One of the most prominent features of the GPR158 ectodomain is the dimerization of the Cache domains which occurs through helices α1 and α2 of each Cache domain which cross at an angle to create a four-helix bundle at the dimer-interface (fig. S9E). The loops connecting helices α1 and α2 are also likely involved in inter-subunit interaction. The dimer interface is stabilized by an extensive network of hydrophobic and hydrophilic interactions, with a buried surface area of 2178.3 Å2 (fig. S9F).
RGS7 recruitment is reminiscent of GPCR interactions with signal transducers. Indeed, the RGS binding surface on GPR158 substantially overlaps with the GPCR surface that binds heterotrimeric G proteins and β-arrestin (fig. S11, A and B). Our modeling using structures of Gα complexes with diverse GPCRs shows that the RGS7 DHEX domain occludes the G protein binding site from the TM3 and TM5 side where the α5 helix of Gα inserts into 7TM central cavity and creates steric clashes with the Ras domain of Gα subunits (fig. S11, C to E). Thus, recruitment of RGS7-Gβ5 would preclude GPR158 from productively interacting with G proteins, supporting lack of G protein activation (fig. S8). We further detect bidirectional allosteric effects resulting from the GPR158-RGS7 binding similar to what is observed upon GPCR-Gα interaction. These include inward shift of the cytoplasmic end of TM3, as seen in the GABAB-Gi structure and modulation of the ligand-binding ectodomain upon RGS7 binding. The interaction of GPR158 with RGS7-Gβ5 complex is quite distinct and mutagenesis at the GPR158 dimerization interface that constutitvely activate class C GPCRs failed to change RGS activity in the absence of a ligand (fig. S12).
To further investigate conformational dynamics resulting from RGS7-Gβ5 recruitment to GPR158 we performed biochemical experiments. First, we studied the impact of binding to a synthetic C-terminal peptide that comprises the CT-CC module by gel filtration. Complexing with this peptide was sufficient to induce a large change in hydrodynamic behavior of the RGS7-Gβ5 complex consistent with substantial conformational changes in RGS7-Gβ5 upon binding to GPR158 (fig. S13A). We further refined these investigations using HDX-MS which showed that the C-terminal peptide induced significant changes in solvent accessibility within the DEP-DHEX domain, specifically in Dα1, Eα3 and Eα4 helices, β-hairpin, Eα3Eα4 and DEP-DHEX loops of RGS7 (fig. S13, B to D).
In this work, we present high-resolution structures of an unusual receptor assembly that involves an orphan GPCR complexed with a signaling regulator- RGS protein. The RGS protein binds the same elements that GPCRs use for engaging their signal transducers: G proteins and β-arrestins. In the present structure we observe constitutive engagement of RGS7/Gβ5 complex by GPR158 in the absence of a G protein. We speculate that binding of a ligand to the extracellular ECD would activate GPR158 rearranging of the cytoplasmic domains that engage RGS to alter its activity. Given that RGS binding precludes GPR158 from canonical activation of G proteins, one can describe it as an RGS-coupled receptor.
In addition to providing information on the GPCR-RGS structure we show the role of two phospholipids in organizing the dimerization interface of GPCRs. These lipids staple the protomers and provide intriguing possibilities for GPCR modulation. We also identify a Cache domain raising the possibility that GPR158 detects a small molecule ligand that could regulate of the RGS module, an avenue to be explored in future studies. In conclusion, we hope our findings will spur further progress in understading the regulatory and signaling mechanisms of GPR158 by facilitating the structure-based discovery of its ligands and by guiding exploration of GPR158-mediated control of RGS proteins in the endogenous neuronal setting.