Fig. 1. GPR158 forms dimers at the cell surface. (A) Western blot analysis using three different commercially available antibodies, recognizing the Flag epitope (M2 clone), the N-terminal (Anti N-term, SAB4502509 Sigma), or the C-terminal domain (anti C-term, HPA013185 Sigma) of N-terminal Flag-tagged GPR158 (Flag-GPR158), revealed two major bands, at around 150 and 300 kDa, in HEK293 cells transiently expressing Flag-GPR158 (+), but not in mock-transfected cells (−). (B) Coimmunoprecipitation of Flag- and HA-GPR158 coexpressed in HEK293 cells. Note that HA-GPR158 did not coimmunoprecipitate with N-terminal–tagged Flag-mGlu2 or Flag-GB2 (GABAB2), whereas a positive control showed a coimmunoprecipitation between the two subunits Flag-GB1a and HA-GB2 of the dimeric GABAB receptor. (C) TR-FRET analysis of GPR158 dimerization. A TR-FRET signal was recorded between Flag- and HA-GPR158, using TR-FRET donor and acceptor fluorophores labeled anti-Flag and anti-HA antibodies. Similarly, a strong TR-FRET signal was obtained between the two subunits of GABAB receptor HA-GB1a and Flag-GB2; however, no HA-Flag TR-FRET signal was detected in cells expressing Flag-GPR158 and HA-GB2. (D) HA-GPR158, but not HA-GB2, can dimerize with Flag-GPR158. In cells expressing a constant level of Flag-GPR158, the Flag-HA TR-FRET signal increased when the amount of HA-GPR158 increased up to saturation, in contrast to HA-GB2 increasing expression. (E) HA-GPR158, but not HA-GB2, competed for dimerization with Flag-GPR158. In cells expressing a constant amount of Flag-GPR158, an increasing amount of HA-GPR158 decreased Flag-Flag TR-FRET signal, indicating a competition in the dimer formation between the Flag- and the HA- versions of GPR158, whereas no competition was observed when coexpressing an increasing amount of HA-GB2. In (A–C) panels, data are representative of three independent experiments. In (D and E), data from three independent experiments are pooled. Data are means ± S.E.M. of triplicate determinations. N- and C-terminal stand for N- and C-terminal domain, respectively.