Importance of the γ-Aminobutyric AcidB Receptor C-Termini for G-Protein Coupling

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Abstract
Functional γ-aminobutyric acidB (GABAB) receptors assemble from two subunits, GABAB(1) and GABAB(2). This heteromerization, which involves a C-terminal coiled-coil interaction, ensures efficient surface trafficking and agonist-dependent G-protein activation. In the present study, we took a closer look at the implications of the intracellular C termini of GABAB(1) and GABAB(2) for G-protein coupling. We generated a series of C-terminal mutants of GABAB(1) and GABAB(2) and tested them for physical interaction, surface trafficking, coupling to adenylyl cyclase, and G-protein-gated inwardly rectifying potassium channels in human embryonic kidney (HEK) 293 cells as well as on endogenous calcium channels in sympathetic neurons of the superior cervical ganglion (SCG). We found that the C-terminal interaction contributes only partly to the heterodimeric assembly of the subunits, indicating the presence of an additional interaction site. The described endoplasmic reticulum retention signal within the C terminus of GABAB(1)functioned only in the context of specific amino acids, which constitute part of the GABAB(1) coiled-coil sequence. This finding may provide a link between the retention signal and its shielding by the coiled coil of GABAB(2). In HEK293 cells, we observed that the two well-known GABAB receptor antagonists [S-(R*,R*)]-[3-[[1-(3,4-dichlorophenyl)ethyl]amino]-2-hydroxypropyl](cyclohexylmethyl) phosphinic acid (CGP54626) and (+)-(2S)-5,5-dimethyl-2-morpholineacetic acid (SCH50911) CGP54626 and SCH50911 function as inverse agonists. The C termini of GABAB(1) and GABAB(2) strongly influenced agonist-independent G-protein coupling, although they were not necessary for agonist-dependent G-protein coupling. The C-terminal GABAB receptor mutants described here demonstrate that the active receptor conformation is stabilized by the coiled-coil interaction. Thus, the C-terminal conformation of the GABABreceptor may determine its constitutive activity, which could be a therapeutic target for inverse agonists.