Dimerization of the Human Receptors for Prostacyclin and Thromboxane Facilitates Thromboxane Receptor-mediated cAMP Generation

Stephen J Wilson,Aoife M Roche,Ekaterina Kostetskaia,Emer M Smyth

doi:10.1074/jbc.m405002200

Stephen J Wilson, Aoife M Roche + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m405002200

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Abstract

Prostacyclin (PGI(2)) and thromboxane (TxA(2)) are biological opposites; PGI(2), a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA(2), a vasoconstrictor and platelet activator. The molecular mechanisms involved in the counterregulation of PGI(2)/TxA(2) signaling are unclear. We examined the interaction of the receptors for PGI(2) (IP) and TxA(2) (TPalpha). IP-induced cAMP and TP-induced inositol phosphate generation were unaltered when the receptors were co-expressed in HEK 293 cells (IP/TPalpha-HEK). TP-cAMP generation, in response to TP agonists or a TP-dependent isoprostane, iPE(2)III, was evident in IP/TPalpha-HEK and in aortic smooth muscle cells, but not in cells expressing either receptor alone, or in IP-deficient aortic smooth muscle cells. Augmentation of TP-induced cAMP generation, with the IP agonist cicaprost, was ablated in IP-deficient cells and was independent of direct IP signaling. IP/TPalpha heterodimers were formed constitutively when the receptors were co-expressed, with no overt changes in ligand binding to the individual receptor sites. However, despite inefficient binding of iPE(2)III to either the IP or TPalpha, expressed alone or in combination, robust cAMP generation was evident in IP/TPalpha-HEK, suggesting the formation of an alternative receptor site. Thus, IP/TPalpha dimerization was coincident with TP-cAMP generation, promoting a "PGI(2)-like" cellular response to TP activation. This represents a previously unknown mechanism by which IP may limit the cellular effects of TP.

Highlights

Maintenance of the PGI2/TxA2 balance appears to be a critical regulator of vascular disease; the molecular mechanisms underlying the counterregulation of PGI2/TxA2 signaling have not been fully elucidated
We examined the biological relevance of this relationship in a cell model that endogenously expresses both IP and TP. cAMP production in response to IBOP or iPE2III was quantified in aortic smooth muscle cells isolated from humans or WT mice
PGI2 and TxA2 are important regulators of vascular homeostasis, and their respective levels dictate the response to vascular injury

Summary

EXPERIMENTAL PROCEDURES

Materials—Cyclic AMP radioimmunoassay kit, enhanced chemiluminescence kits, protein G-Sepharose, and all radiochemicals were purchased from Amersham Biosciences. HASMC and mASMC were pretreated overnight with 3 ␮M indomethacin to inhibit endogenous eicosanoid generation, followed by the addition of medium containing isobutylmethylxanthine (0.01 M) 30 min prior to agonist treatment. HEK 293 cells stably expressing hTP␣ or hIP or both were treated with 3 mM dithiobis(succinimidylpropionate) (Pierce) for 30 min and lysed in buffer A (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 10% glycerol, and a mixture of protease inhibitors) for 2 h at 4 °C. MychIP or HAhTP␣ was immunoprecipitated from precleared lysates by adding 150 ␮l of anti-Myc- or anti-HA-protein G-Sepharose to each lysate and rotating for 16 h. Immunoblotting for HA or Myc was carried out as described above, using the appropriate biotinylated antibody (1:500) followed by peroxidase-labeled streptavidin

RESULTS

DISCUSSION

IP binding