Biology subjects

Fanelli, F.

Publications and source records attributed to Fanelli, F..

3 recordsLinked to original sources

bioRxiv2024

Stabilised D2R G-protein coupled receptor oligomers identify multi-state β-arrestin complexes

The G-protein coupled receptor (GPCR) superfamily directs central roles in many physiological and pathophysiological processes via diverse and complex mechanisms. GPCRs can exhibit signal pleiotropy via formation of di/oligomers both with themselves and other GPCRs. A deeper understanding of the molecular basis and functional role of oligomerization would facilitate rational design of activity-selective ligands. A structural model of the D2 dopamine receptor (D2R) homomer identified distinct combinations of substitutions likely to stabilize protomer interactions. Molecular modelling of {beta}-arrestin-2 ({beta}arr2) bound to predicted dimer models suggests a 2:2 receptor:{beta}arr2 stoichiometry, with the dimer favouring {beta}arr2 over Gi coupling. A combination of biochemical, biophysical and super-resolution, single molecule imaging approaches demonstrated that the D2R mutant homomers exhibited greater stability. The mutant D2R homomers also exhibited bias towards recruitment of the GPCR adaptor protein {beta}arr2 with either faster or ligand-independent {beta}arr2 recruitment, increased internalization and reprogrammed regulation of ERK signaling. Through GPCR dimer-stabilization, we propose that D2R di/oligomerization has a role in {beta}arr2-biased signaling.

cell biology↗

bioRxiv2023

Engineered blue-shifted melanopsins for subcellular optogenetics

Melanopsin (MeOp) is a G protein-coupled Receptor (GPCR) family photopigment, expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) that display remarkable functional diversity. In addition to non-image-forming visual functions, MeOp also controls signaling underlying the retina development, circadian clock, mood, and behavior. MeOp is bistable, recycles retinal, and can function under low retinaldehyde availability. It also activates multiple G protein heterotrimers. Though MeOp could be a versatile optogenetic tool, its potential, especially its utility for subcellular signaling control, is hampered by the broader spectral sensitivity spanning the entire visible range. Here, we use a recently reported in silico technology called Automatic Rhodopsin Modeling (ARM) to identify blue-shifting mutations of MeOp and, ultimately, allow for imaging biosensors with red light without activating the opsin. Accordingly, ARM was used to construct validated quantum mechanics/molecular mechanics (QM/MM) models for mouse MeOp (mMeOp) to search and optimize a set of mutants featuring a blue-shifted light absorption. We demonstrate that four mutants of such can be successfully expressed and display the required resistance to activation by red light; however, they are activated by yellow, green, and blue light. Localized subcellular optical activation of these mutants in macrophage cells showed localized PIP3 generation and cell migration. Further characterization showed that MeOp blue-shifted mutants are also bistable. Altogether, our data demonstrate the computer-aided engineering feasibility of opsins with desired spectral properties for subcellular optogenetic applications.

biochemistry↗

bioRxiv2020

Membrane estrogen receptor (GPER) and follicle-stimulating hormone receptor heteromeric complexes promote human ovarian follicle survival

Classically, follicle stimulating hormone receptor (FSHR) driven cAMP-mediated signaling boosts human ovarian follicle growth and would be essential for oocyte maturation. However, contradicting in vitro suggest a different view on physiological and clinical significance of FSHR-mediated cAMP signaling. We found that the G protein coupled estrogen receptor (GPER) heteromerizes with FSHR, reprogramming cAMP/death signals into proliferative stimuli fundamental for sustaining oocyte survival. In human granulosa cells, survival signals are effectively delivered upon equal expression levels of both receptors, while they are missing at high FSHR:GPER ratio, which negatively impacts follicle maturation and strongly correlates with FSH responsiveness of patients undergoing controlled ovarian stimulation. Consistent with high FSHR expression levels during follicular selection, cell viability is dramatically reduced in FSHR overexpressing cells due to preferential coupling to the Gs protein/cAMP pathway. In contrast, FSHR/GPER heteromer formation resulted in FSH-triggered anti-apoptotic/proliferative signaling delivered via the G{beta}{gamma} dimer while heteromer impairment or GPER-associated Gs inhibitory protein complexes resulted in cell death. GPER-depleted granulosa cells have an amplified FSH-dependent decrease in cell viability and steroidogenesis, consistent with the requirement of estrogen signaling for successful oocyte growth. Therefore, our findings indicate how oocyte maturation depends on the capability of GPER to shape FSHR selective signals, indicating hormone receptor heteromers may be a marker of cell proliferation. One Sentence SummaryFSHR/GPER heteromers block cAMP-dependent selection of ovarian follicles and target tumor growth and poor FSH-response in women.

physiology↗