Multiplexed Mapping of the Interactome of GPCRs with Receptor Activity-Modifying Proteins

Receptor activity-modifying proteins (RAMPs) have been described to form complexes with G protein-coupled receptors (GPCRs). Here, we present a comprehensive GPCR-RAMP interactome based on a library of 215 Dual Epitope-Tagged (DuET) GPCRs representing all GPCR subfamilies and co-expressed each GPCR with each of the three RAMPs.

The provided data describes multiplexed affinity-based measurements of the GPCRs and RAMPs.

The GPCRs were engineered to carry 1D4 and FLAG epitope tags, while the RAMPS carried OLLAS and HA tags. The receptors were co-overexpressed in Expi293F cells and solubilized for the interaction assays using Luminex's suspension bead array (SBA) technology. The platform is based on color-coded beads to distinguish the different assays. GPCRs and/or RAMPs were captured by antibodies on the beads, and another fluorescently labeled antibody was used for detection. The epitope tag capture and detection combinations allowed us to determine the interactions between GPCRs and RAMPs. We also used antibodies validated for GPCRs and RAMPs from a previous study (10.1126/sciadv.aaw2778) with open-access data (10.17044/scilifelab.21907995).

The resulting output data are the median fluorescence intensity (MFI) values for each sample and bead. MFI represents a relative measurement of the receptors being captured by the antibody-conjugated beads. The submission also contains the data processed by Z-scores and robust Z-scores.

The resource also contains data from

• a small pilot study to test the system,
• interaction SBA-screening of untransfected cells,
• interaction analysis in native membranes from the MolBoolean assay (https://doi.org/10.1038/s41467-022-32395-w).

File descriptions (version 2024-05-29)

• `antibodies.csv` contains information about antibodies used in the GPCR-RAMP interaction overexpression screen.
• `endog_antibodies.csv` contains information about antibodies used in the screen for endogenous interactions.
• `endog_mfi.csv` contains data (signal-to-background ratios of MFI) for the assays of the endogenous interaction screening.
• `endog_well_info.csv` contains sample information for the assays of the endogenous interaction screening.
• `long_data.csv` contains sample information and measurements (MFI, Z-scores, and robust Z-scores) for samples in the GPCR-RAMP interaction over-expression screen.
• `molboolean.xlsx` contains measurements (RCPs/cell) from the MolBoolean native membrane interaction assays.
• `pilot_plate.xlsx` contains sample info and measurements (MFI) from a small-scale pilot plate for GPCR-RAMP interactions.