γ-Aminobutyric acid type B (GABAB) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABAB receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity α-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABAB receptors in live cells. To simultaneously study R1a and R2 trafficking, a new α-bungarotoxin binding site-labeling technique was used, allowing α-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABAB receptor.

␥-Aminobutyric acid type B (GABA B ) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABA B receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity ␣-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABA B receptors in live cells. To simultaneously study R1a and R2 trafficking, a new ␣-bungarotoxin binding site-labeling technique was used, allowing ␣-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABA B receptor.Metabotropic GABA B 2 receptors mediate a slow and prolonged phase of synaptic inhibition in the CNS. Their importance for neuronal function is evident when they become dysfunctional, which can lead to a range of diseases that includes epilepsy, sleep disorders, stress, depression, and substance abuse (1-3).Native GABA B receptors are considered to function as heterodimers formed from R1 and R2 subunits (4, 5) with the possibility that some higher order oligomeric assemblies (e.g. dimer of dimers) may also retain functionality (6, 7). Heterodimerization of GABA B receptors has profound consequences for their structural and functional properties, impacting on the efficiency of cell surface expression and the linkage between agonist binding and G-protein activation.Functional GABA B receptors require both R1 and R2 to coassemble because an endoplasmic reticulum (ER) retention motif, RSR, in the C-terminal coiled-coil domain of R1 subunits has to be masked by the R2 subunit to ensure surface expression (8, 9). Substitution of the retention motif with ASA allows R1 subunits to exit the ER and travel alone to the cell surface (8). Although the role of R2 in heterodimerization for forward trafficking has been reported, how heterodimerization, and in particular the R2 subunit, affects the cell surface stability and internalization of GABA B receptor subunits remains unresolved.This aspect is important because the cell surface stability and mobility of GA...