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

Area of Science:

• Immunology
• Molecular Biology
• Cancer Research

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, producing 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 investigate the functional consequences of CCR7 isoform expression and cross-regulation.
• To elucidate the role of CCR7 splicing in fine-tuning chemokine responses.

Main Methods:

• Cloning and expression of CCR7 isoforms in mammalian cells under different promoters.
• Variant-specific RT-PCR to analyze transcript expression.
• Confocal microscopy, EGFP imaging, and HiBiT/NanoBiT assays for localization and dimerization.
• Signaling assays measuring Ca²⁺ mobilization, ERK phosphorylation, and protein recruitment (GRK3, β-arrestin1).

Main Results:

• CCR7A and CCR7B transcripts are predominant; CCR7C variants are weakly expressed.
• CCR7A efficiently targets 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, reducing its surface expression and signaling.
• MDA-MB-231 breast cancer cells expressing CCR7A/B showed impaired chemokine-induced migration.

Conclusions:

• CCR7A is the primary functional isoform mediating chemokine responses.
• CCR7C is a less active, CCL19-specific receptor with inefficient membrane targeting.
• CCR7B acts as a dominant-negative regulator, attenuating CCR7A function.
• CCR7 alternative splicing provides a mechanism to modulate immune and cancer cell responses to chemokines.