The Sushi Domains of Secreted GABAB1 Isoforms Selectively Impair GABAB Heteroreceptor Function

Abstract

GABA B receptors are the G-protein-coupled receptors for ␥-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA B receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA B1a and GABA B1b . GABA B1a differs from GABA B1b in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA B1a contributes to heteroreceptors at glutamatergic terminals, whereas both GABA B1a and GABA B1b contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA B1j , a secreted GABA B1 isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA B heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA B autoreceptors nor impairs the activity of postsynaptic GABA B receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA B1a -containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA B1 isoforms like GABA B1j may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA B -mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA B heteroreceptors by targeting their SDs.

The Sushi Domains of GABABReceptors Function as Axonal Targeting Signals

“…Specifically, we addressed whether functional GABA B heteroreceptors are present in cultured pyramidal neurons of 1b Ϫ/ Ϫ mice, but absent in neurons of 1a Ϫ/ Ϫ mice. Activation of GABA B heteroreceptors by baclofen, a GABA B receptor agonist, inhibits the spontaneous release of glutamate and as a result reduces the miniature EPSC (mEPSC) frequency (Yamada et al, 1999;Tiao et al, 2008). We found that baclofen strongly reduced the mEPSC frequencies in wild-type (WT) and 1b Ϫ/ Ϫ neurons, while baclofen only marginally reduced the mEPSC frequency in 1a Ϫ/ Ϫ neurons ( Fig.…”

“…The tertiary structure of SDs is fixed by two intramolecular disulfide bridges that are critical for interaction with other proteins (Soares and Barlow, 2005). Consistent with their role as interaction motifs, the SDs of GABA B1a recognize binding sites in neuronal membranes (Tiao et al, 2008).…”

Sushi domains confer distinct trafficking profiles on GABA B receptors

“…SD1 interacts with the extracellular matrix protein fibulin-2 in vitro (23), and there is evidence that CCAAT/enhancer-binding protein homologous protein (CHOP) differentially interacts with R1a (31). In addition, a soluble excreted isoform of part of R1a, defined as R1j, is mostly composed from the two SDs (32,33), and this inhibits the function of GABA B heteroreceptors and recognizes binding partners on neuronal membranes. However, the sites of attachment have not been identified (33).…”

“…In addition, a soluble excreted isoform of part of R1a, defined as R1j, is mostly composed from the two SDs (32,33), and this inhibits the function of GABA B heteroreceptors and recognizes binding partners on neuronal membranes. However, the sites of attachment have not been identified (33). It is, therefore, likely that the SDs interact with other proteins that could modulate the signaling and trafficking of GABA B R1aR2 receptors.…”

GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

“…Because the sushi repeats are the only difference between GABA B1a and GABA B1b , it is to be expected that proteins interacting with these domains target or retain the GABA B1a isoform to specific microdomains. Consistently, a soluble recombinant protein of the two sushi repeats binds to neuronal membranes with low nanomolar affinity (10). An interaction of one of the two sushi repeats with fibulin-2 has been described (11), but the functional implication remains elusive.…”