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J Biol Chem, Vol. 273, Issue 35, 22753-22760, August 28, 1998

Expression, Characterization, and Purification of C-terminally Hexahistidine-tagged Thromboxane A₂ Receptors

Siddharama Pawate, Kevin L. Schey, G. Patrick Meier^§, Michael E. Ullian^¶, Dale E. Mais, and Perry V. Halushka^¶

From the Departments of  Cell and Molecular Pharmacology and Experimental Therapeutics, ^¶ Medicine, and ^§ Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425 and  Ligand Pharmaceutical Co., San Diego, California 92121

Thromboxane A₂ (TxA₂) receptors belong to the class of G-protein-coupled receptors. Knowledge of the relationship of structure to function for TxA₂ receptors is limited because of their low levels of expression, lengthy purification procedures and poor recoveries. A C-terminal hexahistidine-tag (C-His) was ligated to the -isoform of TxA₂ receptors and expressed in COS-7 and Chinese hamster ovary cells. The C-His-TxA₂ receptors bound the radioligands ¹²⁵I-7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-benzenesulfonylamino)bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid, an antagonist, and ¹²⁵I-[1S-1,2(5Z),3(1E,3S*),4]-7-[3[(3-hydroxy-4-(4'-phenoxy)-1butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptanoic acid, an agonist, with affinities not significantly different from those of the wild type (wt)-TxA₂ receptors. LipofectAMINE transfection of the cDNAs resulted in high levels of expression (B_max = 95 ± 6 pmol/mg) of the C-His-TxA₂ receptors. In competition binding studies the IC₅₀ values of five different ligands were not significantly different between C-His-TxA₂ and wt-TxA₂ receptors. Agonist-induced stimulation of cAMP and total inositol phosphate formation were not significantly different between the two receptors. Purification on a Ni²⁺-NTA column resulted in a rapid (within 4 h) purification with a 36 ± 2% recovery and a 30 ± 6-fold purification (n = 5). The partially purified receptors were resolved on SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, dissolved in acetone/trifluoroacetic acid/hexafluoroisopropanol/sinapinic acid, and successfully subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. The results suggest that the combination of a high level of expression of C-His-TxA₂ receptors and a rapid purification procedure followed by SDS- polyacrylamide gel electrophoresis may provide a useful approach for mass-spectrometry based structure-function and other studies of TxA₂ receptors.

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