Functional analysis of the dimerization potential of the P2Y12 receptor and two naturally-occuring mutant variants
Date
2017
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Publisher
University of Delaware
Abstract
An expanding body of evidence on GPCR oligomerization has challenged the classical concept that GPCRs are non-interacting or monomeric membrane receptors (Bouvier, 2001; Ferre et al., 2014; Milligan, 2008; Pin et al., 2007; Salahpour et al.,2000). Furthermore, a numerous number of evidence that state that G-Protein-Coupled-Receptor dimers are required for effective G protein coupling have challenged the belief that one GPCR interacts to one G protein. Therefore, the monomeric one GPCR: one G protein pattern cannot be generalized to all the receptors (Maurice et al., 2011). ☐ For the Class A family of GPCRs, there is some evidence that some family members form dimers; however, it has been more challenging to combine the observed oligomerization status with whether a direct homomeric association of GPCRs is required for function, given that the interactions are sometimes transient or dynamic in nature and other times constitutive. It has been observed that these interactions sometimes correlate with function and sometimes not. The suggested use of dopamine D2 and adenosine A2A receptor heteromer-specific tools as an approach to treat some diseases (Armentero et al., 2011) possibly best represents the pharmacological advantages of further understanding GPCR dimerization and their functional relevance. Thus, we set out to investigate GPCR dimerization and functional relevance for the platelet GPCR, P2Y12, in this thesis. ☐ ADP receptors P2Y1 and P2Y12 belong to class A GPCRS and are essential for normal platelet function, hemostasis and thrombosis (Gachet, 2008; Lin et al., 2013; Nakata et al., 2010). However, the role of P2Y12-P2Y12 dimer or oligomer status in receptor function has never been elucidated. This work assesses the ability of the WT P2Y12 receptors to form homo-dimers and demonstrates the ability of P2Y12 mutants reported to occur concurrently with bleeding diatheses to exhibit a notably changed dimerization ratio compared to the WT receptor. ☐ P2Y12 is a crucial target of anti-platelet drugs, but the role of its dimer/oligomerization in receptor function has not been clarified, although it is in vitro oligomeric status has been reported. In this project, I investigated the ability of P2Y12 protomers to homodimerize using Fluorescence Correlation Spectroscopy (FCS) and Photon Counting Histogram (PCH) analyses of confocal microscopy. Previous studies from our lab demonstrated that P2Y12 receptors could specifically interact in a saturable manner in a saturation Bi-fluorescence complementation (BiFC) assay. To determine the importance of oligomerization to receptor physiological relevance, we chose to evaluate the dimerization potential of two mutant variants of P2Y12, P2Y12-R256Q and -R265W, which were detected in a patient with a mild bleeding disorder and was found to have compound heterozygous expression of the mutations (Cattaneo et al., 2003). The platelets from the patient expressing these mutant variants had compromised Gi function, although the platelets displayed normal ADP binding and membrane expression (Mao et al., 2010). It is unknown how Arg256 and Arg265 affect the structural integrity of the receptor and how they may influence Gi function of the receptor. The existence of an asymmetric model in which one G-protein may couple to two GPCRs has been hypothesized and there is evidence for this stoichiometry for various class A GPCRs. ☐ Taking these models into account, we hypothesized that the mutant variants (R256Q and R265W) might weaken the ability of P2Y12 receptors to dimerize which in turn could affect their ability to couple to Gi, leading to the observed functional defect. WT P2Y12 and mutant variants R256Q and R265W did not show altered expression or altered trafficking when observed by live cell imaging (Khan,2015). However, in contrast to my hypothesis, both R256Q and R265W mutants displayed significantly high molecular brightness, indicating enhanced dimerization abilities compared to WT P2Y12. ☐ Such a modified oligomerization profile of these receptors due to the R265W / R256Q mutations might alter the abilities or proportion with which Gi might associate with receptors, thus affecting the net Gi function. We have shown that the R256Q mutant variant of P2Y12, in fact, fails to activate Gi optimally. The change in oligomerization status of this variant receptor may affect Gi activation either by disturbing the surface interface for receptor association to G protein or by altering the conformation which allows G protein to exchange GDP for GTP. These possibilities will be discriminated in future work. In addition, altered internalization, recycling or membrane stability of receptors, could be impacted by enhanced oligomerization, hence reducing the net Gi function of the mutant variants of P2Y12. These future lines of investigation will help us further understand the potential role of P2Y12 oligomerization in receptor structure/function and dynamics.
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Biological sciences