Pharmacologic and molecular evidence conflicts in regard to the existence of tissue-specific subtypes of thromboxane A2 receptors (TXR). The full length TXR complementary DNA (cDNA) was cloned from a platelet-like cell line. It was expressed and its pharmacology was characterized. Northern analysis of TXR transcripts in multiple tissues showed strong hybridization to K562 chronic myelogenous leukemia messenger RNA. Therefore, a K562 cDNA library was screened and a full-length TXR cDNA (K562TXR) was isolated. K562TXR encodes a protein identical to the previously characterized placenta TXR cDNA, except for a single amino acid substitution (Glu21 → Lys). Similar to thromboxane receptors on K562 cells, K562TXR transiently expressed in HEK 293 cells (K562TXR/293) bound the thromboxane agonist 125I-labeled [1S-(1α,2β(5Z),3α-(1E,3S),4α]-7-[3-(3-hydroxy-4-(p-iodophenoxy)-1- butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5-heptenoic acid ([125I]BOP) with a K(d) of 5.5 ± 1.1 nM, a B(max) of 289,056 ± 60,220 sites/cell and a Hill coefficient of -0.94 ± 0.01 (n = 6). K562TXR/293 cells also demonstrated concentration-dependent increases in intracellular calcium in response to the thromboxane agonist (15S-hydroxy-11α,9α(epoxymethano)- prosta-5Z,13E-dienoic acid. In contrast to the single [125I]BOP binding site observed in K562TXR/293, [125I]BOP binding to placental membranes resulted in a Hill coefficient significantly less than unity with a statistically superior two-site model for binding [K(dH) 0.63 ± 0.18 nM and K(dL) of 12.5 ± 5.0 nM, with B(max)s of 29 ± 9 and 212 ± 41 fmol/mg of protein, respectively (n = 7)]. Addition of the nonhydrolyzable GTP analog, GTPγS, to placental membranes transferred [125I]BOP binding to a single site with a K(d) of 10.2 ± 2.2 nM (n = 6). GTPγS had no effect on [125I]BOP binding to K562TXR/293 cell membranes. These data suggest that placenta and K562TXR/293 cells expresses similar thromboxane receptors, which in the case of placenta, displays a second higher affinity state when coupled to G-proteins.
|Number of pages||8|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|State||Published - 1994|