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Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

ABSTRACT: Receptor activity-modifying proteins (RAMPs) form complexes with G protein-coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening the GPCR-RAMP pairs with customized multiplexed suspension bead array (SBA) immunoassays, we identified 122 GPCRs that showed strong evidence for interaction with at least one RAMP. We screened for interactions in three cell lines and found 23 endogenously expressed GPCRs that formed complexes with RAMPs. Mapping the GPCR-RAMP interactome expands the current system-wide functional characterization of RAMP-interacting GPCRs to inform the design of selective therapeutics targeting GPCR-RAMP complexes.

SUBMITTER: Kotliar IB

PROVIDER: S-EPMC11290489 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

Kotliar Ilana B IB Bendes Annika A Dahl Leo L Chen Yuanhuang Y Saarinen Marcus M Ceraudo Emilie E Dodig-Crnković Tea T Uhlén Mathias M Svenningsson Per P Schwenk Jochen M JM Sakmar Thomas P TP

Science advances 20240731 31

Receptor activity-modifying proteins (RAMPs) form complexes with G protein-coupled receptors (GPCRs) and may regulate their cellular trafficking and pharmacology. RAMP interactions have been identified for about 50 GPCRs, but only a few GPCR-RAMP complexes have been studied in detail. To elucidate a comprehensive GPCR-RAMP interactome, we created a library of 215 dual epitope-tagged (DuET) GPCRs representing all GPCR subfamilies and coexpressed each GPCR with each of the three RAMPs. Screening t ...[more]

PMID: 39083597

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Project description:G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein-protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor conformational states, explaining the pharmacological preferences of calcitonin receptor-RAMP complexes. This provides novel insight into our understanding of G protein-coupled receptor-protein interaction that is likely broadly applicable for this receptor class.

Dataset Information

Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

Publications

Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins.

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