Rapid flow-induced activation of Gαq/11 is independent of Piezo1 activation

Endothelial cell (EC) mechanochemical transduction is the process by which mechanical stimuli are sensed by ECs and transduced into biochemical signals and ultimately into physiological responses. Identifying the mechanosensor/mechanochemical transducer(s) and describing the mechanism(s) by which they receive and transmit the signals has remained a central focus within the field. The heterotrimeric G protein, Gαq/11, is proposed to be part of a macromolecular complex together with PECAM-1 at EC junctions and may constitute the mechanochemical transducer as it is rapidly activated within seconds of flow onset. The mechanically activated cation channel Piezo1 has recently been implicated due to its involvement in mediating early responses, such as calcium and ATP release. Here, we investigate the role of Piezo1 in rapid shear stress-induced Gαq/11 activation. We show that flow-induced dissociation of Gαq/11 from PECAM-1 in ECs at 15 s is abrogated by BIM-46187, a selective inhibitor of Gαq/11 activation, suggesting that Gαq/11 activation is required for PECAM-1/Gαq/11 dissociation. Although siRNA knockdown of Piezo1 caused a dramatic decrease in PECAM-1/Gαq/11 association in the basal condition, it had no effect on flow-induced dissociation. Interestingly, siRNA knockdown of Piezo1 caused a marked decrease in PECAM-1 expression. Additionally, selective blockade of Piezo1 with ion channel inhibitors had no effect on flow-induced PECAM-1/Gαq/11 dissociations. Lastly, flow onset caused increased association of Gβ1 with Piezo1 as well as with the p101 subunit of phosphoinositide 3-kinase, which were both blocked by the Gβγ inhibitor gallein. Together, our results indicate that flow-induced activation of Piezo1 is not upstream of G protein activation.

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Faculty Opinions recommendation of Re-examining the 'Dissociation Model' of G protein activation from the perspective of Gβγ signaling.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.738875770.793579982 ◽

2020 ◽

Author(s):

Philip Wedegaertner

Keyword(s):

G Protein ◽

Protein Activation ◽

G Protein Activation ◽

Dissociation Model

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DAPLE and MPDZ bind to each other and cooperate to promote apical cell constriction

Molecular Biology of the Cell ◽

10.1091/mbc.e19-02-0091 ◽

2019 ◽

Vol 30 (16) ◽

pp. 1900-1910 ◽

Cited By ~ 6

Author(s):

Arthur Marivin ◽

Mikel Garcia-Marcos

Keyword(s):

G Protein ◽

Cultured Cells ◽

Apical Cell ◽

Cell Junctions ◽

Binding Motif ◽

Apical Constriction ◽

Protein Activation ◽

Neuroepithelial Cells ◽

Two Factors ◽

Apical Junctions

Dishevelled-Associating Protein with a high frequency of LEucines (DAPLE) belongs to a group of unconventional activators of heterotrimeric G-proteins that are cytoplasmic factors rather than membrane proteins of the G-protein–coupled receptor superfamily. During neurulation, DAPLE localizes to apical junctions of neuroepithelial cells and promotes apical cell constriction via G-protein activation. While junctional localization of DAPLE is necessary for this function, the factors it associates with at apical junctions or how they contribute to DAPLE-mediated apical constriction are unknown. MPDZ is a multi-PDZ (PSD95/DLG1/ZO-1) domain scaffold present at apical cell junctions whose mutation in humans is linked to nonsyndromic congenital hydrocephalus (NSCH). DAPLE contains a PDZ-binding motif (PBM) and is also mutated in human NSCH, so we investigated the functional relationship between both proteins. DAPLE colocalized with MPDZ at apical cell junctions and bound directly to the PDZ3 domain of MPDZ via its PBM. Much like DAPLE, MPDZ is induced during neurulation in Xenopus and is required for apical constriction of neuroepithelial cells and subsequent neural plate bending. MPDZ depletion also blunted DAPLE-mediated apical constriction of cultured cells. These results show that DAPLE and MPDZ, two factors genetically linked to NSCH, function as cooperative partners at apical junctions and are required for proper tissue remodeling during early stages of neurodevelopment.

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Opioid-Activated Postsynaptic, Inward Rectifying Potassium Currents in Whole Cell Recordings in Substantia Gelatinosa Neurons

Journal of Neurophysiology ◽

10.1152/jn.1998.80.6.2954 ◽

1998 ◽

Vol 80 (6) ◽

pp. 2954-2962 ◽

Cited By ~ 49

Author(s):

S. P. Schneider ◽

W. A. Eckert ◽

A. R. Light

Keyword(s):

Membrane Potential ◽

G Protein ◽

Potassium Currents ◽

Substantia Gelatinosa ◽

Opioid Agonist ◽

Membrane Hyperpolarization ◽

Whole Cell ◽

Protein Activation ◽

G Protein Activation ◽

Whole Cell Recordings

Schneider, S. P., W. A. Eckert III, and A. R. Light. Opioid-activated postsynaptic, inward rectifying potassium currents in whole cell recordings in substantia gelatinosa neurons. J. Neurophysiol. 80: 2954–2962, 1998. Using tight-seal, whole cell recordings from isolated transverse slices of hamster and rat spinal cord, we investigated the effects of the μ-opioid agonist (d-Ala2, N-Me-Phe4,Gly5-ol)-enkephalin (DAMGO) on the membrane potential and conductance of substantia gelatinosa (SG) neurons. We observed that bath application of 1–5 μM DAMGO caused a robust and repeatable hyperpolarization in membrane potential ( V m) and decrease in neuronal input resistance ( R N) in 60% (27/45) of hamster neurons and 39% (9/23) of rat neurons, but significantly only when ATP (2 mM) and guanosine 5′-triphosphate (GTP; 100 μM) were included in the patch pipette internal solution. An ED50 of 50 nM was observed for the hyperpolarization in rat SG neurons. Because G-protein mediation of opioid effects has been shown in other systems, we tested if the nucleotide requirement for opioid hyperpolarization in SG neurons was due to G-protein activation. GTP was replaced with the nonhydrolyzable GTP analogue guanosine-5′- O-(3-thiotriphosphate) (GTP-γ-S; 100 μM), which enabled DAMGO to activate a nonreversible membrane hyperpolarization. Further, intracellular application of guanosine-5′- O-(2-thiodiphosphate) (GDP-β-S; 500 μM), which blocks G-protein activation, abolished the effects of DAMGO. We conclude that spinal SG neurons are particularly susceptible to dialysis of GTP by whole cell recording techniques. Moreover, the depletion of GTP leads to the inactivation of G-proteins that mediate μ-opioid activation of an inward-rectifying, potassium conductance in these neurons. These results explain the discrepancy between the opioid-activated hyperpolarization in SG neurons observed in previous sharp electrode experiments and the more recent failures to observe these effects with whole cell patch techniques.

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A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein βγ subunits

Cell ◽

10.1016/0092-8674(94)90237-2 ◽

1994 ◽

Vol 77 (1) ◽

pp. 83-93 ◽

Cited By ~ 411

Author(s):

L. Stephens ◽

A. Smrcka ◽

F.T. Cooke ◽

T.R. Jackson ◽

P.C. Sternweis ◽

...

Keyword(s):

G Protein ◽

Kinase Activity ◽

Phosphoinositide 3 Kinase

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G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1418515112 ◽

2014 ◽

Vol 112 (2) ◽

pp. 590-595 ◽

Cited By ~ 43

Author(s):

Timothy Connelly ◽

Yiqun Yu ◽

Xavier Grosmaitre ◽

Jue Wang ◽

Lindsey C. Santarelli ◽

...

Keyword(s):

G Protein ◽

Sensory Neurons ◽

Ectopic Expression ◽

Host Cells ◽

Odorant Receptors ◽

Olfactory Sensory Neurons ◽

Mechanical Stimuli ◽

Genetic Ablation ◽

Mechanical Responses ◽

G Protein Coupled

Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction.

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