Fig. 7
From: Functionally distinct and selectively phosphorylated GPCR subpopulations co-exist in a single cell
PKA-pβ2AR is necessary for activation of β2AR-α11.2 complex in hippocampal neurons. WT or mutant β2AR lacking either PKA sites (PKAmut) or GRK sites (GRKmut) was expressed in hippocampal neurons lacking both β1AR and β2AR genes (DKO). a Neurons were either not stimulated (ND) or stimulated with 1 μM ISO for 5 min. The total α11.2 was immunoprecipitated; the phosphorylation of α11.2 at S1928 and S1700 was probed with phospho-specific antibodies and normalized to total α11.2. Representative of three independent experiments. Molecular weight markers (in kDa) are indicated on the left. b The endogenous α11.2 was pulled down in SiMPull as depicted; representative images show that mYFP-β2AR pulled down together with α11.2. Scale bar, 5 μm. Quantification of the numbers of mYFP-β2AR bound to α11.2 shows that PKA-phosphorylation of β2AR is required for dissociation of the β2AR-α11.2 complex. Representative of n = 13/12, 13/15, and 12/12 images for WT, PKAmut, and GRKmut β2AR groups, four independent experiments. c DKO hippocampal neurons at 7–14 DIV expressing WT and mutant β2AR were subjected to single-channel recording of LTCC currents using the same method as Fig. 6. d The overall channel activity (nPo) of LTCC Cav1.2 was quantified from c (n = 11, 10, 14, 10, 12, and 10 cells, respectively). Error bars denote s.e.m., exact P values are computed by Student’s t-test in a and b, and by Mann–Whitney test in d
