Elizebeth C. Turner

Identification of an Interaction between the TPα and TPβ Isoforms of the Human Thromboxane A2 Receptor with Protein Kinase C-related Kinase (PRK) 1 IMPLICATIONS FOR PROSTATE CANCER

In humans, thromboxane (TX) A2 signals through the TPα and TPβ isoforms of the TXA2 receptor or TP. Here, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPα and ΤPβ involving common and unique sequences within their respective C-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1640–942). Although the interaction with PRK1 is constitutive, agonist activation of TPα/TPβ did not regulate the complex per se but enhanced PRK1 activation leading to phosphorylation of its general substrate histone H1 in vitro. Altered PRK1 and TP expression and signaling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist activation of TPα/TPβ led to phosphorylation of histone H3 at Thr11 (H3 Thr11), a previously recognized specific marker of androgen-induced chromatin remodeling, in the prostate LNCaP and PC-3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen-responsive LNCaP but not in nonresponsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPα/TPβ in both LNCaP and PC-3 cells, and targeted disruption of PRK1 impaired TPα/TPβ-mediated H3 Thr11 phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA2 as a potent mediator of RhoA signaling, the identification of PRK1 as a bona fide interactant of TPα/TPβ, and leading to H3 Thr11 phosphorylation to regulate cell migration, has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated.

Interaction of the Human Prostacyclin Receptor with Rab11 CHARACTERIZATION OF A NOVEL Rab11 BINDING DOMAIN WITHIN α-HELIX 8 THAT IS REGULATED BY PALMITOYLATION

The human prostacyclin receptor (hIP) undergoes agonist-induced internalization and subsequent recyclization in slowly recycling endosomes involving its direct physical interaction with Rab11a. Moreover, interaction with Rab11a localizes to a 22-residue putative Rab11 binding domain (RBD) within the carboxyl-terminal tail of the hIP, proximal to the transmembrane 7 (TM7) domain. Because the proposed RBD contains Cys308 and Cys311, in addition to Cys309, that are known to undergo palmitoylation, we sought to identify the structure/function determinants of the RBD, including the influence of palmitoylation, on agonist-induced trafficking of the hIP. Through complementary approaches in yeast and mammalian cells along with computational structural studies, the RBD was localized to a 14-residue domain, between Val299 and Leu312, and proposed to be organized into an eighth α-helical domain (α-helix 8), comprising Val299–Val307, adjacent to the palmitoylated residues at Cys308–Cys311. From mutational and [3H]palmitate metabolic labeling studies, it is proposed that palmitoylation at Cys311 in addition to agonist-regulated deacylation at Cys309 > Cys308 may dynamically position α-helix 8 in proximity to Rab11a, to regulate agonist-induced intracellular trafficking of the hIP. Moreover, Ala-scanning mutagenesis identified several hydrophobic residues within α-helix 8 as necessary for the interaction with Rab11a. Given the diverse membership of the G protein-coupled receptor superfamily, of which many members are also predicted to contain an α-helical 8 domain proximal to TM7 and, often, adjacent to palmitoylable cysteine(s), the identification of a functional role for α-helix 8, as exemplified as an RBD for the hIP, is likely to have broader significance for certain members of the superfamily.

Interaction of the human prostacyclin receptor with the PDZ adapter protein PDZK1: role in endothelial cell migration and angiogenesis

Prostacyclin is widely implicated in re-endothelialization and angiogenesis but through unknown mechanisms. Herein the HDL scavenger receptor class B, type 1 adapter PDZK1 was identified as a direct, functional interactant of the human prostacyclin receptor and was found to influence prostacyclin-mediated endothelial migration and in vitro angiogenesis.

Transcriptional Regulation of the Human Prostacyclin Receptor Gene Is Dependent on Sp1, PU.1 and Oct-1 in Megakaryocytes and Endothelial Cells

Prostacyclin plays a central role in hemostasis, inflammation and nociception. However, the factors regulating expression of the prostacyclin receptor (IP) gene in humans and in other species have not been identified. In this study, we sought to identify the key trans-acting factors and cis-acting elements regulating IP expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 and vascular endothelial EA.hy 926 cell lines. With the use of deletion and genetic reporter analyses, the essential core promoter, termed PrmIP, was localized to the -1022 to -895 region proximal to the transcription initiation site, while an upstream repressor region, localized to -1502 to -1271, was also identified. Bioinformatic analysis revealed evolutionarily conserved Sp1, PU.1 and Oct-1 sites within the core PrmIP, and disruption of these elements each led to substantial reductions in PrmIP-directed gene expression in both HEL and EA.hy 926 cells. Electrophoretic mobility shift and supershift assays established that Sp1, PU.1 and Oct-1 can bind to elements within the core promoter in vitro, while chromatin immunoprecipitation assays confirmed their specific binding to chromatin in vivo. Furthermore, combination mutations of the Sp1, PU.1 and Oct-1 elements revealed that they act independently to co-regulate basal transcription of the IP gene, while ectopic expression of each of the trans-acting factors led to substantial increases in PrmIP-directed gene expression and IP mRNA expression in both HEL and EA.hy 926 cells. While electrophoretic mobility shift and antibody supershift assays established that the Ets family member Fli1, but not Ets-1, is capable of binding to the PU.1 element within PrmIP in vitro, chromatin immunoprecipitation analysis established that neither Fli1 nor Ets-1 binds to that element in vivo. Collectively, these data provide critical insights into the transcriptional regulation of the IP gene in human megakaryocytic and endothelial cells, identifying Sp1, PU.1 and Oct-1 as the critical factors involved in its basal regulation in humans.

Regulation of the human prostacyclin receptor gene by the cholesterol-responsive SREBP1

Prostacyclin and its prostacyclin receptor, the I Prostanoid (IP), play essential roles in regulating hemostasis and vascular tone and have been implicated in a range cardio-protective effects but through largely unknown mechanisms. In this study, the influence of cholesterol on human IP [(h)IP] gene expression was investigated in cultured vascular endothelial and platelet-progenitor megakaryocytic cells. Cholesterol depletion increased human prostacyclin receptor (hIP) mRNA, hIP promoter-directed reporter gene expression, and hIP-induced cAMP generation in all cell types. Furthermore, the constitutively active sterol-response element binding protein (SREBP)1a, but not SREBP2, increased hIP mRNA and promoter-directed gene expression, and deletional and mutational analysis uncovered an evolutionary conserved sterol-response element (SRE), adjacent to a known functional Sp1 element, within the core hIP promoter. Moreover, chromatin immunoprecipitation assays confirmed direct cholesterol-regulated binding of SREBP1a to this hIP promoter region in vivo, and immunofluorescence microscopy corroborated that cholesterol depletion significantly increases hIP expression levels. In conclusion, the hIP gene is directly regulated by cholesterol depletion, which occurs through binding of SREBP1a to a functional SRE within its core promoter. Mechanistically, these data establish that cholesterol can regulate hIP expression, which may, at least in part, account for the combined cardio-protective actions of low serum cholesterol through its regulation of IP expression within the human vasculature.

Regulation of the human prostacyclin receptor gene in megakaryocytes: Major roles for C/EBPδ and PU.1

The prostanoid prostacyclin plays a central role in haemostasis and vascular repair. Recent studies investigating the regulation of the human prostacyclin receptor (hIP) gene identified an upstream repressor region (URR) within its regulatory promoter, herein termed the PrmIP. This study aimed to identify the main trans-acting factors that bind within the URR to transcriptionally repress PrmIP-directed gene expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 cell line. Of the putative cis-acting elements examined, disruption of C/EBP and PU.1 elements within the URR substantially increased PrmIP-directed gene expression. Chromatin immunoprecipitation (ChIP) confirmed that C/EBPδ and PU.1, but not C/EBPβ, bind to the URR in vivo, while ectopic expression of C/EBPδ substantially reduced hIP mRNA levels and PrmIP-directed gene expression. Phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation increased hIP mRNA and PrmIP-directed reporter gene expression and hIP-mediated cAMP generation in HEL cells. Two PMA-responsive regions, termed PRR1 and PRR2, were identified within PrmIP. Disruption of C/EBPδ and PU.1 cis-elements within the overlapping PRR1/URR and of Sp1, PU.1 and Oct-1 cis-elements within the overlapping PRR2/core PrmIP, revealed that both PRR1 and PRR2 contribute to the PMA- induction of hIP mRNA and gene expression in HEL cells. Furthermore, ChIP analysis established that induction of PrmIP-directed gene expression during megakaryocytic differentiation is largely regulated by PMA-induced dissociation of C/EBPδ and enhanced binding of PU.1 to PRR1 in addition to increased binding of Sp1, PU.1 and Oct-1 to elements within the core promoter/PRR2 in vivo. Taken together, these data provide critical insights into the transcriptional regulation of the hIP gene within the vasculature, including during megakaryocytic differentiation.