On the Expression of the Gi-coupled ADP-Receptor P2RY12 in Human Platelets

Abstract

P2RY12 is an important platelet receptor for ADP and an important drug target. Little is known about the inter- and intra-individual variability of the receptor on both, mRNA- and protein levels. Moreover, the tertiary and quaternary structure of the intact receptor is only partially known at present. The aims of this study were: 1. to characterize the transcript variants expressed in human platelets; 2. to establish a method for the quantitation of the protein in human platelets; 3. to characterize the specificity of commercial antibodies against P2Y12; 4. to establish a cell line with stable overexpression of a functional P2Y12-receptor. Methods and Results: As shown by RT-PCR, human platelets only expressed the shorter transcript variant of P2Y12, whereas in brain RNA both the longer and shorter transcript were detectable. After optimizing the procedure developed for the preparation of platelet membrane proteins (comprising differential centrifugation and detergent-extraction), P2Y12 was entirely contained in one fraction. Under denaturing reducing conditions, the protein migrated as a single broad band with an apparent molecular weight of ~ 50 kD. After deglycosylation with PNGase F the protein shifted to a condensed band with an apparent molecular weight of ~37 kD which is in good accordance with the predicted weight (39 kD). An HA-tagged P2Y12 receptor was transiently and stably expressed in HEK 293 cells, which had similar characteristics as the protein detected in platelets. The overexpression was used to analyse the specificity of two commercial antibodies which both yielded no correct signal (Anti-P2RY12, APR-012 from Alomone labs and P2RY12, P-14 from Santa Cruz). The only antibody tested which correctly identified both, the native and the overexpressed HA-tagged protein was a non-commercial antibody (gift of Dr. P. Savi). The functionality of the overexpressing was proven by concentration-dependent ADP-induced phosphorylation of the MAP-kinases p42/p44. Discussion: Human platelets express only the shorter of the two known RNA transcript variants (NM_176876). Future studies on differences in expression levels should firstly address the promoter of the shorter transcript variant. We were able to develop a fairly rapid method for the quantitation of P2RY12 mRNA and protein from human platelets enabling future studies on differences in the mRNA and protein expression in dependence on genetic or pathophysiological factors.