Evidence For Subtypes

Hie observation of two TP receptor binding sites on platelets, that correlated with function, (See IV. TP RECEPTOR FUNCTION, A. LIGAND BINDING, 3. EQUILIBRIUM BINDING above) suggested that receptor subtypes might be responsible. Another reason to consider the possibility that TP receptor subtypes existed was the fact that subtypes of prostaglandin E (EP) receptors were recognized (2). Support for the existence of receptor subtypes derived primarily from data obtained from studies of the pharmacological characteristics of TP receptors on platelets compared to those on blood vessels (See 1 for review). Equilibrium binding studies performed with a large series of 13 azapinane TXA2 analogs demonstrated differences between TP receptors on platelets and saphenous veins (1). CTAj, 13-azapinane analogs and difluorinated TXA2 derivatives acted as antagonists to platelets receptors, but they were agonists to blood vessel TP receptors (115-117). Although some of the differences between platelets and vascular tissues could be attributed to species differences, studies of the same ligands revealed significant differences in rank order potency between platelets and vascular tissues within the same species (108,118). Other studies observed differences between platelets and vascular tissues when antagonist but not agonist binding was studied (119).


Although some investigators found differences in the pharmacological character-istics of platelet TP receptors compared to those on blood vessels, others did not (119-122). As noted above, multiple EP receptor subtypes exist. However, the occurrence of EP receptor subtypes does not necessarily imply that TP receptors subtypes exist, since EP receptor subtypes are products of separate genes, while only one TP receptor gene has been identified. Another possible explanation for variable TP receptor function is tissue-specific differential expression of receptor isoforms. Isoforms of EP3 receptors that are derived from alternative splicing have been characterized (123,124). Evidence for TP receptor isoforms (TP-a_and TP-P) attributable to alternative splicing has been presented (See III. TP RECEPTOR STRUCTURE, B. MOLECULAR BIOLOGY, 6. TP RECEPTOR ISOTYPES above). If the ligand binding characteristics of these isotypes differed, and expression of one of these splice variants predominated in a tissue-specific manner, it would be theoretically possible to explain tissue-specific functional receptor differences on the basis of receptor isotypes. However, no differences in ligand binding to TP-cc_and TP-P or displacement by agonists or antagonists were observed when their respective cDNAs were expressed in COS cells (60,69). In addition, no evidence of alternatively spliced mRNA was found in mouse kidney even though [125I]-BOP bound to two sites on cells obtained from mouse kidney cortex and to only one site on cells obtained from mouse kidney medulla (110). Furthermore, no differences were noted in ligand binding specificities among the four EP3 isoforms (124).


Some of the tissue variability in TP receptor ligand binding can be attributed to species differences, but other factors are probably responsible. Among factors known to influence ligand binding parameters are G proteins. When G protein availability is limiting, total receptor density measured by agonists may differ from that measured by antagonists (125). This phenomenon is observed in recombinant systems where the natural stoichi-ometry in distorted. Allan, et al (53) demonstrated that binding to transfected TP receptors was influenced by cotransfection of G protein constituents, GaQ and Ga„. Thus variable cell-specific expression of G proteins that couple to TP receptors could influence their ligand binding characteristics. This is particularly likely when platelets are compared to other tissues, since platelets lack Gau, a G protein subunit nearly universally expressed in other cells (126). Another G protein that could influence TP receptor binding in platelets, but not in vascular cells, is Galc, since this subunit is expressed only in hematopoietic cells (127). Van der Vuurst, et al (128) have reported the presence of Ga16 in megakaryocytes and platelets, but it is unlikely to be present in vascular tissues. Differences in local membrane environments could also influence ligand access to TP receptors that might be responsible for tissue differences in binding parameters. The demonstration that enrichment of platelet phospholipids with eicosapentaenoic acid or docosahexaenoic acid decreased TP receptor affinity for [3H]-U46619 (See V. ALTERED TP RECEPTOR FUNCTION, B. TP RECEPTOR SIGNAL TRANSDUCTION DEFECTS, 3. DRUG EFFECTS, b. OMEGA-3 FATTY ACIDS below) supports this contention. Differences in G proteins or in membrane constituents could also explain why TP receptors transfected into cells that do not naturally express these receptors have routinely yielded Scatchard plots indicative of one site binding, whereas native cells often yielded two affinity state binding profiles (See IV. TP RECEPTOR FUNCTION, A. LIGAND BINDING, 3. EQUILIBRIUM BINDING above).

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