RGS proteins: Potential regulators of mouse melanopsin's signaling phototransduction cascade across ipRGC subtypes

Melanopsin, a G-protein coupled receptor (GPCR) and photopigment, mediates nonimage-forming and image-forming visual behaviors. There are six subtypes of intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mouse retina; although melanopsin signals through Gα_q in all subtypes, evidence suggests that M1 and M4 ipRGCs may utilize distinct phototransduction pathways. We sought to uncover a mechanism that might enable a single GPCR to initiate different signaling cascades in these ipRGC subtypes. In HEK293 cells heterologously expressing mouse melanopsin, we show that melanopsin couples promiscuously to all four G-protein families and generates cAMP via a Gα_q-Gβγ-dependent mechanism. In the mouse retina, activation of virally expressed DREADD-Gs demonstrated that cAMP signaling drives action potentials in both M1 and M4 ipRGCs, revealing that M1 cells possess the adenylate cyclase and cAMP-sensitive channels required to respond to cAMP. Using a meta-analysis of single-cell transcriptomics together with RNAscope in the retina, we discovered that ipRGC subtypes differ and selectively enrich certain regulators of G-protein signaling (RGS). Rgs16 is enriched in a subset of M1 ipRGCs, whereas M4 ipRGCs preferentially express Rgs5 and Rgs11. The G-protein selectivity of these RGS proteins under heterologous expression revealed that RGS5 is selective for Gα_i/o, and a combination of RGS proteins found in M4 ipRGCs proteins severely attenuates Gα_i/o signaling compared with M1-like RGS. Our heterologous expression data together with experiments in native ipRGCs suggest a model where the subtype-specific repertoire of RGS proteins in M4 ipRGCs might attenuate Gα_i/o signaling, permitting enhanced Gα_q-Gβγ-driven cAMP. This study provides a potential mechanistic explanation of how a single GPCR can initiate distinct phototransduction cascades in separate cell types.