Functional CCR7A-mediated cellular responses are negatively modulated by the splice variant CCR7B

Area of Science:

• Immunology
• Molecular Biology
• Cell Biology

Background:

• C-C chemokine receptor 7 (CCR7) is crucial for immune cell trafficking to lymphoid organs.
• CCR7 ligands CCL19 and CCL21 are implicated in cancer metastasis.
• Human CCR7 undergoes alternative splicing, yielding three protein isoforms (CCR7A, CCR7B, CCR7C) with undefined properties.

Purpose of the Study:

• To characterize the expression, localization, signaling, and interactions of CCR7A, CCR7B, and CCR7C isoforms.
• To elucidate the functional consequences of CCR7 isoform expression and cross-regulation.
• To investigate the role of CCR7 isoforms in immune cell homing and cancer cell migration.

Main Methods:

• Cloning and expression of CCR7 isoforms in mammalian cells under different promoter strengths.
• Variant-specific RT-PCR for transcript analysis.
• EGFP imaging, HiBiT and NanoBiT assays for localization and protein interactions.
• Chemokine-induced signaling assays (Ca²⁺ mobilization, ERK phosphorylation, G protein recruitment).
• Co-immunoprecipitation and confocal microscopy.
• Cell migration assays using MDA-MB-231 breast cancer cells.

Main Results:

• CCR7A and CCR7B transcripts are predominant; CCR7C variants are weakly expressed.
• CCR7A localizes to the plasma membrane and mediates robust chemokine signaling.
• CCR7C shows partial membrane localization and weak, CCL19-biased signaling.
• CCR7B is cytosolic, non-signaling, and forms dimers with CCR7A and CCR7C.
• CCR7B co-expression inhibits CCR7A surface expression and signaling, and impairs cancer cell migration.

Conclusions:

• CCR7A is the primary functional isoform mediating chemokine responses.
• CCR7C is a weakly signaling, inefficiently targeted isoform.
• CCR7B acts as a dominant-negative regulator, dampening CCR7A function and impacting cell migration.
• Alternative splicing of CCR7 fine-tunes chemokine responsiveness in immune and cancer cells.