Fine-tuning GPCR-mediated neuromodulation by biasing signaling through different G-protein subunits

Abstract

GPCRs mediate neuromodulation through activation of heterotrimeric G-proteins (Gαβγ). Classical models depict that G-protein activation leads to a one-to-one formation of Gα-GTP and Gβγ species. Each of these species propagates signaling by independently acting on effectors, but the mechanisms by which response fidelity is ensured by coordinating Gα and Gβγ responses remain unknown. Here, we reveal a paradigm of G-protein regulation whereby the neuronal protein GINIP biases inhibitory GPCR responses to favor Gβγ over Gα signaling. Tight binding of GINIP to Gαi-GTP precludes its association with effectors (adenylyl cyclase) and, simultaneously, with Regulator-of-G-protein-Signaling (RGS) proteins that accelerate deactivation. As a consequence, Gαi-GTP signaling is dampened whereas Gβγ signaling is enhanced. We show that this mechanism is essential to prevent imbalances of neurotransmission that underlie increased seizure susceptibilityin vivo. Our findings reveal an additional layer of regulation within a quintessential mechanism of signal transduction that sets the tone of neurotransmission.

Dissecting the molecular basis for the modulation of neurotransmitter GPCR signaling by GINIP

“…On the G protein side of this interface, the model was also very congruent with previous experimental evidence (Park, 2023). First of all, GINIP's PHD loop 1 docks on a groove formed between the α3 and the switch II of Gαi, a site previously proposed to be essential for GINIP binding based on G protein mutagenesis data (Park, 2023). For example, mutation of Gαi3's W211, F215, K248, L249, S252, N256 or K257, all of which are predicted by the model to contact GINIP's PHD loop 1 (Fig.…”

“…4C), resulted in loss of binding (Park, 2023), whereas mutation of adjacent amino acids like S206 or G42 not predicted by the model to make direct contact (Fig. 4C) had no effect on binding (Park, 2023). Thus, the high congruency between extensive protein-protein binding data after site-directed mutagenesis and the structure model prediction lends confidence to the conclusion that GINIP's PHD loop 1 is a critical structural determinant for the association with G proteins.…”

“…GINIP forms a stable one-to-one complex with Gαi3-GTP Previous work has established that GINIP binds with high affinity (i.e., K D ~65 nM) to active Gαi3 (Park, 2023), but the specific regions of GINIP involved in this protein-protein interaction were not elucidated. To identify these regions, we set out to carry out hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments with GINIP in the presence and absence of Gαi3.…”

“…For example, mutation of Gαi3's W211, F215, K248, L249, S252, N256 or K257, all of which are predicted by the model to contact GINIP's PHD loop 1 (Fig. 4C), resulted in loss of binding (Park, 2023), whereas mutation of adjacent amino acids like S206 or G42 not predicted by the model to make direct contact (Fig. 4C) had no effect on binding (Park, 2023).…”

“…Recent work has established a unique mechanism by which the neuronal protein GINIP acts as a broad regulator of G i -coupled GPCR signaling in neurons that alters the balance of signaling after receptor activation (Park, 2023). GINIP binds to Gαi-GTP, but does not affect directly how nucleotides are handled by the G protein.…”