DISCUSSION
GPR56 is known to be involved in a wide range of physiological processes
in the nervous system, immune system, male reproductive organs,
hematopoietic stem and progenitor cells; functional problems with the
receptor result in pathologies such as Bilateral frontoparietal
polymicrogyria (BFPP) , depression and arguably GPR56 is the most
studied aGPCR in cancer [37]. BFPP is a monogenetic disease
resulting in a severe brain malformation and several mutations in GPR56
were found in patients diagnosed with BFPP [2]. In this work,
five missense BFPP mutations on GPR56 were chosen that were
previously reported to be expressed on the plasma membrane [32].
From those, R38W, Y88C and C91S are found on the NTF of the receptor;
R565W is located on the 2nd extracellular loop and
L640R is on the 3rd extracellular loop.
GPR56 is known to inhibit neural progenitor cell migration by coupling
with Gα12/13 and inducing Rho dependent activation of
the transcription mediated through the serum-response element (SRE) and
NF-κB-responsive element resulting in an actin fiber reorganization
[38]. GPR56 is also known to form a complex with tetraspanin
proteins CD9 and CD81 on the plasma membrane and this complex couples
with Gαq/11 and Gβ subunits [34]. Agonistic
monoclonal antibodies generated towards the NTF of GPR56 resulted in the
inhibition of human glioma cell migration through the
Gαq dependent Rho pathway [35]. GPR56 was also
reported to promote myoblast fusion through SRE and nuclear factor of
activated T-cell (NFAT) mediated signaling [39]. Expression of GPR56
into HEK293 cells is known to lead Gα12/13 activation
and induce Rho dependent stimulation of SRE [38], however the Rho,
SRE or NFAT mediated downstream signaling studies fail to discriminate
between Gα12 and Gα13 signaling, which
were known to have distinct physiological roles [40-42].
Furthermore, Gβγ and Gαq/11 was also reported to induce
Rho activation [43, 44].
GPR56 has numerous interacting partners such as collagen III [6],
tissue transglutaminase (TG2) [45], and laminin [46]; the
receptor was also previously reported to be activated by itsStachel peptide (TYFAVLM ) [20]. In the current work,
the coupling of GPR56 with Gα12, Gα13and Gα11 upon the receptor activation with itsStachel peptide and the effects of BFPP mutants on G
protein coupling was studied. For this, nanoBRET based biosensor system
was chosen that is suitable for untagged receptor, untagged Gα, Venus
tagged Gβ1γ2 and masGRK3ct-Nluc which is composed of G protein-coupled
receptor kinase 3 carrying a myristic acid plasma membrane attachment
peptide tagged with Nluc [28].
Under the biosensor conditions reported in this study, stimulation of
GPR56 with its Stachel peptide showed G protein coupling and
heterotrimeric G protein activation for Gα12,
Gα13 and Gα11. However, GPR56BFPP mutants showed different coupling defects for each G protein
α-subunit. Coupling between GPR56 and Gα12 was disrupted
in both extracellular loop BFPP mutations (GPR56L640R and
GPR56R565W); on the other hand, receptor Gα13 coupling
and heterotrimeric G protein activation was measured for the
extracellular loop receptor mutants. These results indicate the distinct
signaling roles of GPR56 with Gα12 or
Gα13. Stachel peptide stimulation of the receptor
also showed measurable Gα11 activation in the biosensor
assay reported herein. GPR56 activation was shown to lead Gβγ subunit
dissociation from Gα11 and liberated Gβγ activates the
calcium channels [33]. The working principle of the biosensor in
this study relies on the change in nanoBRET upon dissociation of Gβγ
from Gα as a result of receptor activation. While the results confirmed
the previous findings, studying BFPP mutants provided further
insights into Gα11 signaling. The most severe disruption
was observed between GPR56R565W and Gα11 coupling. AllBFPP mutants showed disrupted coupling with Gα11and heterotrimeric G protein activation except for GPR56C91S. In sum,
stimulation of GPR56 with its Stachel peptide resulted in
measurable activation in Gα12, Gα13 and
Gα11 signaling however, BFPP mutations resulted
in distinctly different effects for each signaling pathway. It is
noteworthy to mention at this point that, the data supplied in this
study reflects the activation of GPR56 with its Stachel peptide,
collagen III or TG2 treatment will provide more insights about the
signaling of this receptor.
In the second part of the study, β-arrestin recruitment of GPR56 orBFPP mutants was measured upon activation of the receptor with
its Stachel peptide. For this, a BRET based biosensor suitable
for untagged receptors that measures the translocation of β-arrestin to
the plasma membrane upon receptor activation was utilized. In the
biosensor design, Rluc8 tagged β-arrestin and membrane-bound citrine
fluorescent protein were used and the recruitment of β-arrestin was
measured as a BRET increase in the exogenous expression of G
protein-coupled receptor kinase 2 [30]. It was previously reported
that NTF truncated GPR56 showed enhanced binding with β-arrestin [33,
36]. In accordance with the previous findings, NTF truncated GPR56
gave enhanced β-arrestin recruitment to the plasma membrane, howeverStachel peptide stimulation of neither GPR56 nor BFPPmutants showed any significant change in β-arrestin recruitment. These
results point to a different receptor activation mechanism for
β-arrestin possibly through the activation of GPR56 with other
ligands/interacting partners.
The complex architecture of aGPCRs arises from their extremely large
NTFs. These structures carry various protein domains that are involved
in diverse array of protein interactions including the extracellular
matrix proteins; cell-cell interactions and cell adhesion. Some members
of this enigmatic family of receptors were also shown to be involved in
sensing the mechanical stimuli at the cellular level [18, 47-49].
Adhesion GPCRs also function like classical GPCRs, signaling canonically
through their CTF domains that couple with and activate heterotrimeric G
proteins. Hence, our current knowledge points to the multifaceted and
multi-functional roles of this receptor family in signal transduction
and modulation [14, 15]. The results reported in this study measured
the direct coupling of three G proteins, Gα12,
Gα13 and Gα11 with GPR56 and the
heterotrimeric G protein activation. Previous reports on GPR56 signaling
rely mostly on the Rho dependent activation of further downstream
elements rather than the direct G protein activation. Studying the
downstream signaling defects of the disease associated mutations of
GPR56 indicated that the receptor has distinct activation and signaling
properties for each G protein and mutations located in various
compartments of the receptor showed distinctly different coupling
disruptions. In β-arrestin recruitment assays, NTF truncated receptor
showed enhanced β-arrestin translocation to the plasma membrane, however
stimulation of wild-type GPR56 or BFPP mutants with theStachel peptide did not result in any recruitment. These results
might indicate that β-arrestin recruitment possibly require different
activation mechanisms.
The BRET based biosensors used in this study showed the direct coupling
of GPR56 with Gα12, Gα13 and
Gα11 and the heterotrimeric G protein activation. Also,
BRET based β-arrestin biosensor measured an enhanced recruitment of the
NTF truncated receptor as previously reported. Considering the rich
physiology and related pathologies of GPR56, the biosensors utilized in
this work can further be applied for studying the mechanisms of receptor
activation through different interacting partners and applied for drug
screening studies.