Heteromerization fingerprints between bradykinin B2 and thromboxane TP receptors in native cells

Abstract

Bradykinin (BK) and thromboxane-A 2 (TX-A 2 ) are two vasoactive mediators that modulate vascular tone and inflammation via binding to their cognate “class A” G-protein coupled receptors (GPCRs), BK-B2 receptors (B2R) and TX-prostanoid receptors (TP), respectively. Both BK and TX-A 2 lead to ERK1/2-mediated vascular smooth muscle cell (VSMC) proliferation and/or hypertrophy. While each of B2R and TP could form functional dimers with various GPCRs, the likelihood that B2R-TP heteromerization could contribute to their co-regulation has never been investigated. The main objective of this study was to investigate the mode of B2R and TP interaction in VSMC, and its possible impact on downstream signaling. Our findings revealed synergistically activated ERK1/2 following co-stimulation of rat VSMC with a subthreshold dose of BK and effective doses of the TP stable agonist, IBOP, possibly involving biased agonist signaling. Single detection of each of B2R and TP in VSMC, using in-situ proximity ligation assay (PLA), provided evidence of the constitutive expression of nuclear and extranuclear B2R and TP. Moreover, inspection of B2R-TP PLA signals in VSMC revealed agonist-modulated nuclear and extranuclear proximity between B2R and TP, whose quantification varied substantially following single versus dual agonist stimulations. B2R-TP interaction was further verified by the findings of co-immunoprecipitation (co-IP) analysis of VSMC lysates. To our knowledge, this is the first study that provides evidence supporting the existence of B2R-TP heteromerization fingerprints in primary cultured VSMC.

Vascular Cells Proteome Associated with Bradykinin and Leptin Inflammation and Oxidative Stress Signals

“…The stimulation of RASMC started at the same time and the treatment was stopped at the specified time of each condition to minimize the batch effect in the experiment. The selection of these concentrations was based on previous studies [ 29 , 30 , 31 ].…”

Androgen Effects on the Adrenergic System of the Vascular, Airway, and Cardiac Myocytes and Their Relevance in Pathological Processes

“…VSM constriction is caused by increments in [Ca 2+ ] i [ 171 ]. Vasoconstrictor agonists act on GPCRs coupled to the q α subunit (GPCR- q α ) such as α 1A -, α 1B -, and α 1D -ARs, bradykinin, histamine H 1 , and thromboxane-A 2 receptors, among others [ 172 – 174 ]. These receptors activate the PLC enzyme and IP 3 signaling pathway, inducing the release of Ca 2+ from the sarcoplasmic reticulum (SR) and the influx of this ion through VDCCs [ 175 ].…”

Roles for heterodimerization of APJ and B2R in promoting cell proliferation via ERK1/2-eNOS signaling pathway

“…Although GPCRs were initially thought to perfectly function as monomeric entities, increasing evidence suggests that they can exist as dimers or oligomers which affect ligand binding, receptor traffic and signaling [3]. Numerous GPCRs have been shown to dimerize, and APJ or B2R are no exception [4,5].…”

“…Likewise, the direct interaction between B2R and other GPCRs has been confirmed, causing changes in cell signaling and receptor functions. It has been demonstrated that B2R dimerize with TX-prostanoid receptors [5], dopamine D2 receptor (D2R) [17], AT 1R and AT2R [18,19], P2Y2 ATP receptors [20], as well as with KOR [21].…”