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Detection of Concordance between Transcriptional Levels of GPCRs and Receptor-Activity-Modifying Proteins.

ABSTRACT: A recent phylogenetic analysis showed global co-evolution of G protein-coupled receptors (GPCRs) and receptor-activity-modifying proteins (RAMPs) suggesting global interactions between these two protein families. Experimental validation of these findings is challenging because in humans whereas there are only three genes encoding RAMPs, there are about 800 genes encoding GPCRs. Here, we report an experimental approach to evaluate GPCR-RAMP interactions. As a proof-of-concept experiment, we over-expressed RAMP2 in HEK293T cells and evaluated the effect on the transcriptional levels of 14 representative GPCRs that were selected based on the earlier phylogenetic analysis. We utilized a multiplexed error-correcting fluorescence in situ hybridization (MERFISH) method to detect message levels for individual GPCRs in single cells. The MERFISH results showed changes in GPCR message levels with RAMP2 over-expression in a concordant pattern that was predicted by the earlier phylogenetic analysis. These results provide additional evidence that GPCR-RAMP interactions are more widespread than previously appreciated and that these interactions have functional consequences.

SUBMITTER: Barbash S

PROVIDER: S-EPMC6354700 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Detection of Concordance between Transcriptional Levels of GPCRs and Receptor-Activity-Modifying Proteins.

Barbash Shahar S Persson Torbjörn T Lorenzen Emily E Kazmi Manija A MA Huber Thomas T Sakmar Thomas P TP

iScience 20181231

A recent phylogenetic analysis showed global co-evolution of G protein-coupled receptors (GPCRs) and receptor-activity-modifying proteins (RAMPs) suggesting global interactions between these two protein families. Experimental validation of these findings is challenging because in humans whereas there are only three genes encoding RAMPs, there are about 800 genes encoding GPCRs. Here, we report an experimental approach to evaluate GPCR-RAMP interactions. As a proof-of-concept experiment, we over- ...[more]

PMID: 30660104

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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

Detection of Concordance between Transcriptional Levels of GPCRs and Receptor-Activity-Modifying Proteins.

Publications

Detection of Concordance between Transcriptional Levels of GPCRs and Receptor-Activity-Modifying Proteins.

Similar Datasets

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