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Abstract

Cloning and pharmacologic characterization of a thromboxane A2 receptor from K562 (human chronic myelogenous leukemia) cells.

D D D'Angelo, M G Davis, S Ali and G W Dorn 2nd
Journal of Pharmacology and Experimental Therapeutics November 1994, 271 (2) 1034-1041;
D D D'Angelo
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M G Davis
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S Ali
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G W Dorn 2nd
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Abstract

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-(1 alpha,2 beta(5Z),3 alpha-(1E,3S),4 alpha]-7-[3-(3-hydroxy-4-(p- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5- heptenoic acid ([125I]BOP) with a Kd of 5.5 +/- 1.1 nM, a Bmax 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-11 alpha,9 alpha(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 [KdH 0.63 +/- 0.18 nM and KdL of 12.5 +/- 5.0 nM, with Bmaxs of 29 +/- 9 and 212 +/- 41 fmol/mg of protein, respectively (n = 7)].(ABSTRACT TRUNCATED AT 250 WORDS)

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Journal of Pharmacology and Experimental Therapeutics
Vol. 271, Issue 2
1 Nov 1994
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Abstract

Cloning and pharmacologic characterization of a thromboxane A2 receptor from K562 (human chronic myelogenous leukemia) cells.

D D D'Angelo, M G Davis, S Ali and G W Dorn
Journal of Pharmacology and Experimental Therapeutics November 1, 1994, 271 (2) 1034-1041;

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Abstract

Cloning and pharmacologic characterization of a thromboxane A2 receptor from K562 (human chronic myelogenous leukemia) cells.

D D D'Angelo, M G Davis, S Ali and G W Dorn
Journal of Pharmacology and Experimental Therapeutics November 1, 1994, 271 (2) 1034-1041;
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