Protein kinase A-dependent coupling of mouse prostacyclin receptors to Gi is cell-type dependent
Introduction
The prostacyclin (IP) receptor is a G protein-coupled receptor belonging to the family of prostanoid receptors (Narumiya et al., 1999). IP receptors couple primarily to activation of adenylyl cyclase via Gs, and can also couple to activation of phospholipase C leading to phosphatidyl inositol turnover and mobilization of intracellular calcium ([Ca2+]i) (see Wise and Jones, 2000). Whether or not IP receptor coupling to Gq/11 is physiologically relevant remains uncertain, since to date, activation of this pathway in native cells has only been demonstrated using the non-specific IP receptor agonist iloprost in piglet cerebral microvascular smooth muscle cells (Parkinson et al., 2000) and in isolated rat dorsal root ganglion cells in vitro (Smith et al., 1998). Activation of multiple signalling pathways by IP receptor agonists (observed as increases in cyclic AMP, inositol trisphosphate or [Ca2+]i) has been reported in transformed cell lines such as BNu2cl3 mouse mast cells, Ob1771 mouse pre-adipocytes, human erythroleukaemia (HEL) cells and human megakaryoblastic leukaemia (MEG-01) cells (for references, see Wise and Jones, 1996).
A recent report has shown that the IP receptor endogenously expressed in mouse erythroleukaemia (MEL) cells and the cloned mouse (mIP) prostacyclin receptor stably overexpressed in human embryonic kidney 293 (HEK 293) cells can couple to Gi (Lawler et al., 2001). Furthermore, the mIP receptor in HEK 293 cells couples to both Gq/11 and Gi in a protein kinase A (PKA)-dependent manner (Lawler et al., 2001). In contrast, the cloned human (hIP) prostacyclin receptor stably overexpressed in HEK 293 cells couples independently to Gs and Gq/11 and does not couple to Gi (Miggin and Kinsella, 2002). We have previously demonstrated that IP receptor agonist-stimulated [3H]cyclic AMP production in Chinese hamster ovary (CHO) cells transiently expressing mIP receptors and in human neuroblastoma SK-N-SH cells was not affected by pertussis toxin treatment, suggesting a lack of coupling of both mIP and human IP receptors to Gi (Kam et al., 2001). Therefore, we have extended our studies here to examine the G protein coupling capacity of human and rodent IP receptors in a variety of cell lines stably or transiently expressing IP receptors. Our studies fail to support the hypothesis that mouse IP receptor switching from Gs to Gi is a universal phenomenon.
Section snippets
Cell culture
CHO cells were cultured in Ham's F-12 medium supplemented with 10% foetal bovine serum. HEK 293 cells were cultured in Dulbecco's modified Eagle medium (DMEM) medium supplemented with 10% foetal bovine serum, and assays were performed in cell culture dishes treated with poly-d-lysine. Rat/mouse neuroblastoma-glioma (NG108-15) cells were cultured in DMEM medium supplemented with 6% foetal bovine serum, 0.1 mM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine and 2 mM l-glutamine. SK-N-SH
Coupling of IP receptors to Gs-mediated signalling pathways
Cicaprost stimulated [3H]cyclic AMP production in a concentration-dependent manner in CHO and HEK 293 cells transiently expressing hIP and mIP receptors, and in NG108-15 and SK-N-SH cells which endogenously express IP receptors (Table 1). The pEC50 values for cicaprost were similar for hIP and mIP receptors when transiently expressed in HEK 293 cells, but were significantly higher for hIP compared with mIP receptors transiently expressed in CHO cells (P<0.01). The affinity of human IP receptors
Discussion
When iloprost was first demonstrated to increase inositol phosphate production in CHO cells stably expressing the mIP receptor, it was also demonstrated that this process was pertussis toxin-insensitive and was not evoked by dibutyryl cyclic AMP (Namba et al., 1994). Furthermore, while pretreatment of these mIP receptor-expressing CHO cells with cholera toxin to degrade Gs resulted in a loss of iloprost-induced cyclic AMP production, it did not affect the inositol phosphate response. Together
Acknowledgments
This work was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region (CA99/00.SC01).
References (30)
- et al.
Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites
J. Mol. Biol.
(1999) - et al.
Recombinant Gqα
J. Biol. Chem.
(1992) - et al.
Prostanoid receptor with a novel pharmacological profile in human erythroleukemia cells
Biochem. Pharmacol.
(1997) - et al.
Factors affecting prostacyclin receptor agonist efficacy in different cell types
Cell. Signal.
(2001) - et al.
Cloning and expression of a cDNA for the human prostacyclin receptor
FEBS Lett.
(1994) - et al.
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
J. Biol. Chem.
(2001) - et al.
Alternative coupling of receptors to Gs and Gi in pancreatic and submandibular gland cells
J. Biol. Chem.
(1999) - et al.
cDNA cloning of a mouse prostacyclin receptor
J. Biol. Chem.
(1994) - et al.
Characterization of prostaglandin and thromboxane receptors expressed on a megakaryoblastic leukemia cell line, MEG-01s
Blood
(1991) Characterization of chimeric prostacyclin/prostaglandin D2 receptors
Eur. J. Pharmacol.
(1999)
Focus on prostacyclin and its novel mimetics
Trends Pharmacol. Sci.
Protein kinase A-mediated phosphorylation of the β2-adrenergic receptor regulates its coupling to Gs and Gi
J. Biol. Chem.
Elevation of intracellular calcium ion by prostaglandin E1 and its inhibition by protein kinase C in a human megakaryocyte leukemia cell line
Cancer Res.
Forskolin binding to intact S49 lymphoma cells
Second Messengers Phosphoprot.
Turnover of adenosine 3′,5′-monophosphate in WI-38 cultured fibroblasts
Biochemistry
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