Prostacyclin (PGI2), a vascular protector with vasodilation and antithrombotic properties, is synthesized by coupling reactions of cyclooxygenase (COX, the first enzyme) with PGI2 synthase (PGIS, the second enzyme) using arachidonic acid (AA) as an initial substrate. The first COX product, prostaglandin H2 (PGH2) is also a command substrate for other prostanoid enzymes that produce distinct eicosanoids, such as thromboxane A2 (TXA2). The actions of TXA2 to cause vasoconstriction and platelet aggregation oppose the vasodilatory and anti-aggregatory effects of PGI2. Specifically upregulating PGI2 biosynthesis is an ideal model for the prevention and treatment of the TXA2-mediated thrombosis involved in strokes and myocardial infarctions. Here, we report that a single protein was constructed by linking COX-2 and PGIS together to form a new fusion enzyme through a transmembrane domain with 10 or 22 residues. The engineered protein expressed in HEK293 and COS-7 cells was able to continually convert AA to prostaglandin (PG) G2 (catalytic step 1), PGH2 (catalytic step 2), and PGI2 (catalytic step 3). The studies first demonstrate that a single protein with three catalytic functions could directly synthesize PGI2 from AA with a Km of approximately 3.2 microM. Specific upregulation of PGI2 biosynthesis through expression of the engineered single protein in the cells has shown strong activity in inhibiting platelet aggregation induced by AA in vitro, which creates a great potential for the fusion enzyme to be used as one of the new therapeutic interventions for strokes and heart attacks. The studies have also provided a model linking COX with its downstream enzymes to specifically regulate biosynthesis of eicosanoids which have potent biological functions.

Thromboxane A(2) receptor (TP receptor), a prostanoid receptor, belongs to the G protein-coupled receptor family, composed of three intracellular loops and three extracellular loops connecting seven transmembrane helices. The highly conserved extracellular domains of the prostanoid receptors were found in the second extracellular loop (eLP(2)), which was proposed to be involved in ligand recognition. The 3D structure of the eLP(2) would help to further explain the ligand binding mechanism. Analysis of the human TP receptor model generated from molecular modeling based on bacteriorhodopsin crystallographic structure indicated that about 12-14 A separates the N- and C-termini of the extra- and intracellular loops. Synthetic loop peptides whose termini are constrained to this separation are presumably more likely to mimic the native loop structure than the corresponding loop region peptide with unrestricted ends. To test this new concept, a peptide corresponding to the eLP(2) (residues 173-193) of the TP receptor has been made with the N- and C-termini connected by a homocysteine disulfide bond. Through 2D nuclear magnetic resonance (NMR) experiments, complete (1)H NMR assignments, and structural construction, the overall 3D structure of the peptide was determined. The structure shows two beta-turns at residues 180 and 185. The distance between the N- and C-termini of the peptide shown in the NMR structure is 14.2 A, which matched the distance (14.5 A) between the two transmembrane helices connecting the eLP(2) in the TP receptor model. This suggests that the approach using the constrained loop peptides greatly increases the likelihood of solving the whole 3D structures of the extra- and the intracellular domains of the TP receptor. This approach may also be useful in structural studies of the extramembrane loops of other G protein-coupled receptors.

The second extracellular loop (eLP2) of the thromboxane A 2 receptor (TP) had been proposed to be involved in ligand binding. Through two-dimensional 1 H NMR experiments, the overall three-dimensional structure of a constrained synthetic peptide mimicking the eLP2 had been determined by our group (Ruan, K.-H., So, S.-P., Wu, J., Li, D., Huang, A., and Kung, J. (2001 Thromboxane A 2 (TXA 2 ) 1 is a potent platelet aggregatory and vasoconstrictive mediator (2). The function of TXA 2 is mediated by specific cell surface receptor, thromboxane A 2 receptor (TP) (3). The understanding of the structure and function of TP receptor can greatly explain how the ligand binds to its receptor and initiates the following cell signaling.TP receptor was first purified from platelet in 1989, and the cDNA of TP receptor was cloned from placenta in (4, 5). Other human prostanoid receptor cDNAs have also been cloned by homology screening. All of the prostanoid receptors belong to the G-protein-coupled receptor family that share a basic seven transmembrane segments and couple to different signal transduction systems to play diverse physiological and pathological roles (6 -14). TXA 2 binds to TP receptor and triggers an increase of intracellular calcium. There were two TP receptor isoforms with different C-terminal tails, resulting from alternative splicing that the last 15 amino acids of the C terminus were replaced by 79 amino acids (15, 16). The two TP receptor isoforms coupled to the same signal transduction, but endothelium expressed only the spliced form and placenta expressed both types of the TP receptors (15-17).Based on the sequence alignment, the second extracellular loop (eLP2) and the third and seventh transmembrane domains of the prostanoid receptors are highly conserved and are proposed to be involved in ligand binding (18). in the eLP2 of the EP3 receptor have been reported as an essential determinant of ligand selectivity (19). These results suggest that the extracellular domains of other prostanoid receptors are involved in the initial specific ligand interaction. The residues responsible for specific ligand recognition within eLP2 of the TP receptor have not been thoroughly examined. The mutations based on alignment only are controversial and will need structural information to support. The structures of the transmembrane domains of prostanoid receptors may be similar, but the specific recognition sites on extracellular domains will be different because the ligand structures are different. Thus, structural characterization of the extracellular functional domains of prostanoid receptors could help in understanding the specificities of ligand binding. In our current study, the structure of the highly conserved eLP2 has been characterized by high resolution NMR using a synthetic eLP2 peptide with constrained loop ends (1). To identify which residues make up the ligand recognition site of the receptor, SQ29,548 was added to the peptide to determine the interaction using high resolution two-dimensional 1 H NMR technique...

To overcome the difficulty of characterizing the structures of the extracellular loops (eLPs) of G protein-coupled receptors (GPCRs) other than rhodopsin, we have explored a strategy to generate a three-dimensional structural model for a GPCR, the thromboxane A 2 receptor. This three-dimensional structure was completed by the assembly of the NMR structures of the computation-guided constrained peptides that mimicked the extracellular loops and connected to the conserved seven transmembrane domains. The NMR structure-based model reveals the structural features of the eLPs, in which the second extracellular loop (eLP 2 ) and the disulfide bond between the first extracellular loop (eLP 1 ) and eLP 2 play a major role in forming the ligand recognition pocket. The eLP 2 conformation is dynamic and regulated by the oxidation and reduction of the disulfide bond, which affects ligand docking in the initial recognition. The reduced form of the thromboxane A 2 receptor experienced a decrease in ligand binding activity due to the rearrangement of the eLP 2 conformation. The ligand-bound receptor was, however, resistant to the reduction inactivation because the ligand covered the disulfide bond and stabilized the eLP 2 conformation. This molecular mechanism of ligand recognition is the first that may be applied to other prostanoid receptors and other GPCRs.

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