intermedin (AM2/IMD) have demonstrated protective effects in disease processes that include ischemia/reperfusion injury and inflammatory bowel disease. The actions of these peptides are mediated by the calcitonin‐like receptor (CLR), which is a class B G protein‐coupled receptor (GPCR) that forms heterodimers with any one of three receptor activity‐modifying proteins (RAMPs 1/2/3). The RAMPs modulate its peptide binding preferences and may also alter signaling output, but the latter remains poorly understood. We hypothesized that different RAMP and peptide combinations stabilize distinct CLR conformations that alter transducer interactions. We adapted a native PAGE method as an inexpensive and efficient way to visualize detergent‐solubilized, fluorescently‐tagged RAMP:CLR complexes expressed in mammalian cells and to measure their interactions with transducer proteins. Addition of agonist peptides and the purified engineered minimal Gs protein (MiniGs) prior to solubilization resulted in agonist‐dependent coupling of MiniGs to each receptor visible as a quaternary complex. Using the MiniG chimeras MiniGs/i and MiniGs/q along with MiniGs we characterized the coupling preferences of each RAMP:CLR complex in the presence of each agonist peptide. For each of the three agonist peptides MiniGs coupled best to each RAMP:CLR complex, followed by MiniGs/q and finally MiniGs/i. Overall, all three MiniG proteins exhibited better coupling to the RAMP1:CLR complex consistent with RAMP‐dependent modulation of MiniG coupling. At each RAMP:CLR complex, we also observed peptide‐dependent effects with CGRP exhibiting distinct behavior from AM and AM2/IMD. This work demonstrates that the native gel assay is a powerful tool for screening and characterizing membrane protein stabilities and ligand interactions and supports our hypothesis that the RAMPs and agonist peptides alter CLR transducer interactions. Our findings may aid development of therapeutics that exploit the beneficial effects of these peptide hormones. Support or Funding Information This work was supported by grants NIH R01GM104251 (AAP) and NIH predoctoral MD/PhD fellowship 1F30 HL142232 (AMR).

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