A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.
We investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A(2) receptor (TxA(2)R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA(2)R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA(2)R, U46619 did not increase cytosolic free Ca(2+) concentration, indicating loss of receptor function. The TxA(2)R antagonist [(3)H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA(2)R. This is the second naturally occurring TxA(2)R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA(2)R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA(2)R function in vivo.
Mumford
Andrew D
AD
Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom. a.mumford@bristol.ac.uk
Dawood
Ban B
BB
Daly
Martina E
ME
Murden
Sherina L
SL
Williams
Michael D
MD
Protty
Majd B
MB
Spalton
Jennifer C
JC
Wheatley
Mark
M
Mundell
Stuart J
SJ
Watson
Steve P
SP
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000293:Adolescent; D020816:Amino Acid Motifs / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D005231:Fatty Acids, Unsaturated; D005260:Female; D015870:Gene Expression; D006474:Hemorrhagic Disorders / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006834:Hydrazines / Q000494:pharmacology; D008024:Ligands; D008297:Male; D020125:Mutation, Missense; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D044244:Receptors, Thromboxane A2, Prostaglandin H2 / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D014662:Vasoconstrictor Agents / Q000494:pharmacology
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D008024:Ligands; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D014662:Vasoconstrictor Agents; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; C045749:SQ 29548; D000255:Adenosine Triphosphate; D002118:Calcium