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Identification of cell surface receptors for the novel adipokine CTRP3
Author(s) -
Peterson Jonathan M
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1249.2
Subject(s) - receptor , gene knockdown , flow cytometry , nonalcoholic fatty liver disease , adipose tissue , insulin receptor , fatty liver , cell , adipokine , insulin , biology , chemistry , medicine , endocrinology , insulin resistance , immunology , biochemistry , disease , apoptosis
C1q TNF Related Protein 3 (CTRP3) is a member of a family of secreted proteins that exert a multitude of biological effects throughout the body. Our initial work shows promise in the development of CTRP3‐induced cellular processes as a means to combat nonalcoholic fatty liver disease (NAFLD). Clinically, NAFLD is defined as the excessive accumulation of fat in the liver, usually due to obesity, and NAFLD effects 1 in 10 Americans. Our previous data show that when high fat fed mice are treated with CTRP3 they are protected from developing NAFLD. However, the mechanism for this effect remains unclear. The purpose of this project was to identify the unknown receptor that mediates the hepatic actions of CTRP3. METHODS We used Ligand‐receptor glycocapture technology with TriCEPS™‐based ligand‐receptor capture (LRC‐TriCEPS; Dualsystems Biotech AG). The LRC‐TriCEPS experiment with CTRP3‐FLAG protein as ligand and Insulin as the control ligand was performed on H4IIE rat hepatoma cell line. Additional analysis using siRNA induced knockdown of the identified receptors was used to validate the findings. RESULTS Initial analysis demonstrated efficient coupling of TriCEPS to CTRP3. Further, flow cytometry analysis demonstrated successful oxidation and crosslinking of CTRP3‐TriCEPS and Insulin‐TriCEPS to the cell surface glycans. In total, an enrichment of glycopeptides of 11% (261 peptides) was observed. Under these conditions, INSR (Insulin receptor) could be identified and quantified in the control dataset. For the ligand sample two unique receptors were identified. Flow cytometry analysis with siRNA induced knockdown of these proteins confirmed that the presence of the protein is needed for CTRP3 binding to occur. CONCLUSION The LRC‐TriCEPS methodology was successful in identifying the receptor for CTRP3. RELEVANCE The identification of the receptors for CTRP3 and CTRP3‐induced cellular signaling pathways are important prerequisites for the development of small molecule drug candidates that exert effects beneficial to human health. Support or Funding Information East Tennessee State University Research Development Committee National Institute On Alcohol Abuse And Alcoholism of the National Institutes of Health under Award Number R03AA023612