Fig. 2: The pinKE mutation impedes G protein subunit dissociation.
From: A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state
A Agonist-induced dissociation of Gα and Gβγ subunits (left). HEK293T cells transfected with the indicated receptor, WT or mutant Gα-RLuc8 donor, Gβ and Gγ-GFP acceptor proteins (middle). Representative concentration-response measurements using the μ-opioid (MOR; Gi1), neurotensin (NT1R; Gq, G13), or β2-adrenergic receptor (β2AR; Gs) receptors, presented as fold decrease in dynamic range measured by comparing the energy transfer from donor to acceptor and reported as ΔBRET (GFP/Rluc8 per well minus Basal BRET, or GFP/Rluc8, at lowest dose of agonist) in comparison to basal activity, presented as raw BRET values prior to stimulus (right). Data are means ± SEM from 3 independent experiments, 2 measurements each. B Agonist-induced dissociation of Gβγ subunits (left). HEK293FT cells transfected with MOR or D2R, either empty vector (pcDNA3.1) or vector with wild-type or mutant Gαo, masGRK3ct-Nluc-HA donor and Venus-Gβγ acceptor proteins. Representative time-course measurements for D2R after dopamine addition, presented as ΔBRET (ratio of emission by Venus at 535 nm and Nluc at 475 nm; recorded prior to agonist stimulation and subtracted from the experimental BRET values, middle). Effect of mutations, quantified as maximum amplitude relative to wild type (right). Data are means ± SEM from 4 (vector) or 5 (Gαo or GαoK46E) independent experiments, 3 measurements each. C Dominant-negative inhibition of subunit release (left). HEK293 cells transfected as in B but with the addition of an equal amount of wild-type Gαo (middle), done by transfecting equivalent amounts of mutant and wild-type DNA. Effect of mutations quantified as maximum amplitude (right). BRET data are means ± SEM from 4 (vector) or 5 (Gαo or GαoK46E) independent experiments, 3 measurements each. D Agonist-induced association of GPCRs and G proteins (left). HEK293FT cells transfected with MOR or D2R fused to myc-SmBiT, Gαo, Gβ fused to LgBiT, and Gγ. Representative time-course measurements after dopamine addition leading to reconstitution of functional Nluc, presented as arbitrary luminesence units (ΔAU, middle). Effect of mutations, quantified as maximum amplitude (right). Data are means ± SEM from 6 independent experiments, 3 measurements each. Statistical analysis performed with 2-tailed unpaired t test; ****p < 0.0001. Source data are provided as a Source Data file.
