Detection and Quantitative Analysis of Dynamic GPCRs Interactions Using Flow Cytometry-Based FRET

Chruścicka, Barbara; Fitzsimons, Shauna E. Wallace; Druelle, Clémentine; Dinan, Timothy G.; Schellekens, Harriët

doi:10.1007/978-1-4939-8576-0_14

Cited by 3 publications

(4 citation statements)

References 65 publications

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“…Prior to analysis, cells were passed through a 100 μm nylon mesh cell strainer (#10199-658; VWR) and collected in 5 mL round-bottom polystyrene tubes (#352054; Corning). The fcFRET analysis was performed on a FACS AriaII cytometer (BD Biosciences) according to the protocol optimized in our group . Briefly, EGFP was excited at 488 nm from a blue laser and detected with a 525/50 nm bandpass filter, whereas tRFP was excited at 561 nm from a yellow/green laser and detected with a 610/20 nm bandpass filter.…”

Section: Methodsmentioning

confidence: 99%

“…The fcFRET analysis was performed on a FACS AriaII cytometer (BD Biosciences) according to the protocol optimized in our group. 65 Briefly, EGFP was excited at 488 nm from a blue laser and detected with a 525/50 nm bandpass filter, whereas tRFP was excited at 561 nm from a yellow/green laser and detected with a 610/20 nm bandpass filter. The FRET signal between EGFP and tRFP was measured by excitation at 488 nm from blue laser and detection with a 610/20 nm bandpass filter located on the same laser.…”

Section: ■ Methodsmentioning

confidence: 99%

“…Next, the formation of these specific heteroreceptor complexes is investigated ex vivo , in rat brain sections with the use of Proximity Ligation Assay (PLA) . Finally, functional cellular-based assays, including intracellular calcium mobilization, IP-One accumulation, and ligand-mediated trafficking, , are used to demonstrate the significant changes in the Gαq-dependent signaling and trafficking of both receptors. Overall, these data demonstrate for the first time compelling evidence for the formation of a functional OTR-5HTR 2A heteroreceptor complex, which may represent a novel molecular mechanism underpinning the OT and 5-HT signaling crosstalk in the brain.…”

Section: Introductionmentioning

confidence: 99%

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Attenuation of Oxytocin and Serotonin 2A Receptor Signaling through Novel Heteroreceptor Formation

Chruścicka

Fitzsimons

Borroto-Escuela

et al. 2019

ACS Chem. Neurosci.

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The oxytocin receptor (OTR) and the 5-hydroxytryptamine 2A receptor (5-HTR2A) are expressed in similar brain regions modulating central pathways critical for social and cognition-related behaviors. Signaling crosstalk between their endogenous ligands, oxytocin (OT) and serotonin (5-hydroxytryptamine, 5-HT), highlights the complex interplay between these two neurotransmitter systems and may be indicative of the formation of heteroreceptor complexes with subsequent downstream signaling changes. In this study, we assess the possible formation of OTR-5HTR2A heteromers in living cells and the functional downstream consequences of this receptor–receptor interaction. First, we demonstrated the existence of a physical interaction between the OTR and 5-HTR2A in vitro, using a flow cytometry-based FRET approach and confocal microscopy. Furthermore, we investigated the formation of this specific heteroreceptor complex ex vivo in the brain sections using the Proximity Ligation Assay (PLA). The OTR-5HTR2A heteroreceptor complexes were identified in limbic regions (including hippocampus, cingulate cortex, and nucleus accumbens), key regions associated with cognition and social-related behaviors. Next, functional cellular-based assays to assess the OTR-5HTR2A downstream signaling crosstalk showed a reduction in potency and efficacy of OT and OTR synthetic agonists, carbetocin and WAY267464, on OTR-mediated Gαq signaling. Similarly, the activation of 5-HTR2A by the endogenous agonist, 5-HT, also revealed attenuation in Gαq-mediated signaling. Finally, altered receptor trafficking within the cell was demonstrated, indicative of cotrafficking of the OTR/5-HTR2A pair. Overall, these results constitute a novel mechanism of specific interaction between the OT and 5-HT neurotransmitters via OTR-5HTR2A heteroreceptor formation and provide potential new therapeutic strategies in the treatment of social and cognition-related diseases.

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Section: Methodsmentioning

confidence: 99%

Section: ■ Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Attenuation of Oxytocin and Serotonin 2A Receptor Signaling through Novel Heteroreceptor Formation

Chruścicka

Fitzsimons

Borroto-Escuela

et al. 2019

ACS Chem. Neurosci.

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“…Furthermore, co-location of the two receptor protomers was observed in primary hippocampal and hypothalamic cultures of postnatal day 1 (P1) Sprague Dawley rats. Wallace Fitzsimons et al ( 2019 ) showed increased trafficking of the OXTR/GHS-R1a pair following co-expression (Chruścicka et al, 2018 ). Notably, the most significant finding was that the formation of the GHS-R1a-OXTR heterocomplex led to an attenuation of oxytocin-induced calcium mobilization mediated via G alpha q signaling.…”

Section: Oxytocin Heteroreceptor Complexesmentioning

confidence: 99%

The oxytocin receptor represents a key hub in the GPCR heteroreceptor network: potential relevance for brain and behavior

Borroto‐Escuela

Cuesta-Marti

López-Salas

et al. 2022

Front. Mol. Neurosci.

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In the last 10 years, it has become increasingly clear that large numbers of axon collaterals extend from the oxytocin (OXT) hypothalamic axons, especially the parvocellular components, to other brain regions. Consequently, the OXT signaling system forms, like other monoamine axons, a rich functional network across several brain regions. In this manuscript, we review the recently indicated higher order G-protein coupled heteroreceptor complexes of the oxytocin receptor (OXTR), and how these, via allosteric receptor-receptor interactions modulate the recognition, signaling, and trafficking of the participating receptor protomers and their potential impact for brain and behavior. The major focus will be on complexes of the OXTR protomer with the dopamine D2 receptor (D2R) protomer and the serotonin 2A (5-HT2AR) and 2C (5-HT2CR) receptor protomers. Specifically, the existence of D2R-OXTR heterocomplexes in the nucleus accumbens and the caudate putamen of rats has led to a postulated function for this heteromer in social behavior. Next, a physical interaction between OXTRs and the growth hormone secretagogue or ghrelin receptor (GHS-R1a) was demonstrated, which consequently was able to attenuate OXTR-mediated Gαq signaling. This highlights the potential of ghrelin-targeted therapies to modulate oxytocinergic signaling with relevance for appetite regulation, anxiety, depression, and schizophrenia. Similarly, evidence for 5-HT2AR-OXTR heteromerization in the pyramidal cell layer of CA2 and CA3 in the dorsal hippocampus and in the nucleus accumbens shell was demonstrated. This complex may offer new strategies for the treatment of both mental disease and social behavior. Finally, the 5-HT2CR-OXTR heterocomplexes were demonstrated in the CA1, CA2, and CA3 regions of the dorsal hippocampus. Future work should be done to investigate the precise functional consequence of region-specific OXTR heteromerization in the brain, as well across the periphery, and whether the integration of neuronal signals in the brain may also involve higher order OXTR-GHS-R1a heteroreceptor complexes including the dopamine (DA), noradrenaline (NA) or serotonin (5-HT) receptor protomers or other types of G-protein coupled receptors (GPCRs).

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Molecular, biochemical and behavioural evidence for a novel oxytocin receptor and serotonin 2C receptor heterocomplex

et al. 2021

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Detection and Quantitative Analysis of Dynamic GPCRs Interactions Using Flow Cytometry-Based FRET

Cited by 3 publications

References 65 publications

Attenuation of Oxytocin and Serotonin 2A Receptor Signaling through Novel Heteroreceptor Formation

Attenuation of Oxytocin and Serotonin 2A Receptor Signaling through Novel Heteroreceptor Formation

The oxytocin receptor represents a key hub in the GPCR heteroreceptor network: potential relevance for brain and behavior

Molecular, biochemical and behavioural evidence for a novel oxytocin receptor and serotonin 2C receptor heterocomplex

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