Biochemical characterization of G protein coupling to calcitonin gene–related peptide and adrenomedullin receptors using a native PAGE assay

• Published in: The Journal of biological chemistry 2020-07, Vol.295 (28), p.9736-9751
• Main Authors: Roehrkasse, Amanda M., Warner, Margaret L., Booe, Jason M., Pioszak, Augen A.
• Format: Article
• Language: English
• Subjects: Animals; Calcitonin Gene-Related Peptide - chemistry; Calcitonin Gene-Related Peptide - genetics; Calcitonin Gene-Related Peptide - metabolism; Chlorocebus aethiops; COS Cells; Cyclic AMP - metabolism; G protein; G protein-coupled receptor (GPCR); HEK293 Cells; hormone receptor; hrCNE; Humans; in-gel fluorescence; Methods and Resources; mini-G; molecular pharmacology; Native Polyacrylamide Gel Electrophoresis; peptide hormone; Protein Domains; receptor structure/function; Receptors, Adrenomedullin - chemistry; Receptors, Adrenomedullin - genetics; Receptors, Adrenomedullin - metabolism; Second Messenger Systems; thermostability
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA120.013854

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.