Functional Analysis of CXCR3 Splicing Variants and Their Ligands Using NanoBiT-Based Molecular Interaction Assays
Huong Thi Nguyen1,2, Sunghoon Hurh1,2, Lan Phuong Nguyen1
1Department of Biomedical Sciences, College of Medicine, Korea University, Seoul 02841, Korea.
Insights
CXCR3 isoforms interact to modulate cell surface expression and signaling. CXCR3Alt isoform negatively impacts CXCR3A and CXCR3B function, affecting leukocyte trafficking.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- CXCR3 (C-X-C chemokine receptor type 3) is crucial for leukocyte trafficking and is generated in three human isoforms via alternative splicing.
- Previous research on CXCR3 isoforms lacks correlation between biochemical data and biological relevance.
- Co-expression of CXCR3 isoforms suggests potential mutual effects on chemokine binding and cellular responses.
Purpose of the Study:
- To investigate the functional relationships among CXCR3 splicing variants and their chemokine-dependent signaling pathways.
- To analyze how CXCR3 isoforms interact to influence cell surface expression, ligand binding, and cellular responses.
Main Methods:
- Utilized NanoBiT live cell protein interaction assays for integrative analysis of CXCR3 isoform interactions.
- Employed RT-PCR to assess expression patterns of CXCR3 splicing variants.
- Used Jurkat cells for evaluating endogenous and exogenous CXCR3 isoform effects on chemotaxis.
Main Results:
- CXCR3 N-terminal region influences cell surface expression and ligand activation.
- CXCR3A shows high plasma membrane expression and responds to I-TAC, IP-10, and MIG chemokines.
- CXCR3B exhibits low plasma membrane expression, responding to I-TAC, while CXCR3Alt is poorly expressed and inhibits CXCR3A/B function and responses.
- CXCR3A enhances chemotaxis, but CXCR3B and CXCR3Alt co-expression diminishes this effect; PF-4 chemokine shows no CXCR3-mediated response.
Conclusions:
- NanoBiT technology is effective for studying CXCR3-mediated cell signaling and molecular interactions among isoforms.
- CXCR3 isoform interactions significantly impact cell surface expression, ligand-dependent activation, and chemotaxis.
- Understanding these interactions is vital for elucidating CXCR3's role in pathophysiological conditions and leukocyte trafficking.
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.
