CLONING AND PHARMACOLOGICAL CHARACTERIZATION OF A THROMBOXANE A(2) RECEPTOR FROM K562 (HUMAN CHRONIC MYELOGENOUS LEUKEMIA) CELLS

Pharmacologic and molecular evidence conflicts in regard to the existe nce of tissue-specific subtypes of thromboxane A(2) receptors (TXR). T he full length TXR complementary DNA (cDNA) was cloned from a platelet -like cell line. It was expressed and its pharmacology was characteriz ed. Northern analysis of TXR transcripts in multiple tissues showed st rong hybridization to K562 chronic myelogenous leukemia messenger RNA. Therefore, a K562 cDNA library was screened and a full-length TXR cDN A (K562TXR) was isolated. K562TXR encodes a protein identical to the p reviously characterized placenta TXR cDNA, except for a single amino a cid substitution (Glu(21) --> Lys). Similar to thromboxane receptors o n K562 cells, K562TXR transiently expressed in HEK 293 cells (K562TXR/ 293) bound the thromboxane agonist I-125-labeled[1S(1 alpha,2 beta(5Z ),3 alpha-(1E,3S),4 alpha]-7-[3-(3-hydroxy-4-(p-iodophenoxy)- nyl)-7-o xabicyclo-[2.2.1]heptane-2-yl]-5-heptenoic acid ([I-125]BOP) with a K- d of 5.5 +/- 1.1 nM, a B-max of 289,056 +/- 60,220 sites/cell and a Hi ll coefficient of -0.94 +/- 0.01 (n = 6). K562TXR/293 cells also demon strated concentration-dependent increases in intracellular calcium in response to the thromboxane agonist (15S-hydroxy-11 alpha,9 alpha(epox ymethano)-prosta-5Z,13E-dienoic acid. In contrast to the single [I-125 ]BOP binding site observed in K562TXR/293, [I-125]BOP binding to place ntal 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, GTP gamma S, to placental membranes tr ansferred [I-125]BOP binding to a single site with a K-d of 10.2 +/- 2 .2 nM (n = 6). GTP gamma S had no effect on [I-125]BOP binding to K562 TXR/293 cell membranes. These data suggest that placenta and K562TXR/2 93 cells expresses similar thromboxane receptors, which in the case of placenta, displays a second higher affinity state when coupled to G-p roteins.