Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Sakmar Laboratory

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 solubilized cell lysates and validated anti-GPCR Abs in hand, we proceeded to map the GPCR-RAMP interactome. First, we conducted a proof-of-concept study with 23 GPCRs, mainly belonging to the secretin family, and successfully mapped their interactions with each RAMP. Then we scaled up the procedure and mapped the interactions between the 215 GPCRs in our library and the three RAMPs. We detected novel GPCR-RAMP interactions across all subfamilies, as detected by both anti-epitope tag capture methods and anti- GPCR capture methods. We then applied the anti-GPCR Ab library to detect native GPCRRAMP complexes in solubilized membranes from neuroepithelioma and neuroblastoma cells. To follow up on our findings, we selected one newly identified RAMP-interacting GPCR and functionally characterized the consequences of RAMP interaction on its pharmacology. We studied Mas-related GPCR subtype X4 (MRGPRX4), which has been recently identified as a receptor for bile acids and has been implicated in itch in cholestatic liver diseases. Using the SBA assay and a proximity ligation assay, we first showed that MRGPRX4 interacts with RAMPs. We then found that the interaction of MRGPRX4 with RAMP2 causes attenuation of both basal and agonist-dependent signaling, which correlates with a decrease of MRGPRX4 cell surface expression as measured using a quantitative NanoBRET pulse-chase assay. We then used AlphaFold Multimer to predict the structure of the MRGPRX4-RAMP2 complex. The discovery that RAMP2 downregulates MRGPRX4 surface expression may have direct implications for future drug development for cholestatic itch. The application of the multiplexed SBA assay methodology to study membrane proteinprotein interactions suggests that RAMPs interact with many more GPCRs than had been previously known. These findings, especially when combined with recent structural studies of membrane protein complexes, have significant implications for advancing GPCR-targeted drug discovery and the understanding of GPCR pharmacology, biology and regulation. Moreover, the multiplexed platform that we established can be readily adapted for a range of basic and translational applications, including screening of pathological anti-GPCR autoantibodies and interrogation of other protein-protein interactions.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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