MUTAGENIC ANALYSIS OF PLATELET THROMBOXANE RECEPTOR CYSTEINES - ROLESIN LIGAND-BINDING AND RECEPTOR-EFFECTOR COUPLING

The human platelet thromboxane A(2) receptor is a member of the G-prot ein-coupled superfamily of receptors. Previous pharmacologic studies e xamining the effects of biochemical reduction, oxidation, or sulfhydry l alkylation on thromboxane receptors have suggested a role for cystei nes in determining receptor binding characteristics. To characterize t he roles of individual cysteines, we employed site-directed mutagenesi s to substitute serines for cysteines at seven positions throughout th e human K562 thromboxane receptor and analyzed mutant receptor radioli gand ([1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S),4 alpha]-7-[3-(3- nyl)-7 -oxabicyclo-[2.2.1]heptane-2-yl]-5-heptenoic acid) binding and calcium signaling. Replacing cysteines in the amino terminus (amino acid posi tion 11), and transmembrane domains two and six (positions 68 and 257) had little effect on thromboxane receptor binding or signaling. Intro duction of serines for cysteines in the first (position 105) or the se cond (position 183) extracellular loop eliminated thromboxane receptor binding, consistent with the existence of a critical disulfide bond b etween these positions. Mutation of a second cysteine in extracellular loop one (position 102) resulted in a receptor with decreased binding affinity and low binding capacity that transduced only a low amplitud e calcium signal, suggesting the involvement of a free sulfhydryl grou p at this location in receptor-ligand interactions. Finally, mutation of the cysteine at position 223, located in intracellular loop three, resulted in a receptor with normal ligand binding characteristics, but which did not transduce a calcium signal. Some additional amino acid substitutions in this region of the receptor (Cys-223 --> Ala, Thr-221 --> Met) resulted in receptors that had normal binding but transduced low amplitude calcium signals, while other mutations in the same regi on (His-224 --> Arg and His-227 --> Arg) exhibited normal binding and calcium signaling characteristics. These findings demonstrate that cys teines in extracellular loops one and two contribute to proper ligand binding to thromboxane receptors and show the importance of discrete a mino acid sequences in the third intracellular loop, especially cystei ne 223, in thromboxane receptor-effector coupling.