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Biochemical Characterization of Receptor Activity‐Modifying Protein and Peptide Agonist Effects on G protein Coupling to the Calcitonin‐Like Receptor
Author(s) -
Roehrkasse Amanda,
Warner Margaret,
Booe Jason,
Pioszak Augen
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.08732
Subject(s) - adrenomedullin , agonist , calcitonin gene related peptide , receptor , calcitonin , g protein coupled receptor , peptide , chemistry , calcitonin receptor , biophysics , biochemistry , microbiology and biotechnology , endocrinology , biology , neuropeptide
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 G s protein (MiniG s ) prior to solubilization resulted in agonist‐dependent coupling of MiniG s to each receptor visible as a quaternary complex. Using the MiniG chimeras MiniG s/i and MiniG s/q along with MiniG s we characterized the coupling preferences of each RAMP:CLR complex in the presence of each agonist peptide. For each of the three agonist peptides MiniG s coupled best to each RAMP:CLR complex, followed by MiniG s/q and finally MiniG s/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).