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.