Multiplexed Mapping of the Interactome of G Protein-Coupled Receptors and Receptor Activity-Modifying Proteins
type: CreativeWork
description: Receptor activity-modifying proteins (RAMPs) have emerged as modulators of many aspects of G protein-coupled receptor (GPCR) biology and pharmacology. The RAMP family was discovered more than two decades ago and GPCR-RAMP interactions and their functional consequences on receptor trafficking and ligand selectivity have been documented for several GPCRs, mostly belonging to the secretin family. However, the pervasiveness of GPCR-RAMP interactions and the mechanisms of their effects are not well understood. Recent bioinformatics and experimental studies suggest that GPCR-RAMP interactions might be much more widespread than previously anticipated. However, potential direct interactions among the three known RAMPs (RAMP1, RAMP2 and RAMP3) and hundreds of GPCRs have never been investigated. To address this gap in knowledge about the GPCR-RAMP interactome, we developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized cell lysates. To enable multiplexing, we engineered a library of GPCRs and validated a library of anti- GPCR antibodies (Abs). We first prepared a library of dual epitope-tagged GPCR clones and complementary dual epitope-tagged RAMPs. We expressed each clone in the library in cultured cells to generate solubilized membranes harboring each GPCR alone, or together with each RAMP. We next tested >400 anti-GPCR Abs from the Human Protein Atlas targeting our customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that ~61% of Abs tested were selective for their intended target, ~11% bound offtarget, and ~28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeutic Abs. With the libraries of solubilize…
abstract: Receptor activity-modifying proteins (RAMPs) have emerged as modulators of many aspects of G protein-coupled receptor (GPCR) biology and pharmacology. The RAMP family was discovered more than two decades ago and GPCR-RAMP interactions and their functional consequences on receptor trafficking and ligand selectivity have been documented for several GPCRs, mostly belonging to the secretin family. However, the pervasiveness of GPCR-RAMP interactions and the mechanisms of their effects are not well understood. Recent bioinformatics and experimental studies suggest that GPCR-RAMP interactions might be much more widespread than previously anticipated. However, potential direct interactions among the three known RAMPs (RAMP1, RAMP2 and RAMP3) and hundreds of GPCRs have never been investigated. To address this gap in knowledge about the GPCR-RAMP interactome, we developed a multiplexed suspension bead array (SBA) immunoassay to detect GPCR-RAMP complexes from detergent-solubilized cell lysates. To enable multiplexing, we engineered a library of GPCRs and validated a library of anti- GPCR antibodies (Abs). We first prepared a library of dual epitope-tagged GPCR clones and complementary dual epitope-tagged RAMPs. We expressed each clone in the library in cultured cells to generate solubilized membranes harboring each GPCR alone, or together with each RAMP. We next tested >400 anti-GPCR Abs from the Human Protein Atlas targeting our customized library of 215 expressed and solubilized GPCRs representing all GPCR subfamilies. We found that ~61% of Abs tested were selective for their intended target, ~11% bound offtarget, and ~28% did not bind to any GPCR. Antigens of on-target Abs were, on average, significantly longer, more disordered, and less likely to be buried in the interior of the GPCR protein than the other Abs. These results provide important insights into the immunogenicity of GPCR epitopes and form a basis for designing therapeutic Abs. With the libraries of solubilize…
author: Kotliar, Ilana Beth
keywords: GPCR-RAMP interactions, G protein-coupled receptors (GPCRs), multiplexed protein screening, GPCR pharmacology, RAMP2 regulation, cholestatic itch therapy, Life Sciences
date published: 2023-01-01T08:00:00Z
date modified: 2026-07-24T04:11:21Z
provider: Rockefeller
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Rockefeller