Helen M. Reid

The α, but not the β, isoform of the human thromboxane A2 receptor is a target for nitric oxide-mediated desensitization. INDEPENDENT MODULATION OF TPα SIGNALING BY NITRIC OXIDE AND PROSTACYCLIN.

In humans, thromboxane A2 signals through two thromboxane A2 receptor (TP) isoforms termed TPα and TPβ. Signaling by TPα, but not TPβ, is subject to prostacyclin-induced desensitization mediated by direct protein kinase (PK) A phosphorylation where Ser329 represents the phosphotarget (Walsh, M. T., Foley, J. F., and Kinsella, B. T. (2000) J. Biol. Chem. 275, 20412-20423). In the current study, the effect of the vasodilator nitric oxide (NO) on intracellular signaling by the TP isoforms was investigated. The NO donor 3-morpholinosydnonimine, HCl (SIN-1) and 8-bromo-guanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) functionally desensitized U46619-mediated calcium mobilization and inositol 1,4,5-trisphosphate generation by TPα whereas signaling by TPβ was unaffected by either agent. NO-mediated desensitization of TPα signaling occurred through a PKG-dependent, PKA- and PKC-independent mechanism. TPα, but not TPβ, was efficiently phosphorylated by PKG in vitro and underwent NO/PKG-mediated phosphorylation in whole cells. Deletion/site-directed mutagenesis and metabolic labeling studies identified Ser331 as the target residue of NO-induced PKG phosphorylation of TPα. Although TPαS331A was insensitive to NO/PKG-desensitization, similar to wild type TPα its signaling was fully desensitized by the prostacyclin receptor agonist cicaprost occurring through a PKA-dependent mechanism. Conversely, signaling by TPαS329A was insensitive to cicaprost stimulation whereas it was fully desensitized by NO/PKG signaling. In conclusion, TPα undergoes both NO- and prostacyclin-mediated desensitization that occur through entirely independent mechanisms involving direct PKG phosphorylation of Ser331, in response to NO, and PKA phosphorylation of Ser329, in response to prostacyclin, within the unique carboxyl-terminal tail domain of TPα. On the other hand, signaling by TPβ is unaffected by either NO or prostacyclin.

Recycling of the human prostacyclin receptor is regulated through a direct interaction with Rab11a GTPase

The 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 cicaprost stimulation. 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 Rab11(S25N) 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.

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.

Differential regulation of RhoA-mediated signaling by the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor: independent modulation of TPalpha signaling by prostacyclin and nitric oxide.

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.

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.

Agonist-dependent internalization and trafficking of the human prostacyclin receptor : A direct role for Rab5a GTPase

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.

The effects of KATP channel modulators on counterregulatory responses and cognitive function during acute controlled hypoglycaemia in healthy men: a pilot study

Aims To examine the effects of agents that alter potassium adenosine triphosphate (KATP) channel activity in β‐cells on cognitive function and counterregulatory hormone responses during acute hypoglycaemia, given the physiological similarities between the pancreatic β‐cell and the hypothalamic glucose‐sensitive neurones (GSN) and the widespread distribution of sulphonylurea receptors in neuronal cells throughout the brain.

Palmitoylation of the TPbeta isoform of the human thromboxane A2 receptor. Modulation of G protein: effector coupling and modes of receptor internalization.

Palmitoylation is a prevalent feature amongst G protein-coupled receptors. In this study we sought to establish whether the TPα and TPβ isoforms of the human prostanoid thromboxane (TX) A2 receptor (TP) are palmitoylated and to assess the functional consequences thereof. Consistent with the presence of three cysteines within its unique carboxyl-terminal domain, metabolic labelling and site-directed mutagenesis confirmed that TPβ is palmitoylated at Cys347 and, to a lesser extent, at Cys373,377 whereas TPα is not palmitoylated. Impairment of palmitoylation did not affect TPβ expression or its ligand affinity. Conversely, agonist-induced [Ca2+]i mobilization by TPβC347S and the non-palmitoylated TPβC347,373,377S, but not by TPβC373S or TPβC373,377S, was significantly reduced relative to the wild type TPβ suggesting that palmitoylation at Cys347 is specifically required for efficient Gq/phospholipase Cβ effector coupling. Furthermore, palmitoylation at Cys373,377 is critical for TPβ internalization with TPβC373S, TPβC373,377S and TPβC347,373,377S failing to undergo either agonist-induced or temperature-dependent tonic internalization. On the other hand, whilst TPβC347S underwent reduced agonist-induced internalization, it underwent tonic internalization to a similar extent as TPβ. The deficiency in agonist-induced internalization by TPβC347S, but not by TPβC373,377 nor TPβC347,373,377S, was overcome by over-expression of either β-arrestin1 or β-arrestin2. Taken together, data herein suggest that whilst palmitoylation of TPβ at Cys373,377 is critical for both agonist- and tonic-induced internalization, palmitoylation at Cys347 has a role in determining which pathway is followed, be it by the β-arrestin-dependent agonist-induced pathway or by the β-arrestin-independent tonic internalization pathway.

IHG-1 Increases Mitochondrial Fusion and Bioenergetic Function

Induced in high glucose-1 (IHG-1) is a conserved mitochondrial protein associated with diabetic nephropathy (DN) that amplifies profibrotic transforming growth factor (TGF)-β1 signaling and increases mitochondrial biogenesis. Here we report that inhibition of endogenous IHG-1 expression results in reduced mitochondrial respiratory capacity, ATP production, and mitochondrial fusion. Conversely, overexpression of IHG-1 leads to increased mitochondrial fusion and also protects cells from reactive oxygen species–induced apoptosis. IHG-1 forms complexes with known mediators of mitochondrial fusion—mitofusins (Mfns) 1 and 2—and enhances the GTP-binding capacity of Mfn2, suggesting that IHG-1 acts as a guanine nucleotide exchange factor. IHG-1 must be localized to mitochondria to interact with Mfn1 and Mfn2, and this interaction is necessary for increased IHG-1–mediated mitochondrial fusion. Together, these findings indicate that IHG-1 is a novel regulator of both mitochondrial dynamics and bioenergetic function and contributes to cell survival following oxidant stress. We propose that in diabetic kidney disease increased IHG-1 expression protects cell viability and enhances the actions of TGF-β, leading to renal proximal tubule dedifferentiation, an important event in the pathogenesis of this devastating condition.