Pharmacological characterization of GABAB receptor subtypes assembled with auxiliary KCTD subunits.

GABAB receptors (GABABRs) are considered promising drug targets for the treatment of mental health disorders. GABABRs are obligate heteromers of principal GABAB1 and GABAB2 subunits. GABABRs can additionally associate with auxiliary KCTD8, 12, 12b and 16 subunits, which also bind the G-protein and differentially regulate G-protein signaling. It is unknown whether the KCTDs allosterically influence pharmacological properties of GABABRs. Here we show that KCTD8 and KCTD16 slightly but significantly increase GABA affinity at recombinant receptors. However, KCTDs clearly do not account for the 10-fold higher GABA affinity of native compared to recombinant GABABRs. The positive allosteric modulator (PAM) GS39783, which binds to GABAB2, increases both potency and efficacy of GABA-mediated G-protein activation ([(35)S]GTPγS binding, BRET between G-protein subunits), irrespective of whether KCTDs are present or not. Of note, the increase in efficacy was significantly larger in the presence of KCTD8, which likely is the consequence of a reduced tonic G-protein activation in the combined presence of KCTD8 and GABABRs. We recorded Kir3 currents to study the effects of GS39783 on receptor-activated G-protein ßγ-signaling. In transfected CHO cells and cultured hippocampal neurons GS39783 increased Kir3 current amplitudes activated by 1 µM of baclofen in the absence and presence of KCTDs. Our data show that auxiliary KCTD subunits exert marginal allosteric influences on principal GABABR subunits. PAMs at principal subunits will therefore not be selective for receptor subtypes owing to KCTD subunits. However, PAMs can differentially modulate the responses of receptor subtypes because the KCTDs differentially regulate G-protein signaling.

Auxiliary GABAB receptor subunits uncouple G protein ßγ subunits from effector channels to induce desensitization.

Activation of K(+) channels by the G protein ßγ subunits is an important signaling mechanism of G-protein-coupled receptors. Typically, receptor-activated K(+) currents desensitize in the sustained presence of agonists to avoid excessive effects on cellular activity. The auxiliary GABAB receptor subunit KCTD12 induces fast and pronounced desensitization of the K(+) current response. Using proteomic and electrophysiological approaches, we now show that KCTD12-induced desensitization results from a dual interaction with the G protein: constitutive binding stabilizes the heterotrimeric G protein at the receptor, whereas dynamic binding to the receptor-activated Gßγ subunits induces desensitization by uncoupling Gßγ from the effector K(+) channel. While receptor-free KCTD12 desensitizes K(+) currents activated by other GPCRs in vitro, native KCTD12 is exclusively associated with GABAB receptors. Accordingly, genetic ablation of KCTD12 specifically alters GABAB responses in the brain. Our results show that GABAB receptors are endowed with fast and reversible desensitization by harnessing KCTD12 that intercepts Gßγ signaling.

Opposite effects of KCTD subunit domains on GABA(B) receptor-mediated desensitization.

GABA(B) receptors assemble from principle and auxiliary subunits. The principle subunits GABA(B1) and GABA(B2) form functional heteromeric GABA(B(1,2)) receptors that associate with homotetramers of auxiliary KCTD8, -12, -12b, or -16 (named after their K(+) channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and -12b generate desensitizing receptor responses while KCTD8 and -16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABA(B2), and a H1 homology domain. KCTD8 and -16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and -12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and -16. In addition, the H2 domains in KCTD8 and -16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and -12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif.

De novo helical peptides as target sequences for a specific, fluorogenic protein labelling strategy.

New methods are needed to selectively label proteins in a manner that minimally perturbs their structures and functions. We have developed a 'small molecule'-based labelling technique that relies on the use of dimaleimide fluorogens that react with a target peptide sequence that presents appropriately spaced, solvent-exposed Cys residues. The thiol addition reaction between target sequence and dimaleimide fluorogen restores the latent fluorescence of the latter and results in the covalent fluorescent labelling of the protein of interest (J. Guy, K. Caron, S. Dufresne, S. W. Michnick, W. G. Skene and J. W. Keillor, J. Am. Chem. Soc., 2007, 129, 11969-11977). We demonstrated the proof-of-principle of this method previously, using a dicysteine mutant of the helical protein Fos (S. Girouard, M.-H. Houle, A. Grandbois, J. W. Keillor and S. W. Michnick, J. Am. Chem. Soc., 2005, 127, 559-566). Herein, we present the design of a novel peptide sequence presenting two Cys residues separated by two turns of an alpha-helix. The secondary structure of this sequence was confirmed by CD spectroscopy, before and after the fluorescent labelling reaction. A new series of di(3-methylmaleimide) fluorogens was prepared and kinetically evaluated, tuning their reactivity toward the target sequence. Attempts were made to increase the reactivity of the parent target sequence by rational design; however, the introduction of basic His residues in the vicinity of one or more Cys residues did not have the desired effect. Finally, epidermal growth factor receptors bearing the de novo target sequence were specifically labelled with a di(3-methylmaleimide) fluorescein fluorogen, validating our method for specific cell-surface labelling of proteins. A wide variety of fluorogen and peptide designs can be envisioned with potential applications to multiplexed labelling for the study of temporal and spatial dynamics of protein expression.

Characterization of an extended receptive ligand repertoire of the human olfactory receptor OR17-40 comprising structurally related compounds.

Molecular properties of odorant compounds essential for activation of the human olfactory receptor hOR17-40 were investigated using a collection of 23 variants of its cognate ligand helional. Coupling receptor activation to an optically detectable intracellular Ca(2+) ion flux allowed dose-dependent screening of different odorant molecules in human embryonic kidney (HEK)293 cells. We found an extended collection of activating ligands and provide first evidence for hOR17-40-specific antagonists. The C-terminal fusion of enhanced green fluorescent protein to the hOR17-40 retained full receptor function and permitted the selection of cells with defined receptor expression levels, which was an essential step for optimizing our screening protocol. Interestingly, cells with a low EGFP fluorescence intensity exhibited efficient hOR17-40 cell surface targeting and odorant-evoked signal transduction; in contrast, highly fluorescent cells displayed mainly incorrectly targeted, intracellular receptors. Fluorescence-activated cell sorting was used to separate hOR17-40-expressing cells on the basis of their endogenous EGFP fluorescence intensity, thereby increasing the fraction of odorant-responsive cells to up to 80% of the total cell number.

Seeing is believing.

Modern visualization techniques are affording a peek into complex cellular processes. A recent paper describes an automated fluorescence microscopy method to map the subcellular localization of up to 100 different proteins in the same sample.