Fig. 4
From: Functionally distinct and selectively phosphorylated GPCR subpopulations co-exist in a single cell
Subpopulations of β2AR display distinct structural properties. a Schematic of SiMPull with phospho-specific antibody against FLAG-mYFP-β2AR and examples of fluorescent time traces depicting one- or two-step photobleaching of mYFP-β2ARs, which provides stoichiometric information of β2AR. The composition of β2AR was calculated based on photobleaching steps in different conditions described in b and c. b Cells are stimulated with different concentrations of ISO for 5 min, the total β2ARs were pulled down for photobleaching analysis, displaying a composition of ~40% dimers and ~60% monomers independent of agonist stimulation (mean ± s.d.; n = 10, 8, 10, and 8 independent experiments, respectively). c The PKA- and GRK-phosphorylated β2ARs were pulled down with phospho-specific antibodies against S261/262 (PKA-pβ2AR) and S355/356 (GRK-pβ2AR) with different concentrations of ISO as indicated. At 1 nM, ISO only induces PKA-phosphorylation of β2AR but not GRK-phosphorylation of β2AR; higher concentrations of ISO induce both PKA- and GRK-phosphorylation of β2ARs. PKA-phosphorylated β2ARs display a composition of nearly 70% dimers and 30% monomers upon stimulation with all tested concentration of ISO (mean ± s.d.; n = 6, 6, and 14 independent experiments, respectively), whereas GRK-phosphorylated β2ARs display a composition of 12% dimers and 88% monomers at high concentrations of ISO stimulation (mean ± s.d.; n = 12 and 21 independent experiments, respectively). N.A. not available
