Proteomic identification and validation of novel interactions of the putative tumor suppressor PRELP with membrane proteins including IGFI-R and p75NTR

Hirofumi Kosuge,Makoto Nakakido,Satoru Nagatoishi,Tetsuya Fukuda,Yasuhiko Bando,Shin-Ichi Ohnuma,Kouhei Tsumoto

doi:10.1016/j.jbc.2021.100278

Abstract

Proline and arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich repeat proteoglycans (SLRPs) family. Levels of PRELP mRNA are downregulated in many types of cancer, and PRELP has been reported to have suppressive effects on tumor cell growth, although the molecular mechanism has yet to be fully elucidated. Given that other SLRPs regulate signaling pathways through interactions with various membrane proteins, we reasoned that PRELP likely interacts with membrane proteins to maintain cellular homeostasis. To identify membrane proteins that interact with PRELP, we carried out coimmunoprecipitation coupled with mass spectrometry (CoIP-MS). We prepared membrane fractions from Expi293 cells transfected to overexpress FLAG-tagged PRELP or control cells and analyzed samples precipitated with anti-FLAG antibody by mass spectrometry. Comparison of membrane proteins in each sample identified several that seem to interact with PRELP; among them, we noted two growth factor receptors, insulin-like growth factor I receptor (IGFI-R) and low-affinity nerve growth factor receptor (p75NTR), interactions with which might help to explain PRELP’s links to cancer. We demonstrated that PRELP directly binds to extracellular domains of these two growth factor receptors with low micromolar affinities by surface plasmon resonance analysis using recombinant proteins. Furthermore, cell-based analysis using recombinant PRELP protein showed that PRELP suppressed cell growth and affected cell morphology of A549 lung carcinoma cells, also at micromolar concentration. These results suggest that PRELP regulates cellular functions through interactions with IGFI-R and p75NTR and provide a broader set of candidate partners for further exploration.

Highlights

Protein–protein interactions are crucial for biological functions
Human Proline and arginine-rich end leucine-rich repeat protein (PRELP) with a FLAGtag was overexpressed in Expi293 cells, a human cell line in which proteins can be expressed at high levels
The addition of recombinant PRELP (rPRELP) resulted in a change of cell morphology to round shapes, as observed by overexpression of PRELP [20]. This change of cell morphology was observed only in the micromolar range, namely low concentration of PRELP appears not to affect the morphology of A549 cells. It remains unclear whether these phenomena were triggered via the interactions of PRELP with insulin-like growth factor I receptor (IGFI-R), p75NTR, or other membrane proteins that we identified, our results indicate that PRELP functions in the micromolar range of concentration, which is consistent with the micromolar affinities of the interaction of PRELP with IGFI-R and p75NTR

Summary

Introduction

Protein–protein interactions are crucial for biological functions. Membrane proteins are important as they mediate cellular responses to the environment [1, 2]. To identify novel membrane proteins that interact with PRELP, we used coimmunoprecipitation coupled with mass spectrometric analysis (CoIP-MS). We identified various membrane proteins, including two growth factor receptors, IGFI-R and low-affinity nerve growth factor receptor (p75NTR), as interacting partners.

Results

Conclusion