Student Theses and Dissertations

Date of Award

2020

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Sakmar Laboratory

Abstract

G protein-coupled receptors (GPCRs) are known to interact with several other classes of integral membrane proteins. However, the extent of these interactions and their role in regulating GPCR-mediated transmembrane signaling is not well understood. For example, receptor activitymodifying proteins (RAMPs), a family of single transmembrane proteins with only three members, are ubiquitously expressed and have been shown to interact with several different GPCRs. Most research to date has focused on the ability of RAMPs to modulate the function of several GPCRs in the secretin-like GPCR family. GPCR-RAMP interactions were shown to affect the ligand binding affinity of two different secretin-like GPCRs, causing the functional diversity of the GPCRs to be driven by an interacting protein. Yet, potential direct interactions among the three known RAMPs and hundreds of non-olfactory GPCR has never been investigated and whether RAMP-GPCR interactions are widespread remains an open question. To determine the breadth of GPCR-RAMP interactions, we first investigated the global coexpression and coevolution between GPCRs and RAMPs. On the one hand, if many GPCRs interact with RAMPs we would except to see statistically significant coexpression and coevolution in comparison to random gene pairs. On the other hand, if GPCR-RAMP interactions are limited to a small number of genes, then averaged coexpression and coevolution would be similar to that expected by chance. To calculate coexpression, we analyzed an RNASeq database of human transcriptomes across 53 different tissues and found that coexpression between non-olfactory GPCRs and RAMPs is significantly higher than random gene pairs. We also analyzed genomic data from all currently available sequenced organisms to calculate the coevolution between non-olfactory GPCRs and RAMPs. We discovered that GPCRs and RAMPs have a significant percentage of shared species and significantly correlated phylogenetic trees. Our results support the hypothesis that GPCRs interact globally with RAMPs. Only a handful of GPCR-RAMP interactions have been reported to date, but our coexpression and coevolution analysis suggested that additional GPCRs interact with RAMPs. To begin to address the potential for direct interactions among the three known RAMPs and hundreds of GPCRs, we developed a highly multiplexed immunoassay using a suspension bead array (SBA) assay designed to detect RAMP-GPCR complexes. We engineered three epitopetagged RAMPs and 23 epitope-tagged GPCRs, including all members of the secretin-like family of GPCRs, as well as eight other GPCRs. We then used 64 antibodies raised against native RAMPs and GPCRs, along with four antibodies targeting the epitope tags, to multiplex the SBA assay to detect and measure all possible combinations of interaction among the 23 GPCRs and three RAMPs from detergent-solubilized lysates. We also used the epitope-tagged constructs to verify a collection of antibodies that target native GPCRs and RAMPs. We validated nearly all previously reported secretin-like GPCR-RAMP interactions, and also found previously unidentified RAMP interactions with additional secretin-like GPCRs, chemokine receptors, and orphan receptors. Using in situ proximity ligation assay, we verified a subset of these novel GPCR-RAMP interactions in cell membranes. The results of the SBA assay provide a complete interactome of secretin-like GPCRs with RAMPs. GPCR-RAMP interactions are more common than previously appreciated, and the SBA strategy will be useful to search for additional GPCRRAMP complexes and other interacting membrane protein pairs in cell lines and tissues.

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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