Mouse Prostacyclin Receptor Research Articles

Endothelial Progenitor Cells and Angiogenesis Join the PPARty

See related article, pages 80–88 Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors that belong to the nuclear hormone receptor superfamily.1 In mammals, the PPAR family consists of 3 subtypes of proteins encoded by separate genes: PPARα (NR1C1), PPARγ (NR1C3), and PPARδ (also known as β or NR1C2).2 They act as heterodimers with the retinoid X receptor and regulate gene transcription by binding to specific response elements in the promoter of the target genes.3 The classical biological activity of PPARα is the regulation of the rate of fatty acid uptake and their esterification into triglyceride or oxidation,4–7 whereas PPARγ is classically involved in adipocyte differentiation, regulation of fat storage, and maintenance of glucose homeostasis.5 The physiological functions of PPARδ are instead still unclear, although it is known that this receptor contributes to an inflammatory switch through its association and disassociation with transcriptional repressors.8 The clinical importance of PPARs originates with fibrates and thiazolidinediones (TZDs), which respectively act on PPARα and PPARγ and are used to ameliorate hyperlipidemia and hyperglycemia in subjects with type 2 diabetes mellitus (T2DM). Fibrates, such as gemfibrozil, clofibrate, fenofibrate, and bezofibrate, are drugs that effectively reduce triglycerides (TG) and free …

Agonists can discriminate between cloned human and mouse prostacyclin receptors

The ability of prostacyclin analogues to stimulate adenylyl cyclase (AC) and phospholipase C (PLC) in Chinese hamster ovary (CHO) cells expressing cloned human (hIP) or cloned mouse (mIP) prostacyclin receptors has been compared. For hIP, the order of potency (pEC 50) for stimulating AC and PLC pathways was similar: AFP-07 (9.3, 8.4)>cicaprost (8.3, 6.9), iloprost (7.9, 6.8)>taprostene (7.4, 6.8)>carbacyclin (6.9, 6.6), PGE 1 (6.6, 5.1). Although the standard IP agonists cicaprost and iloprost behaved similarly in both hIP and mIP receptor-expressing cells, carbacyclin and PGE 1 showed significantly higher potency at the mIP receptor, suggesting that the agonist recognition sites on hIP and mIP receptors are not identical. A further distinction between hIP and mIP receptors was found with taprostene, which had greater efficacy at hIP receptors (AC 94%, PLC 14%) than at mIP receptors (AC 77%, PLC 0%) (cicaprost=100% in each assay).

Protein kinase A-dependent coupling of mouse prostacyclin receptors to G i is cell-type dependent

The ability of the prostacyclin (IP) receptor agonist cicaprost to activate G s-, G q/11- and G i-mediated cell signalling pathways has been examined in Chinese hamster ovary (CHO) cells and human embryonic kidney 293 (HEK 293) cells expressing the cloned human (hIP) or mouse (mIP) prostacyclin receptor, and compared with data from NG108-15 and SK-N-SH cells that endogenously express rat/mouse and human IP receptors, respectively. Cicaprost stimulated [ 3H]cyclic AMP production with EC 50 values of 1.5–22 nM, and stimulated [ 3H]inositol phosphate production (EC 50 values 49–457 nM) in all but the SK-N-SH cells. Cicaprost failed to inhibit forskolin-stimulated [ 3H]cyclic AMP production in any of these cell lines. Therefore, although both human and mouse IP receptors couple to G s and G q/11-mediated signalling pathways in a cell type-dependent manner, we could find no evidence for IP receptor coupling to G i.

Investigation of the Mechanisms of G Protein: Effector Coupling by the Human and Mouse Prostacyclin Receptors: IDENTIFICATION OF CRITICAL SPECIES-DEPENDENT DIFFERENCES

We recently identified a novel mechanism explaining how the mouse (m) prostacyclin receptor (IP) couples to Galpha(s), Galpha(i), and Galpha(q) (Lawler, O. A., Miggin, S. M., and Kinsella, B. T. (2001) J. Biol. Chem. 276, 33596-33607) whereby mIP coupling to Galpha(i) and Galpha(q) is dependent on its initial coupling to Galpha(s) and subsequent phosphorylation by cAMP-dependent protein kinase A (PKA) on Ser(357). In the current study, the generality of that mechanism was investigated by examining the G protein coupling specificity of the human (h) IP. The hIP efficiently coupled to Galpha(s)/adenylyl cyclase and to Galpha(q)/phospholipase C activation but failed to couple to Galpha(i). Coupling of the hIP to Galpha(q), or indeed to Galpha(s) or Galpha(i), was unaffected by the PKA or protein kinase C (PKC) inhibitors H-89 and GF 109203X, respectively. Thus, mIP and hIP exhibit essential differences in their coupling to Galpha(i) and in their dependence on PKA in regulating their coupling to Galpha(q). Analysis of their primary sequences revealed that the critical PKA phosphorylation site within the mIP, at Ser(357), is replaced by a PKC site within the hIP, at Ser(328). Conversion of the PKC site of the hIP to a PKA site generated hIP(QL325,326RP) that efficiently coupled to Galpha(s) and to Galpha(i) and Galpha(q); coupling of hIP(QL325,326RP) to Galpha(i) but not to Galpha(s) or Galpha(q) was inhibited by H-89. Abolition of the PKC site of the hIP generated hIP(S328A) that efficiently coupled to Galpha(s) and Galpha(q) but failed to couple to Galpha(i). Finally, conversion of the PKA site at Ser(357) within the mIP to a PKC site generated mIP(RP354,355QL) that efficiently coupled to Galpha(s) but not to Galpha(i) or Galpha(q). Collectively, our data highlight critical differences in signaling by the mIP and hIP that are regulated by their differential phosphorylation by PKA and PKC together with contextual sequence differences surrounding those sites.

Factors affecting prostacyclin receptor agonist efficacy in different cell types

Octimibate and related nonprostanoid prostacyclin mimetics are partial agonists displaying highly tissue-specific responses. Octimibate demonstrated considerably greater efficacy for stimulation of adenylyl cyclase activity in Chinese hamster ovary cells transiently expressing mouse prostacyclin receptors (mIP-CHO cells) when compared to human SK-N-SH neuroblastoma cells, which endogenously express prostacyclin (IP) receptors. Pretreatment of both cell types with pertussis toxin (PTx) failed to influence IP agonist efficacy or potency, indicating a lack of involvement of an agonist-stimulated inhibitory G i-coupled pathway. Although stimulation of mIP-CHO cells with the full agonist cicaprost increased both [ 3H]cyclic AMP and [ 3H]inositol phosphate ([ 3H]IP) accumulation (pEC 50 values of 8.35 and 6.82, respectively), IP receptor signalling through G q in SK-N-SH cells was absent. Inhibition of protein kinase C (PKC) in mIP-CHO cells increased [ 3H]IP accumulation but had no effect on [ 3H]cyclic AMP accumulation. Therefore, the poor coupling of the IP receptor in SK-N-SH cells to G q is unlikely to explain the relatively low efficacy of octimibate for stimulating adenylyl cyclase in these cells. Furthermore, protein kinase A (PKA) inhibition appears to enhance IP receptor signalling through both G s and G q in mIP-CHO cells.

Protein Kinase A-mediated Phosphorylation of Serine 357 of the Mouse Prostacyclin Receptor Regulates Its Coupling to Gs-, to Gi-, and to Gq-coupled Effector Signaling

The prostacyclin receptor (IP) is primarily coupled to G alpha(s)-dependent activation of adenylyl cyclase; however, a number of studies indicate that the IP may couple to other secondary effector systems perhaps in a species-specific manner. In the current study, we investigated the specificity of G protein:effector coupling by the mouse (m) IP overexpressed in human embryonic kidney 293 cells and endogenously expressed in murine erythroleukemia cells. The mIP exhibited efficient G alpha(s) coupling and concentration-dependent increases in cAMP generation in response to the IP agonist cicaprost; however, mIP also coupled to G alpha(i) decreasing the levels of cAMP in forskolin-treated cells. mIP coupling to G alpha(i) was pertussis toxin-sensitive and was dependent on protein kinase (PK) A activation status. In addition, the mIP coupled to phospholipase C (PLC) activation in a pertussis toxin-insensitive, G alpha(i)-, G beta gamma-, and PKC-independent but in a G alpha(q)- and PKA-dependent manner. Whole cell phosphorylation assays demonstrated that the mIP undergoes cicaprost-induced PKA phosphorylation. mIP(S357A), a site-directed mutant of mIP, efficiently coupled to G alpha(s) but failed to couple to G alpha(i) or to efficiently couple to G alpha(q):PLC. Moreover, mIP(S357A) did not undergo cicaprost-induced phosphorylation confirming that Ser(357) is the target residue for PKA-dependent phosphorylation. Finally, co-precipitation experiments permitted the detection of G alpha(s), G alpha(i), and G alpha(q) in the immunoprecipitates of mIP, whereas only G alpha(s) was co-precipitated with mIP(S357A) indicating that Ser(357) of mIP is essential for G alpha(i) and G alpha(q) interaction. Moreover, inhibition of PKA blocked co-precipitation of mIP with G alpha(i) or G alpha(q). Taken together our data indicate that the mIP, in addition to coupling to G alpha(s), couples to G alpha(i) and G alpha(q); however, G alpha(i) and G alpha(q) coupling is dependent on initial cicaprost-induced mIP:G alpha(s) coupling and phosphorylation of mIP by cAMP-dependent PKA where Ser(357) was identified as the target residue for PKA phosphorylation.

CDNA cloning of a mouse prostacyclin receptor. Multiple signaling pathways and expression in thymic medulla

A functional cDNA for a mouse prostacyclin receptor was isolated from a mouse cDNA library by reverse transcription polymerase chain reaction and hybridization screening. The cDNA encodes a polypeptide of 417 amino acid residues with putative seven transmembrane domains and an calculated molecular weight of 44,722. The amino acid sequence is 30-40% identical in the transmembrane domains to those of the mouse prostaglandin (PG) E receptor subtypes and thromboxane A2 receptor. [3H]Iloprost, a specific prostacyclin receptor radioligand, specifically bound to the membrane of Chinese hamster ovary cells permanently expressing the cDNA with Kd of 4.6 nM. This binding was displaced with unlabeled prostanoids in the order of cicaprost > iloprost, both prostacyclin agonists > PGE1 > carbacyclin >> PGD2 approximately STA2, a thromboxane A2 agonist approximately PGE2 > PGF2 alpha. Iloprost in a concentration-dependent fashion increased cAMP level and generated inositol phosphates in these cells, indicating that the receptor couples to multiple signal transduction pathways. Northern blot analysis revealed that the mRNA is expressed most abundantly in thymus, followed by spleen, heart, and lung. In situ hybridization of thymus showed that it is expressed exclusively in medulla and not in cortex.

O-344 - Tissue Distribution and Cellular Localization of The Mouse Prostacyclin Receptor

O-344 - Tissue Distribution and Cellular Localization of The Mouse Prostacyclin Receptor