Functional Analysis of CXCR3 Splicing Variants and Their Ligands Using NanoBiT-Based Molecular Interaction Assays

Huong Thi Nguyen, Sunghoon Hurh, Lan Phuong Nguyen, Thai Uy Nguyen, Hee Kyung Park, Jae Young Seong, Cheol Soon Lee, Byung Joo Ham, Jong Ik Hwang

Research output: Contribution to journalArticlepeer-review

Abstract

CXCR3 regulates leukocyte trafficking, maturation, and various pathophysiological conditions. Alternative splicing generates three CXCR3 isoforms in humans. Previous studies investigated the roles of CXCR3 isoforms, and some biochemical data are not correlated with biological relevance analyses. RT-PCR analyses indicate that most cells express all three splicing variants, suggesting that they may mutually affect the chemokine binding and cellular responses of other splicing variants. Here, we performed an integrative analysis of the functional relations among CXCR3 splicing variants and their chemokine-dependent signaling using NanoBiT live cell protein interaction assays. The results indicated that the CXCR3 N-terminal region affected cell surface expression levels and ligand-dependent activation. CXCR3A was efficiently expressed in the plasma membrane and responded to I-TAC, IP-10, and MIG chemokines. By contrast, CXCR3B had low plasma membrane expression and mediated I-TAC–stimulated cellular responses. CXCR3Alt was rarely expressed on the cell surface and did not mediate any cell responses to the tested chemokines; however, CXCR3Alt negatively affected the plasma membrane expression of CXCR3A and CXCR3B and their chemokine-stimulated cellular responses. Jurkat cells express endogenous CXCR3, and exogenous CXCR3A expression enhanced chemotactic activity in response to I-TAC, IP-10, and MIG. By contrast, exogenous expression of CXCR3B and CXCR3Alt eliminated or reduced the CXCR3A-induced chemotactic activity. The PF-4 chemokine did not activate any CXCR3-mediated cellular responses. NanoBiT technology are useful to integrative studies of CXCR3-mediated cell signaling, and expand our knowledge of the cellular responses mediated by molecular interactions among the splicing variants, including cell surface expression, ligand-dependent receptor activation, and chemotaxis.

Original languageEnglish
Pages (from-to)281-297
Number of pages17
JournalMolecules and cells
Volume46
Issue number5
DOIs
Publication statusPublished - 2023 May 31

Bibliographical note

Publisher Copyright:
© The Korean Society for Molecular and Cellular Biology.

Keywords

  • CXCR3
  • I-TAC
  • IP-10
  • MIG
  • NanoBiT technology
  • chemotaxis

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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