Figure 7.

Regulation of G protein coupling depends on the dimerization of the GABA_B heterodimers. (A) Schematic representation of the oligomeric GABA_B receptor in the absence (left) or in the presence (right) of a GB2 subunit unable to activate the G protein and carrying a KKXX retention signal motif (GB2(L686P)-KKXX), which can substitute one wild-type GB2 to form tetramers with a single functional heterodimer per tetramer (right). FRET intensity measured on COS-7 cells expressing the indicated constructs: control (black bars) and control with GB2(L686P)-KKXX (white bars) (a) between the Myc-GB1 subunits after labelling with a mixture of K and d2 anti-Myc antibodies, (b) between Myc-GB1 and HA-GB2-KKXX after labelling with K anti-HA and d2 anti-Myc antibodies and finally (c) between Flag-GB2(L686P)-KKXX and Myc-GB1 labelled with K anti-Flag and d2 anti-Myc antibodies. Data are mean±s.e.m. of three independent experiments each performed in triplicates. ^** and ^*** represent P<0.01 and P<0.001, respectively, in a t-test. (B) Calcium dose-response recorded after stimulation by the GABA on cells expressing GB1 and GB2-KKXX (control) (black-filled circles), GB1 and GB2(L686P)-KKXX (negative control) (black triangles) and control with GB2(L686P)-KKXX (grey open squares) in the presence of a Gqi9 chimeric protein. (C) GABA-induced response of the native Gi/o protein by measurement of the calcium formation after pre-stimulation of an endogenous Gq-coupled receptor. For (B, C), representative of three to four independent experiments, each performed in triplicates.