Constitutive internalisation of GABAB receptor R2 subunits in live cells

Saad Hannan, Megan Wilkins, Trevor Smart

University College London, London, UK

The GABAB receptor is a member of the class C family of G-protein coupled receptors. Its activation causes a slow and prolonged component of synaptic inhibition that is mediated by GABA, the main inhibitory neurotransmitter in the CNS. It also plays an important role in synaptic plasticity and in a wide range of diseases, including substance abuse (Enna & Bowery, 2004). To form functional cell surface receptors, both GABABR1 and R2 subunits need to heterodimerise. Although the R1 subunit contains the GABA binding domain and the R2 subunit is important for coupling to G-proteins, the cell surface mobility and trafficking of these receptors remains unclear (Fairfax et al. 2004, Grampp et al. 2007), particularly in real time. This is important since receptor trafficking may play a role in the duration of inhibition caused by GABAB receptor activation. Prior studies have used antibodies and biotinylation as reporters to study GABAB receptor movement. Here, we use a different type of reporter method, based on incorporating the α-bungarotoxin (BTX) binding site (BBS), previously employed for GABABR1a subunits (Wilkins et al., 2008), into the N-terminal domain of R2 subunits to monitor their trafficking.

Using confocal microscopy, we measured α-bungarotoxin-alexa fluor-555 binding to the BBS in the R2 subunit (R2BBS), which is expressed in live HEK293 cells, either with R1a (GABABR1aR2BBS) or alone (GABABR2BBS). These cells also stably express Kir 3.1 and 3.2 potassium channels (GIRK cells). The BBS tag is functionally-silent as the sensitivity to GABA, for activating Kir 3.1 and 3.2, was unchanged for GABABR1aR2BBS receptors (EC50 = 0.42 ± 0.06 μM, mean ± s.e.m, n=6) compared to wild-type GABABR1aR2 receptors (0.43 ± 0.05 μM (n =5-11; P>0.05). GIRK cells expressing just GABABR2BSS did not respond to GABA (n = 3).

Confocal images were analysed for the rate of internalization at different temperatures using ImageJ (ver 1.40g). The rates were described by single exponentials (Table 1) and these were unaffected by the presence of the R1 subunit. R2 internalisation was blocked at 18°C.

We conclude that the GABABR2 subunit constitutively endocytoses from the membrane of GIRK cells and this process is unaffected by co-assembly with R1. The GABABR2BBS can be expressed on the cell surface in the absence of R1, but although it is non-functional, it is still constitutively endocytosed.

This work and the studentship to SH, are supported by the BBSRC and GSK

Enna SJ and Bowery NG (2004). Biochem Pharmacol. 68, 1541-1548

Fairfax BP et al. (2004) J. Biol, Chem. 279, 12565-12573

Grampp T et al. (2007) J. Biol, Chem. 282, 24157-24165

Wilkins ME et al. (2008)J. Biol, Chem. in press