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

• Published in: The Journal of biological chemistry Vol. 296; p. 100278
• Main Authors: Kosuge, Hirofumi, Nakakido, Makoto, Nagatoishi, Satoru, Fukuda, Tetsuya, Bando, Yasuhiko, Ohnuma, Shin-ichi, Tsumoto, Kouhei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.01.2021; American Society for Biochemistry and Molecular Biology
• Subjects: A549 Cells; Carcinoma, Non-Small-Cell Lung - genetics; Carcinoma, Non-Small-Cell Lung - pathology; coimmunoprecipitation; Extracellular Matrix Proteins - genetics; Gene Expression Regulation, Neoplastic - genetics; Glycoproteins - genetics; growth factor receptor; Humans; membrane protein; Nerve Tissue Proteins - genetics; PRELP; protein–protein interaction; proteomics; Proteomics - methods; Receptors, Nerve Growth Factor - genetics; Recombinant Proteins - genetics; RNA, Messenger; small leucine-rich proteoglycan (SLRP); surface plasmon resonance (SPR); p75NTR; rPRELP; LRP1; protein–protein interaction; SLRPs; PRELP; surface plasmon resonance (SPR); DDM; LRR; rsp75NTR; NF-κB; SPR; CoIP-MS; KD; TGF-β; ESI; P1; P2; P3; proteomics; GO; IGFs; growth factor receptor; rsIGFI-R; NGF; mAb; small leucine-rich proteoglycan (SLRP); membrane protein; IGFI-R; coimmunoprecipitation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2021.100278

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.