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

C-C chemokine receptor 7 (CCR7) directs immune cell homing to secondary lymphoid organs and has been implicated in cancer metastasis through its ligands CCL19 and CCL21. Human CCR7 pre-mRNA undergoes alternative splicing to generate five transcripts that encode three protein isoforms with distinct N-termini, termed CCR7A, CCR7B, and CCR7C, but their comparative properties and cross-regulation are not well defined. Here, we cloned these three isoforms and systematically characterized their expression, localization, signaling, and mutual interactions in mammalian cells under both strong (CMV) and weaker (ubiquitin C, UbiC) promoter control to reduce overexpression-related artifacts. Variant-specific RT-PCR revealed that transcripts encoding CCR7A (V1) and CCR7B (V2) predominate in diverse human cell lines, whereas CCR7C-encoding variants (V3-V5) are weakly expressed. EGFP imaging and HiBiT-based assays showed efficient plasma-membrane targeting of CCR7A, partial membrane localization and prominent perinuclear accumulation of CCR7C, and largely cytosolic retention of CCR7B. Under UbiC-driven expression, CCR7A mediated robust CCL19- and CCL21-induced Gi/o and Gq-like activation, intracellular Ca² ⁺ mobilization, ERK phosphorylation, GRK3-dependent Gβ1 recruitment, and β-arrestin1 binding, whereas CCR7C displayed weaker and mainly CCL19-biased signaling. CCR7B did not respond to either chemokine in any signaling readout and thus behaved as a non-signaling isoform. NanoBiT-based assays and co-immunoprecipitation demonstrated that all three isoforms form homo- and heterodimers, with particularly strong association between CCR7A and CCR7B. Co-expression of CCR7B reduced CCR7A surface expression and markedly attenuated chemokine-induced Ca² ⁺ responses, mini-Gi interaction, and β-arrestin1 recruitment, while confocal microscopy revealed redistribution of CCR7A-EGFP from the plasma membrane to intracellular compartments. Moreover, MDA-MB-231 breast cancer cells, which express CCR7A and CCR7B transcripts, did not migrate toward CCL19 or CCL21 despite preserved motility toward low-serum medium. These findings identify CCR7A as the dominant functional isoform, CCR7C as a weak CCL19-biased receptor with inefficient membrane targeting, and CCR7B as a non-signaling dominant-negative isoform that dampens CCR7A-mediated responses, suggesting that CCR7 splicing fine-tunes chemokine responsiveness in immune and cancer cells.