Multiplexed analysis of the secretin-like GPCR-RAMP interactome.

Lorenzen E, Dodig-Crnković T, Kotliar IB, Pin E, Ceraudo E, Vaughan RD, Uhlèn M, Huber T, Schwenk JM, Sakmar TP

Sci Adv 5 (9) eaaw2778 [2019-09-00; online 2019-09-18]

Receptor activity-modifying proteins (RAMPs) have been shown to modulate the functions of several G protein-coupled receptors (GPCRs), but potential direct interactions among the three known RAMPs and hundreds of GPCRs have never been investigated. Focusing mainly on the secretin-like family of GPCRs, we engineered epitope-tagged GPCRs and RAMPs, and developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized lysates. Using 64 antibodies raised against the native proteins and 4 antibodies targeting the epitope tags, we mapped the interactions among 23 GPCRs and 3 RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. The results provide a complete interactome of secretin-like GPCRs with RAMPs. The SBA strategy will be useful to search for additional GPCR-RAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

Affinity Proteomics Stockholm [Collaborative]

PubMed 31555726

DOI 10.1126/sciadv.aaw2778

Crossref 10.1126/sciadv.aaw2778

pii: aaw2778
pmc: PMC6750928