This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project is based upon the observation that PAR4 signaling in platelets is dependent on P2Y12, but the underlying basis for this is not understood. We have shown that PAR4 and P2Y12 can be co-?immunoprecipitated from human platelets following agonist activation and that the two receptors directly associate when expressed in HEK293T cells. We hypothesize that the functional dependence of PAR4 on P2Y12 is at least in part due to the physical association of the two receptors and that their association promotes the recruitment of the scaffolding protein, arrestin-?2, to the PAR4-?P2Y12 complex. We will test this hypothesis in the following 2 specific aims:
Aim 1 is to assess the specificity and structural requirements for PAR4-?P2Y12 heterodimerization in HEK293T cells. We have identified a 3-?amino acid determinant in the fourth transmembrane (TM4) of PAR4 required for interaction with P2Y12 assessed by bioluminescence resonance energy transfer (BRET) in HEK293T cells. We will confirm this result using co-immunoprecipitation studies, characterize membrane expression of the PAR4 mutant receptor using immunofluorescence microscopy and address the interaction specificity of both native PAR4 and our TM4-?PAR4 mutant using BRET and co-immunoprecipitation studies.
Aim 2 is to define the relevance of P2Y12 association with PAR4 to PAR4-?induced signaling responses. By expressing mutant versus native forms of PAR4 together with P2Y12, we will determine whether arrestin-?2 specifically associates with native PAR4 that is capable of interacting with P2Y12, but not a mutant form of PAR4 that fails to interact with P2Y12. We will then evaluate arrestin-?PI3K association and Akt phosphorylation under the same conditions. Finally, we will assess P2Y12-independent signaling of the mutant form of PAR4 to ensure the structural integrity of the mutant receptor. The project has the potential to impact our understanding of the regulation and functional significance of GPCR heterodimerization, and will help us to generate additional preliminary data for an R01 application to understand the functional significance of PAR4-?P2Y12 association in platelets.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Center Core Grants (P30)
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Special Emphasis Panel (ZRR1-RI-2 (01))
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University of Delaware
Engineering (All Types)
Schools of Engineering
United States
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