Identification of Gq as one of the G-proteins which copurify with human platelet thromboxane A2/prostaglandin H2 receptors.
The present study employed ligand affinity and immunoaffinity chromatography to isolate human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor-coupled G-proteins. Purification of TXA2/PGH2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the elution of receptor binding and GTPase activity in the same fraction. GTPase activity of this fraction was enriched (6-fold) relative to solubilized platelet membranes, was stimulated (65%) by 500 nM U46619, and was blocked (74%) by 250 nM SQ29,548. Furthermore, GTP (100 microM) increased [3H]SQ29,548 receptor binding by 48%. Immunoblotting of this fraction against QL antiserum identified a 42-kDa protein as a member of the Gq family. In separate experiments, TXA2/PGH2 receptors were purified by immunoaffinity chromatography using P1Ab, P2Ab, and TxAb affinity columns. QL-immunoreactive proteins at 42 kDa were found in all three column eluates. Studies using G alpha,common antiserum (GA/1) demonstrated immunoblotting of two proteins of approximately 42 and 85 kDa in both the ligand and P2Ab affinity column fractions. On the other hand, the P1Ab and TxAb affinity column eluates contained GA/1 immunoreactivity only in the 42 kDa region. Collectively, these data identify Gq as a TXA2/PGH2 receptor-coupled G-protein and suggest the association of this receptor with additional G alpha subunits.
Knezevic
I
I
Department of Pharmacology, University of Illinois, Chicago 60612.
Borg
C
C
Le Breton
G C
GC
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D005231:Fatty Acids, Unsaturated; D020558:GTP Phosphohydrolases / Q000097:blood; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D006834:Hydrazines / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology; D011982:Receptors, Prostaglandin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017482:Receptors, Thromboxane / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D011450:Prostaglandin Endoperoxides, Synthetic; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D014662:Vasoconstrictor Agents; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D006160:Guanosine Triphosphate; C045749:SQ 29548; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins