Structural Elements Directing G Proteins and β-Arrestin Interactions with the Human Melatonin Type 2 Receptor Revealed by Natural Variants

Abstract

G protein-coupled receptors (GPCRs) can engage distinct subsets of signaling pathways, but the structural determinants of this functional selectivity remain elusive. The naturally occurring genetic variants of GPCRs, selectively affecting different pathways, offer an opportunity to explore this phenomenon. We previously identified 40 coding variants of the MTNR1B gene encoding the melatonin MT 2 receptor (MT 2 ). These mutations differently impact the β-arrestin 2 recruitment, ERK activation, cAMP production, and Gα i1 and Gα z activation. In this study, we combined functional clustering and structural modeling to delineate the molecular features controlling the MT 2 functional selectivity. Using non-negative matrix factorization, we analyzed the signaling signatures of the 40 MT 2 variants yielding eight clusters defined by unique signaling features and localized in distinct domains of MT 2 . Using computational homology modeling, we describe how specific mutations can selectively affect the subsets of signaling pathways and offer a proof of principle that natural variants can be used to explore and understand the GPCR functional selectivity.

CHARMM-GUI Membrane Builder: Past, Current, and Future Developments and Applications

“…MD simulations have been extensively used to study GPCR interactions with signaling molecules − as well as transducers in the cytosolic side. − For example, Park et al recently studied the neuropeptide Y and Y1 receptor, which is enriched in the brain and responsible for neurological processes such as food intake, anxiety, obesity, and cancer. , The cryo-EM structure shows the binding of neuropeptide Y to Y1 receptor, and MD simulations further reveal the stability and dynamics of the neuropeptide Y binding, providing a conformational ensemble of binding interactions (Figure A). Other studies have examined the GPCR-G protein binding and tried to derive a rationale for signaling through different G proteins or β-arrestin. − Zhao et al studied β2 adrenaline receptors and their differential binding interfaces with Gs, Gi, and β-arrestin 1 . Since the binding interface is different for each cellular transducer, the conformational rearrangement in the TM domain is also different, allowing signal transduction to happen.…”

“…GPCRs play a vital role in relaying information from neurotransmitters and hormone by transmitting the binding of these molecules from the extracellular side to signals on the intracellular side via binding with cellular transducers such as G protein or β-arrestin. MD simulations have been extensively used to study GPCR interactions with signaling molecules − as well as transducers in the cytosolic side. − For example, Park et al recently studied the neuropeptide Y and Y1 receptor, which is enriched in the brain and responsible for neurological processes such as food intake, anxiety, obesity, and cancer. , The cryo-EM structure shows the binding of neuropeptide Y to Y1 receptor, and MD simulations further reveal the stability and dynamics of the neuropeptide Y binding, providing a conformational ensemble of binding interactions (Figure A). Other studies have examined the GPCR-G protein binding and tried to derive a rationale for signaling through different G proteins or β-arrestin. − Zhao et al studied β2 adrenaline receptors and their differential binding interfaces with Gs, Gi, and β-arrestin 1 .…”

Molecular Characterization and Pharmacology of Melatonin Receptors in Animals

“…In addition to monitoring cAMP production, the assays of GTP-shift and [ 35 S]-GTPγ binding have also been classically applied to characterize activation of melatonin receptors [46,99]. More recently, with the development of several Bioluminescence Resonance Energy Transfer (BRET)-based sensors for the investigation of GPCR functions [100], assessing several G protein subunits and isoforms' recruitment and activation or ß-arrestin recruitment became part of the repertoire of assays for melatonin receptor and ligands characterization [101,102]. Coupling to both G protein and ß-arrestin contributes to the formation of the high-affinity ternary complex comprising ligand-receptor-effector, typically revealed in the 2[ 125 I]-iodoMLT binding assays.…”