Molecular bases of defective signal transduction in the platelet P2Y12 receptor of a patient with congenital bleeding

We have identified structural attributes required for signal transduction through a seven-transmembrane-domain receptor. Platelets from a patient (AC) with a congenital bleeding disorder had normal shape change but reduced and reversible aggregation in response to 4 μM ADP, similar to normal platele...

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Published in:Proceedings of the National Academy of Sciences - PNAS 2003-02, Vol.100 (4), p.1978-1983
Main Authors: Cattaneo, Marco, Zighetti, Maddalena L, Lombardi, Rossana, Martinez, Constantino, Lecchi, Anna, Conley, Pamela B, Ware, Jerry, Ruggeri, Zaverio M
Format: Article
Language:English
Subjects:
Adult
Alprostadil - metabolism
Base Sequence
Biological Sciences
Blood Platelets - metabolism
Calcium - metabolism
Cyclic AMP - metabolism
DNA Primers
Female
Hemorrhage - congenital
Hemorrhage - genetics
Heterozygote
Humans
Male
Membrane Proteins
Middle Aged
Receptors, Purinergic P2 - genetics
Receptors, Purinergic P2 - metabolism
Receptors, Purinergic P2Y12
Signal Transduction
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Summary:We have identified structural attributes required for signal transduction through a seven-transmembrane-domain receptor. Platelets from a patient (AC) with a congenital bleeding disorder had normal shape change but reduced and reversible aggregation in response to 4 μM ADP, similar to normal platelets with blocked P2Y 12 receptor. The response to 20 μM ADP, albeit still decreased, was more pronounced and was reduced by a P2Y 12 antagonist, indicating some residual receptor function. ADP failed to lower the adenylyl cyclase activity stimulated by prostaglandin E 1 in the patient's platelets, even though the number and affinity of 2-methylthioadenosine 5′-[ 33 P]diphosphate-binding sites was normal. Analysis of the patient's P2Y 12 gene revealed a G-to-A transition in one allele, changing the codon for Arg-256 in the sixth transmembrane domain to Gln, and a C-to-T transition in the other allele, changing the codon for Arg-265 in the third extracellular loop to Trp. Neither mutation interfered with receptor surface expression but both altered function, since ADP inhibited the forskolin-induced increase of cAMP markedly less in cells transfected with either mutant P2Y 12 as compared with wild-type receptor. These studies delineate a region of P2Y 12 required for normal function after ADP binding. ADP‖platelet function disorder‖G-protein coupled receptors‖ platelet aggregation
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0437879100