Lateral Diffusion of the GABAB Receptor Is Regulated by the GABAB2 C Terminus

Abstract

GABA B (␥-aminobutyric acid, type B) is a heterodimeric G-protein-coupled receptor. The GABA B1 subunit, which contains an endoplasmic reticulum retention sequence, is only transported to the cell surface when it is associated with the GABA B2 subunit. Fluorescence recovery after photobleaching studies in transfected COS-7 cells and hippocampal neurons revealed that GABA B2 diffuses slowly within the plasma membrane whether expressed alone or with the GABA B1 subunit. Treatment of cells with brefeldin A revealed that GABA B2 moves freely within the endoplasmic reticulum, suggesting that slow movement of GABA B2 is a result of its plasma membrane insertion. Disruption of the cytoskeleton did not affect the mobility of GABA B2 , indicating that its restricted diffusion is not due to direct interactions with actin or tubulin. To determine whether the C terminus of GABA B2 regulates its diffusion, this region of the subunit was attached to the lymphocyte membrane protein, CD2, which then exhibited a slower rate of lateral diffusion. Furthermore, co-expression of a cytoplasmically expressed soluble form of the GABA B2 C terminus increased movement of the GABA B2 subunit. We constructed forms of GABA B2 with various C-terminal truncations. Truncation of GABA B2 after residue 862, but not residue 886, caused a dramatic increase in its mobility, suggesting that the region between these two residues is critical for restricting GABA B2 diffusion. Finally, we investigated whether activation of GABA B might modulate its movement. Treatment of COS-7 cells with the GABA B receptor agonist baclofen significantly increased its mobile fraction. These data show that the restricted movement of GABA B at the cell surface is regulated by a region within its C terminus.GABA B receptors are metabotropic receptors for the inhibitory neurotransmitter ␥ aminobutyric acid (GABA).2 Pre-and post-synaptic GABA B receptors are coupled to inhibitory G-proteins and can regulate neurotransmission via several mechanisms, including modulation of adenylyl cyclase (1), inhibition of voltage-gated Ca 2ϩ channels (2), and modulation of K ϩ channels (3, 4). Formation of a functional receptor requires the heterodimerization of two subunits, GABA B1 and GABA B2 (5). Previous work has demonstrated that the stable assembly of these subunits occurs, to some extent, via association of coiled-coil domains within their C termini (6). The subunits appear to serve different functions within the fully formed receptor. GABA B1 contains the agonist binding site on its large extracellular N terminus, and the affinity of this site for agonists is increased following heterodimerization with GABA B2 (7). The GABA B2 subunit contains intracellular loops that couple the receptor to the G-protein (8 -10).Heterodimerization of the GABA B subunits is important not only for proper receptor function but also for forward trafficking of the receptor to the cell surface (5, 7). In the absence of GABA B2 , the GABA B1 subunit is retained within the endoplasmic reticulum due to th...

Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag

“…Recently, there is increasing evidence in support of rapid GABA B receptor recycling (11), constitutive endocytosis (9), and lateral diffusion of these receptors in the cell membrane (10). Why these studies reach different conclusions is unclear.…”

“…However, other reports indicate that agonist activation of GABA B receptors may promote internalization and/or rapid recycling (10,11,18). To address the topic of GABA B receptor trafficking, prior studies have used various techniques to monitor receptor movement, including: receptor biotinylation (8,9); antibody labeling of extracellular GABA B receptor epitopes on live cells (9); as well as fluorescence recovery after photobleaching (FRAP) (10). These methods have therefore relied on the use of relatively large reporter molecules, such as antibodies.…”

“…Although, generally, GPCRs are readily internalized from the cell surface following agonist activation and receptor phosphorylation (15)(16)(17); the GABA B receptor was thought to behave differently, being relatively stable in the cell membrane (8,9). However, other reports indicate that agonist activation of GABA B receptors may promote internalization and/or rapid recycling (10,11,18). To address the topic of GABA B receptor trafficking, prior studies have used various techniques to monitor receptor movement, including: receptor biotinylation (8,9); antibody labeling of extracellular GABA B receptor epitopes on live cells (9); as well as fluorescence recovery after photobleaching (FRAP) (10).…”

Differential Regulation of GABAB Receptor Trafficking by Different Modes of N-methyl-d-aspartate (NMDA) Receptor Signaling

“…Neither native nor recombinant GABA B Rs are G protein-coupled receptor-dependent kinase substrates, and they do not undergo agonist-induced internalization (9, 10). They do, however, display constitutive endocytosis (11), and it has been proposed that they undergo both very rapid endocytic recycling (12, 13) and membrane lateral diffusion (14). …”

Complex GABAB receptor complexes: how to generate multiple functionally distinct units from a single receptor

“…Examination of the dynamic lateral diffusion of GABA B receptors at the cell surface revealed restricted mobility of GABA B2 . After activation by baclofen, levels of the mobile fraction were significantly increased (Pooler and McIlhinney, 2007). Furthermore, by using single-molecule analysis of fluorescently labeled GPCR revealed that larger oligomers of GABA B receptor were prevalently organized into ordered arrays (Calebiro et al, 2013).…”