Vm modulates mGlu5-mediated Gq/11activation
We then assessed the influence of Vm on the activation
by mGlu5 receptor of canonical effectors, starting with the
Gq/11 activation. We monitored Gq/11activation by mGlu5 using Effector Membrane Translocation Assay (EMTA)
ebBRET in single-cell BRET imaging (, Figure 4 ) and cell
population (Figure Supp. 3 ). HEK293T cells were transfected
with mGlu5, Gq/11, p63hRhoGEF-RlucII and rGFP-CAAX
coding plasmids. Gq/11 activation triggers the
recruitment of its cytosolic effector, p63RhoGEF-RlucII, to the plasma
membrane, which is reported by an increase in ebBRET with the plasma
membrane-anchored rGFP-CAAX. The ebBRET signal is therefore a measure of
Gq/11 activation (, Figure 4A ). Of note, the
ebBRET signal was not affected by Vdepol compared to
Vrest, validating the use of this biosensor to assess
the influence of Vm on mGlu5-induced
Gq/11 activation (Figure Supp 1C ). Application
of DHPG (100µM) induced a slight but significant increase in ebBRET at
Vrest and Vdepol (Figure 4B and4C ). Paired measurement of the DHPG net effect from individual
cell ebBRET values revealed a mean DHPG-induced increase of 97 ± 9.9
milliBRET units at Vrest, and only 22.8 ± 6.2 at
Vdepol (Figure 4D ). These findings indicate a
significant reduction of mGlu5 receptor-induced Gq/11activation at Vdepol. Similar results were obtained with
ebBRET measurement in cell populations, where the mGlu5 agonist-induced
increase of ebBRET was significantly reduced by Vdepol(agonist-induced ebBRET increase was reduced by 24.8 ± 7.2 % at
Vdepol compared to Vrest, Figure Supp 3A ). The
same protocol applied to AT1 receptor showed no influence of
Vm on AT1 receptor agonist-induced
Gq/11-coupling (Figure Supp 3B ), suggesting
once again that Vdepol specifically affects mGlu5
receptor signaling.