Non-classical ligand-independent regulation of Go protein by an orphan Class C GPCR

The orphan G protein-coupled receptor (GPCR) GPR158 is expressed in the brain, where it is involved in the osteocalcin effect on cognitive processes, and at the periphery where it may contribute to glaucoma and cancers. GPR158 forms a complex with RGS7-[beta]5 leading to the regulation of neighboring GPCR-induced Go protein activity. Intriguingly, GPR158 also interacts with [alpha]o although no canonical Go coupling has been reported. GPR158 displays three VCPWE motifs in its C-terminal domain putatively involved in G protein regulation. Here, we addressed the scaffolding function of GPR158 and its VCPWE motifs on Go. We observed that GPR158 interacted with and stabilized the amount of RGS7-[beta]5 through a 50-residue region downstream of its transmembrane domain and upstream of the VCPWE motifs. We show that two VCPWE motifs are involved in [alpha]o binding. Using a G[alpha]o-β[gamma] BRET sensor we found that GPR158 decreases the BRET signal as observed upon G protein activation. However, no constitutive activity of GPR158 could be detected through the measurement of various G protein-mediated downstream responses. We propose that the effect of GPR158 on Go is unlikely due to a canonical activation of Go, but rather to the trapping of G[alpha]o by the VCPWE motifs possibly leading to its dissociation from [beta][gamma]. Such action of GPR158 is expected to prolong the [beta][gamma]activity as also observed with some Activators of G protein Signaling (AGS). Taken together our data revealed a complex functional scaffolding/signaling role for GPR158 controlling Go through an original mechanism. SIGNIFICANCE STATEMENT The orphan receptor GPR158 is the focus of increasing interest, as illustrated by recent publications on its roles in the central nervous system. Indeed, it plays crucial roles in the control of signal transduction in the retina and in differentiation of synapses in CA3 area of the brain, in addition to be a putative receptor for the well-known hormone osteocalcin. It could also be involved in pathology onset (glaucoma, prostate cancer, glioblastoma, etc.). Its peculiar structure (no known structure for its extracellular domain and a long intracellular domain possessing 3 specific VCPWE motifs) suggests atypical roles and functioning mechanisms. Our manuscript brings three major new findings. First, we demonstrate that the VCPWE motifs in the C-terminal domains of GPR158 are not necessary for RGS7 binding to GPR158 C-terminal domain, while they are key element for G protein o subunit binding. Second, we show that these VCPWE motifs regulate the dissociation of the heterotrimeric [alpha][beta][gamma] Go protein, and likely traps the G protein [alpha]o subunit, leading to reducing its association with the [beta][gamma] complex. This would be of importance, as both entities, [ALPHA]o subunit and [beta][gamma] complex regulate their own effectors, and any trapping of [alpha]o subunit by GPR158 VCPWE motifs would increase the signaling triggered by [beta][gamma] complex. Third, we bring evidence that GPR158 might not display constitutive canonical G protein coupling. This suggests that GPR158 activity is highly and tightly controlled. In absence of ligand, GPR158 can regulate Go protein via interaction with its VCPWE motifs and increase [beta][gamma] signaling, can terminate the Go coupling by interacting with the RGS7 protein known to inhibit Go signaling, and GPR158 may couple to a G protein when activated by osteocalcin. This makes GPR158 a new type of signaling regulatory receptor, whose peculiar features have to be put in line with the fact that the GPR158-related orphan receptor GPR179 possesses 21 VCPWE motifs. GPR179 is expressed in the retina where it controls the shape of the signaling response and mutated GPR179 receptors have been shown to induce night blindness.