Molecular and Structural Determinants of Melanopsin Signaling Specificity

Abstract

Purpose: The core question I address is mechanistic: how can one GPCR choose different G-proteins and second messengers depending on the cell it sits in? The main goal of this work is to identify a mechanism through which melanopsin engages in distinct signaling cascades, which would explain the observed heterogeneity of signaling seen in vivo. An overarching goal is to show, using melanopsin as a prototypical GPCR, that a regulatory protein network unique to each cell type ultimately determines the outcome of GPCR signaling. We hypothesize that RGS proteins act as a downstream filter of G-proteins, inhibiting certain signals in M1 or M4 ipRGCs, enabling melanopsin to diversify its signaling cascade to suit the needs of the individual cell types where it is expressed. Method: A combination of transcriptomics and RNAscope immunohistochemistry that labeled individual subtypes of ipRGC. Bioluminescence resonance energy transfer (BRET) assays were optimized for stoichiometry and stimulus length to achieve optimal and reproducible results with opsin GPCRs. We performed electrophysiology in mouse lines specifically labeling M1 or M4 ipRGCs, with targeted Designer Receptor Exclusively Activated by Designer Drugs (DREADD) expression to evaluate preference for a Gq or Gs cascade. Sequence alignment and computationally derived structural modeling were employed to evaluate a series of melanopsin-DREADD chimeras. Results: We show that RGS proteins are differentially expressed among ipRGC subtypes. When RGS proteins are combined to create an M1- or M4-like profile, the M1-like RGS proteins caused melanopsin to signal Gi greater than Gq. Furthermore, melanopsin can produce cAMP downstream of Gq signaling alone, We tested both Gq- and Gs-DREADDs in M1 and M4 ipRGCs and found that they activated both cell types similarly. Separate experiments focused on the role of PKA phosphorylation of melanopsin produced the expected result of reducing Gq coupling, which we confirmed occurs with Gs signaling of the B2AR as well. Conclusion: Our data show that melanopsin’s sequence and structure provides a broad G-protein signaling potential, while subtype-specific RGS repertoires and activity-dependent PKA phosphorylation filter that signaling to the single pathway observed in each ipRGC subtype. Together, these results support a two-tier scheme. The first is upstream signaling, where sequence and structure of a GPCR defines which G-proteins can be activated. The second is regulation of downstream signaling, where cell-restricted RGS determine which G-protein pathways ultimately drive functional activity, and an activity–driven PKA regulation which tunes the magnitude of signaling. This work demonstrates that G-protein pathway bias can arise solely from intracellular context, without changing ligands or receptor.