The development of an atherosclerotic lesion in an injured vasculature is highly dependent on the proliferative state of vascular smooth muscle cells (VSMC). Bradykinin (BK) and Thromboxane are two G-protein coupled receptor ligands, whose individual binding to their respective receptors, B
2
R and TP, promotes ERK1/2-mediated VSMC proliferation. However, it is not yet known whether receptor-receptor interactions between B
2
R and TP could contribute to their co-regulation. Thus, our work addresses the hypothesis that, in VSMC, B
2
R and TP form functional hetero-complexes, of distinct trafficking and signaling properties. B
2
R-TP signaling crosstalk was first analyzed in rat VSMC by Western Blot analysis and subsequent fold/basal quantification of ERK1/2 phosphorylation. B
2
R-TP cooperation was evident through the synergistic ERK1/2 phosphorylation, in VSMC co-stimulated with optimized combinations of BK and the TP agonist, IBOP (21.05 ± 4.93 fold of basal, n=3; p < 0.001). Interestingly, however, pretreatment with the TP antagonist, SQ29548, totally abolished (BK+IBOP)-induced ERK1/2 (n=3; p < 0.001). Furthermore, knowing that B
2
R, unlike the human TPα isoform, undergoes agonist-induced sequestration and β-arrestin2 recruitment, we conducted immunofluorescence analysis on HEK293T cells overexpressing human B
2
R and human TPα (B
2
R-TPα-HEKs). While stimulation with IBOP failed to mobilize cell membranous B
2
R or TPα, substantial co-internalization of TPα and B
2
R was seen, subsequent to stimulation of B
2
R-TPα-HEKs with BK. Likewise, overexpressing β-arrestin2 in B
2
R-TPα-HEKs resulted in co-localization of B
2
R, TPα, and β-arrestin2 within internalized puncta, only after BK stimulation. Pretreatment with SQ29548 inhibited BK-induced co-internalization and β-arrestin2 recruitment. However, SQ29548 could not reverse BK-induced B
2
R sequestration in HEK293T cells overexpressing B
2
R alone, thus excluding the possibility of direct SQ29548 - B
2
R binding. Finally, results of our ongoing work are the first to show nuclear localization of B
2
R and TP within human and rat VSMC. Thus, our findings favor the likelihood of functional B
2
R-TP heterodimerization in VSMC, which could serve as a novel target for interventional strategies.
