Abstract 133: Mechanistic Insights Into Bradykinin and Thromboxane Receptors Heterodimerization in Vascular Smooth Muscle Cells

Abstract

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.