Coupling of thromboxane A2 receptor isoforms to Galpha13: effects on ligand binding and signalling.
Previous subtyping of thromboxane A2 (TXA2) receptors in platelets and vascular smooth muscle cells was based on pharmacological criteria. Two distinct carboxy-terminal splice variants for TXA2 receptors exist and they couple to several different G protein alpha subunits including Galpha13, but it has not been established whether either or both isoforms interact with and signal through it. We sought to determine: (1) which TXA2 receptor isoforms exist in vascular smooth muscle, (2) if Galpha13 is present in vascular smooth muscle and (3) if Galpha13 interacts with either or both of the two TXA2 receptor isoforms as determined by changes in ligand binding properties and generation of intracellular signals. Both TXA2 receptor isoforms and Galpha13 were found in vascular smooth muscle cells. Both the alpha and beta isoforms of the TXA2 receptors were transiently transfected with or without Galpha13 into COS-7 (radioligand binding assays) or CHO cells (agonist induced Na+/H+ exchange). Co-expression of each receptor isoform with Galpha13 significantly (P<0.05) increased the affinity of each receptor for the two agonists, I-BOP and ONO11113, and decreased the affinity of the receptor for the antagonists, SQ29,548 and L657,925. I-BOP stimulated Na+/H+ exchange in vascular smooth muscle cells. Co-expression of Galpha13 with each TXA2 receptor isoform in CHO cells resulted in a significant (P<0.04) agonist induced increase in Na+/H+ exchange compared to cells not transfected with Galpha13. The results support the possibility that the previous classification of TXA2 receptor subtypes based on pharmacological criteria reflect unique interactions with specific G protein alpha subunits.
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425, USA.
K P
KP
Becker
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M
Garnovskaya
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T
Gettys
P V
PV
Halushka
D000818:Animals; D015153:Blotting, Western; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000096:biosynthesis; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics; D006834:Hydrazines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017482:Receptors, Thromboxane / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D019086:Bridged Bicyclo Compounds, Heterocyclic; D018076:DNA, Complementary; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D020033:Protein Isoforms; D017482:Receptors, Thromboxane; C061750:7-(3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo(2.2.1)heptan-2-yl)-5-heptenoic acid; C045749:SQ 29548; D019204:GTP-Binding Proteins