Structural and functional interactions of platelet thrombin and ADP receptors
Date
2015
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
A growing body of evidence on GPCR oligomerization has changed the
classical notion that GPCRs are monomeric, non-interacting membrane receptors
(Bouvier, 2001; Ferre et al., 2014; Milligan, 2008; Pin et al., 2007; Salahpour et al.,
2000). Moreover, the dogma that one GPCR binds to one G protein has been
challenged by several lines of evidence that show GPCR dimers are required for
efficient G protein coupling and hence the monomeric 1 GPCR: 1 G protein model
cannot be generalized for all GPCRs (Maurice et al., 2011).For members of Class A
GPCRs, there is unequivocal evidence that they form dimers and higher-order
oligomers; however, it has been more challenging to link the observed oligomerization
status with a requirement for function, given that interactions are frequently dynamic
and transient in nature. The proposed use of Adenosine A2A and Dopamine D2
receptor heteromer-specific properties as a strategy for the treatment of Parkinson’s
disease (Armentero et al., 2011) perhaps best exemplifies the pharmacological benefits
of investigating and understanding GPCR oligomerization and their functional and
physiological relevance. Thrombin receptors PAR1 and PAR4 and ADP receptor P2Y1 and P2Y12
belong to class A GPCRS and are essential for normal platelet function, haemostasis
and thrombosis (Gachet, 2008; Lin et al., 2013; Nakata et al., 2010). However the role
of PAR4-P2Y12 and P2Y12-P2Y12 oligomer status in receptor function has never
been elucidated. This work assesses the ability of PAR4-P2Y12 to form inducible
dimers and P2Y12 receptors to form constitutive dimers and further demonstrates that
the ability of P2Y12 mutant variants which have been established to cause bleeding
diatheses also exhibit a significantly altered ability to form oligomeric forms.
Our laboratory has demonstrated that thrombin induces the association of
PAR4 with P2Y12, together with arrestin recruitment to the complex (Li et al., 2011).
Here I show that PAR4 and P2Y12 directly interact to co-regulate Akt signaling
following PAR4 activation. I observed direct and specific interaction of P2Y12 with
PAR4, but not PAR1 by Bioluminescent Resonance Energy Transfer (BRET) when
the receptors were co-expressed in HEK293T cells. PAR4-P2Y12 dimerization was
promoted by PAR4-AP and inhibited by P2Y12 antagonist. Using sequence
comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant
form of PAR4, ‘PAR4SFT’, by replacing LGL194-196 at the base of transmembrane
domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT
supported only 8.74% of PAR4-P2Y12 interaction, abolishing P2Y12-dependent
arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still
supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux
when expressed independently; however, co-expression of increasing concentrations
of PAR4SFT together with PAR4 potentiated PAR4-mediated calcium flux,
suggesting that PAR4 act as homodimers to signal to Gq-coupled calcium responses.
In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4
with P2Y12 and contributes to Gq-coupled calcium responses. PAR4-P2Y12
association supports arrestin-mediated sustained signaling to Akt. Hence, PAR4-
P2Y12 dimerization is likely to be important for the PAR4-P2Y12 dependent
stabilization of platelet thrombi. P2Y12 is a critical target of anti-platelet drugs, but although its oligomeric
nature in platelets has been reported, the role of P2Y12 oligomerization in receptor
function has not been elucidated. Here, I investigated the ability of P2Y12 monomers
to directly interact using Bimolecular Fluorescence Complementation (BiFC) and
visualized these by confocal microscopy. P2Y12 receptors were observed to
specifically interact in a saturable manner in a saturation BiFC assay, as compared to a
no interaction between P2Y12 and prostanoid receptor DP2. In order to extend the
relevance of oligomerization to receptor function and its physiological importance, we
chose two P2Y12 mutant variants R256Q and R265W, which have been reported to
cause a mild bleeding disorder in human subjects (Cattaneo et al., 2003) and examined
their abilities to oligomerize. R256Q and R265W have a Gi function defect, but
exhibit normal membrane expression and ADP binding abilities (Mao et al., 2010). It
is unclear how structural integrity of Arg256 and Arg265 might influence Gi function
of P2Y12. Asymmetric model of one G protein associating with 2 GPCR molecules
has been supported by multiple class A GPCRs. Keeping these models in mind I
hypothesized that mutations R256Q and R265W might impair the ability of P2Y12
receptors to oligomerize which could in turn influence the ability of the mutant
receptors to associate with Gi leading to the observed functional defect. Wildtype
P2Y12 and mutant variants R256Q and R265W did not show altered expression or
altered trafficking when observed by live cell imaging. However, in contrast to our
hypothesis both R256Q and R265W mutants exhibited significantly brighter BiFC
fluorescence, indicating enhanced oligomerization abilities as compared to wild type
P2Y12 receptors. Fluorescence Correlation Spectroscopy and Photon Counting
Histogram analyses were employed to determine the oligomer size of wild type P2Y12
and assess whether R256Q and R265W mutants form higher order oligomers than
wild type P2Y12. BiFC complexes of P2Y12 and the mutant variants R256Q, and
R265W show that these are mainly present at least as dimers since the average
molecular brightness of the receptor pairs was significantly greater than the
monomeric Venus brightness standards. Moreover, the average molecular brightness
of R265W was significantly greater than wildtype P2Y12. We reasoned that the
average molecular brightness was much greater than the monomeric Venus brightness
standard due to the formation of a heterogeneous mixture of P2Y12 dimers/trimers
and tetramers. 2 component PCH analyses were performed to resolve this
heterogeneity of the receptor complexes, and determine the proportions of receptors
present as dimers and as oligomers greater than dimers. Preliminary modeling of the
heterogeneous mixture of receptors into at least two kinds of population showed that
77.25% of the wildtype P2Y12 receptors were present as dimers and 22.75% in
higher-order oligomer clusters. Interestingly, the R265W had a reduced proportion
(53.63%) of dimeric receptor pairs and an increased proportion of higher-order
oligomers (~46.37%). This indicates that R265W mutants have increased selfaffinities,
which might promote the noted formation of higher-order clusters. Such an
altered oligomerization profile of P2Y12 receptors due to the R265W mutation could
modify the abilities and proportion with which Gi would associate with receptor
clusters and thus influence the net Gi function defect. Testing the ability of R265W
mutant variant to associate with Gi will help in establishing a link between
oligomerization and G protein association. Receptor oligomerization moves the
receptor clusters to membrane micro domains which are primary sites for receptor
internalization. Hence altered membrane stability, internalization and recycling of
receptors is another potential facet of receptor life cycle that could be influenced by
enhanced oligomerization, thus reducing the net Gi function of the R265W mutant
variant of P2Y12. Future experiments on these lines will help bring to light the
potential role of P2Y12 oligomerization in receptor function.