In humans, thromboxane (TX) A2 signals through the TPα and TPβ isoforms of the TXA2 receptor that exhibit common and distinct roles. For example, Gq/phospholipase (PL)Cβ signaling by TPα is directly inhibited by the vasodilators prostacyclin and nitric oxide (NO) whereas that signaling by TPβ is unaffected. Herein, we investigated whether TPα and/or TPβ regulate G12/Rho activation and whether that signaling might be differentially regulated by prostacyclin and/or NO. Both TPα and TPβ independently regulated RhoA activation and signaling in clonal cells over-expressing TPα or TPβ and in primary human aortic smooth muscle cells (1° AoSMCs). While RhoA-signaling by TPα was directly impaired by prostacyclin and NO through protein kinase (PK)A- and PKG-dependent phosphorylation, respectively, signaling by TPβ was not directly affected by either agent. Collectively, while TPα and TPβ contribute to RhoA activation, our findings support the hypothesis that TPα is involved in the dynamic regulation of haemostasis and vascular tone, such as in response to prostacyclin and NO. Conversely, the role of TPβ in such processes remains unsolved. Data herein provide essential new insights into the physiologic roles of TPα and TPβ and, through studies in AoSMCs, reveal an additional mode of regulation of VSM contractile responses by TXA2.

In humans, thromboxane (TX)A

Running Title: Role of Rab11 and Rab4 in recycling of the human prostacyclin receptor.Key Words: human prostacyclin receptor, internalization, Rab11a, Rab4a, yeast-two-hybrid, GPCR. -2 - ABSTRACTThe human prostacyclin receptor (hIP) undergoes agonist-induced internalization but the mechanisms regulating its intracellular trafficking and/or recycling to the plasma membrane are poorly understood.Herein, we conducted a yeast-two-hybrid screen to identify proteins interacting with the carboxyl terminal (C)-tail domain of the hIP and discovered a novel interaction with Rab11a. This interaction was confirmed by co-immunoprecipitations in mammalian HEK293 and was augmented by cicaproststimulation. The hIP co-localized to Rab11-containing recycling endosomes in both HEK293 and endothelial EA.hy 926 cells in a time-dependent manner following cicaprost-stimulation. Moreover, over-expression of Rab11a significantly increased recycling of the hIP, while the dominant negative Rab11S25N impaired that recycling. Conversely, while the hIP co-localized to Rab4-positive endosomes in response to cicaprost, ectopic expression of Rab4a did not substantially affect overall recycling nor did Rab4a directly interact with the hIP. The specific interaction between the hIP and Rab11a was dependent on a 22 amino acid (Val 299 -Gln 320 ) sequence within its C-tail domain and was independent of isoprenylation of the hIP. This study elucidates a critical role for Rab11a in regulating trafficking of the hIP and has identified a novel Rab11 binding-domain (RBD) within its C-tail domain that is both necessary and sufficient to mediate interaction with Rab11a.

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The human prostacyclin receptor (hIP) undergoes rapid agonist-induced internalization by largely unknown mechanism(s). Herein the involvement of Rab5 in regulating cicaprost-induced internalization of the hIP expressed in human embryonic kidney 293 cells was investigated. Over-expression of Rab5a significantly increased agonist-induced hIP internalization. Additionally, the hIP co-localized to Rab5a-containing endocytic vesicles in response to cicaprost stimulation and there was a coincident net translocation of Rab5 from the cytosol/soluble fraction of the cell. Co-immunoprecipitation studies confirmed a direct physical interaction between the hIP and Rab5a that was augmented by cicaprost. Whilst the dominant negative Rab5aS34N did not show decreased interaction with the hIP or fully impair internalization, it prevented hIP sorting to endocytic vesicles. Moreover, the GTPase deficient Rab5aQ79L significantly increased internalization and co-localized with the hIP in enlarged endocytic vesicles. While deletion of the carboxyl terminal (C)-tail domain of the hIP did not inhibit agonist-induced internalization, co-localization or co-immunoprecipitation with Rab5a per se, receptor trafficking was altered suggesting that it contains structural determinant(s) for hIP sorting post Rab5-mediated endocytosis. Taken together, data herein and in endothelial EA.hy 926 cells demonstrate a direct role for Rab5a in agonist-internalization and trafficking of the hIP and increases knowledge of the factors regulating prostacyclin signaling.