Multiplexed analysis of the secretin-like GPCR-RAMP interactome
| Autor: | Emily Lorenzen, Tea Dodig-Crnković, Ilana B. Kotliar, Jochen M. Schwenk, Elisa Pin, Emilie Ceraudo, Roger D. Vaughan, Thomas P. Sakmar, Thomas Huber, Mathias Uhlén |
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| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
genetic structures
Computational biology Interactome Biochemistry Epitope Receptor Activity-Modifying Proteins Secretin Receptors G-Protein-Coupled 03 medical and health sciences Chemokine receptor 0302 clinical medicine Protein Interaction Mapping Humans Multiplex Receptor Research Articles 030304 developmental biology G protein-coupled receptor 0303 health sciences Multidisciplinary Chemistry musculoskeletal neural and ocular physiology HEK 293 cells SciAdv r-articles HEK293 Cells Membrane protein Bead array human activities 030217 neurology & neurosurgery hormones hormone substitutes and hormone antagonists Research Article |
| Zdroj: | Science Advances |
| ISSN: | 2375-2548 |
| Popis: | We used a multiplexed suspension bead array immunoassay to map GPCR-receptor activity–modifying protein (RAMP) complexes. 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. |
| Databáze: | OpenAIRE |
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