Abstract
The metabotropic gamma-aminobutyric acid, type B (GABA(B)) receptors mediate the slow component of GABAergic transmission in the brain. Functional GABA(B) receptors are heterodimers of the two subunits GABA(B1) and GABA(B2), of which GABA(B1) exists in two main isoforms, GABA(B1a) and GABA(B1b). The significance of the structural heterogeneity of GABA(B) receptors, the mechanism leading to their differential targeting in neurons as well as the regulation of cell surface numbers of GABA(B) receptors, is poorly understood. To gain insights into these processes, we searched for proteins interacting with the C-terminal domain of GABA(B2). Here, we showed that the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) directly interacts with GABA(B) receptors in a subtype-selective manner to regulate cell surface expression of GABA(B1a)/GABA(B2) receptors upon co-expression in HEK 293 cells. The interaction of CHOP with GABA(B1a)/GABA(B2) receptors resulted in their intracellular accumulation and in a reduced number of cell surface receptors. This regulation required the interaction of CHOP via two distinct domains with the heterodimeric receptor; its C-terminal leucine zipper associates with the leucine zipper present in the C-terminal domain of GABA(B2), and its N-terminal domain associates with an as yet unidentified site on GABA(B1a). In conclusion, the data indicated a subtype-selective regulation of cell surface receptors by interaction with the transcription factor CHOP.
Highlights
GABAB1 harbors the ligand binding site, GABAB2 contains all the molecular determinants for G-protein coupling and is necessary for trafficking of GABAB receptors to the cell surface by shielding the endoplasmic reticulum retention signal of GABAB1
CHOP interacted through its N-terminal domain with an as yet undefined site of GABAB1a but not with GABAB1b
Co-expression of CHOP with heterodimeric GABAB1a/GABAB2 receptors resulted in a perturbed localization of the receptors to intracellular sites along with frequent GABAB2-positive inclusions and a reduction of cell surface receptors
Summary
GABAB1 harbors the ligand binding site, GABAB2 contains all the molecular determinants for G-protein coupling and is necessary for trafficking of GABAB receptors to the cell surface by shielding the endoplasmic reticulum retention signal of GABAB1 (for a review, see Refs. 1– 4). To verify a reduced association of CHOP with heterodimeric GABAB1a/ GABAB2 receptors, GABAB1a, GABAB2, and CHOP were co-expressed in HEK 293 cells and subjected to immunoprecipitation using GABAB1or GABAB2-selective antibodies.
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