GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice

Abstract

GABA B receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter ␥-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligandbinding subunit GABA B1 , are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABA B receptors. The transgenic mice express GABA B1 isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABA B1 complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA B receptors via the GABA B2 subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA B2 . The mice equipped with tags on GABA B1 facilitate validation and identification of native binding partners of GABA B receptors, providing insight into the molecular mechanisms of synaptic modulation.GABA B receptors convey the metabotropic action of GABA, 2 the main inhibitory neurotransmitter in the brain (1). They are concentrated at axonal boutons of GABAergic and glutamatergic neurons as well as in dendritic spines and shafts at extrasynaptic sites (2). Presynaptic GABA B receptors primarily inhibit calcium channels regulating evoked neurotransmitter release, whereas postsynaptic GABA B receptors mainly open G-protein-regulated inwardly rectifying potassium channels and thereby elicit slow inhibitory postsynaptic potentials. Additionally, GABA B receptor activation decreases the local cAMP level. GABA B receptors function as heteromeric G-protein-coupled receptors consisting of two subunits, GABA B1 and GABA B2 . Heterodimerization is a prerequisite for both surface trafficking of the receptor and ligand-stimulated activation of a G i/o -protein (3).Two N-terminal GABA B1 variants, termed GABA B1a and GABA B1b differing only by a pair of sushi repeats that is exclusively present in GABA B1a , have been identified (4). They originate from the use of alternative transcriptional start sites (3, 5, 6) and underlie functional subtypes of the GABA B receptor with differential localization to pre-and postsynaptic sites in hippocampus and cortex (7-9). 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.Further subtypes of the GABA B receptor may be formed by a compartment-specific interaction of additional proteins, e.g. e...

Opposite Effects of KCTD Subunit Domains on GABAB Receptor-mediated Desensitization

“…Association of auxiliary KCTD8, -12, -12b, and -16 subunits with principal GABA B receptor subunits was recently shown to generate molecularly and functionally distinct receptor subtypes (5,6,9,28). The four KCTD proteins are built from obligatory T1 and H1 domains and optional H2 domains.…”

In Vivo Interaction Proteomics in Caenorhabditis elegans Embryos Provides New Insights into P Granule Dynamics

“…On the other hand, stringent purification procedures, such as tandem affinity purification (TAP), reduce nonspecific binding but could lead to loss of transient interactions. Most published studies did not employ quantification, which makes it difficult to distinguish genuine interaction partners from nonspecific contaminants (15)(16)(17)(18). Quantitative affinity purification and mass spectrometry solves this problem by using quantification as an additional filter (19 -21).…”

The tandem affinity purification technology: an overview

“…However, the authors recommended starting with 3 9 10 7 cells for standard purification of protein complexes using the proposed procedure. Recently, using this SBP-HA tag and following a similar protocol, Bartoi et al (2010) and Wyler et al (2011) successfully isolated GABA B receptor complexes from transgenic mice and ribosomal subunit precursors from HEK 293 cells, respectively. Li et al (2011) reported the efficient purification of protein complexes from mammalian cells using a SBP-His tandem tag.…”