Figure 2.
PTH induces the formation and internalization of TβRII–PTH1R complexes. (a) Strategy of YFP-based PCA (YFP-PCA). (b, c) PTH, but not TGF-β, induces an interaction of PTH1R with TβRII as shown with YFP-PCA. HEK293 cells were transfected with indicated plasmids and then treated with PTH or TGF-β for 30 s. The reconstituted YFP fluorescence appears green. The cells were stained with 4,6-diamidino-2-phenylindole (DAPI) (blue)(b), and YFP fluorescence was quantified by fluorimetry normalized to untransfected cells. Scale bars, 10 μm. Two asterisks, P < 0.01 versus untreated (c). (d) Co-internalization of TβRII with PTH/PTH1R. HEK293 cells co-expressing TβRII–YFP1 and PTH1R–YFP2 were treated with PTHTMR for 30 s or 5 min. Reconstituted YFP fluorescence appears green and PTHTMR appears red. Co-localization of PTH and the TβRII–PTH1R complex appears yellow. Scale bars, 10 μm. (e, f) TβRII specifically interacts with PTH1R in response to PTH in immunoprecipitation assays. HEK293 cells co-transfected with indicated plasmids (e) or primary calvarial osteoblasts (f) were treated with PTH for 30 min. Cell extracts were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies. The asterisk indicates that the cells were lysed in IP buffer with 0.5% SDS for immunoblotting. HA, haemagglutinin. (g, h) PTH induces the formation of PTH1R dimer/oligomer as shown with YFP-PCA illustrated in g. HEK293 cells co-expressing PTH1R–YFP1 and PTH1R–YFP2 were treated with PTH and then stained with DAPI (blue). Reconstituted YFP fluorescence on formation of the PTH1R dimer/oligomer appears green (h, upper panel). Co-localization of PTHTMR (red) and the PTH1R dimer/oligomer (green) appears yellow (h, lower panel). Scale bars, 10 μm. (i) TβRII is mainly co-localized with PTH-induced PTH1R dimer/oligomer. HEK293 cells co-expressing PTH1R–YFP1, PTH1R–YFP2 and TβRII–Flag were treated with PTH for 5 min and then immunostained with anti-Flag antibody. Reconstituted YFP fluorescence appears green, TβRII appears red, and co-localization of TβRII and the PTH1R dimer/oligomer appears yellow. Results in panels c are shown as the mean ± s.d. (n = 3). Uncropped images of blots are shown in Supplementary Information, Fig. S9.