Multiplexed analysis of the secretin-like GPCR-RAMP interactome

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 GPCR...

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Detalles Bibliográficos
Autores principales: Lorenzen, Emily, Dodig-Crnković, Tea, Kotliar, Ilana B., Pin, Elisa, Ceraudo, Emilie, Vaughan, Roger D., Uhlèn, Mathias, Huber, Thomas, Schwenk, Jochen M., Sakmar, Thomas P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Association for the Advancement of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750928/
https://www.ncbi.nlm.nih.gov/pubmed/31555726
http://dx.doi.org/10.1126/sciadv.aaw2778
Descripción
Sumario: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.