GABAB receptor auxiliary subunits modulate Cav2.3-mediated release from medial habenula terminals

The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation

Monoallelic de novo AJAP1 loss-of-function variants disrupt trans-synaptic control of neurotransmitter release.

Adherens junction-associated protein 1 (AJAP1) has been implicated in brain diseases; however, a pathogenic mechanism has not been identified. AJAP1 is widely expressed in neurons and binds to γ-aminobutyric acid type B receptors (GBRs), which inhibit neurotransmitter release at most synapses in the brain. Here, we show that AJAP1 is selectively expressed in dendrites and trans-synaptically recruits GBRs to presynaptic sites of neurons expressing AJAP1. We have identified several monoallelic AJAP1 variants in individuals with epilepsy and/or neurodevelopmental disorders. Specifically, we show that the variant p.(W183C) lacks binding to GBRs, resulting in the inability to recruit them. Ultrastructural analysis revealed significantly decreased presynaptic GBR levels in Ajap1-/- and Ajap1W183C/+ mice. Consequently, these mice exhibited reduced GBR-mediated presynaptic inhibition at excitatory and inhibitory synapses, along with impaired synaptic plasticity. Our study reveals that AJAP1 enables the postsynaptic neuron to regulate the level of presynaptic GBR-mediated inhibition, supporting the clinical relevance of loss-of-function AJAP1 variants

Propeller protein containing a FYVE domain, ProF : an adaptor for kinases involved in glucose uptake in adipocytes

Adapterproteine sind wesentliche Bestandteile zahlreicher Signalwege. Obwohl selbst nicht enzymatisch aktiv, können sie wichtige Proteine wie zum Beispiel Proteinkinasen nach deren Aktivation zu Proteinkomplexen oder zu bestimmten subzellulären Bereichen rekrutieren. Um diese Aufgabe zu erfüllen, besitzen Adapterproteine oftmals viele verschiedene Proteindomänen zur koordinierten Interaktion mit Proteinen, Lipiden oder Nukleinsäuren. Diese Arbeit behandelt die Identifizierung und ausführliche Beschreibung eines potentiellen Adapterproteins. Das Protein beinhaltet zwei Domänen, einen WD-repeat Propeller und eine FYVE Domäne, und wurde daher als ProF bezeichnet. Die FYVE Domäne ermöglicht die Bindung von ProF an interne Vesikel, während die WD-repeats für die Wechselwirkung mit anderen Proteinen verantwortlich sind. ProF interagiert mit verschiedenen Proteinen, was in Adipozyten (Fettzellen) und anderen Zellsystemen demonstriert werden konnte. Unter diesen Interaktionspartnern sind zwei Kinasen, Akt und PKCζ, und VAMP2, ein Protein, welches an der Fusion von Vesikeln mit ihren Zielmembranen beteiligt ist. Überexpression und Knockdown von ProF zeigten, dass das Protein die Differenzierung von Präadipozyten und Glukoseaufnahme in Adipozyten reguliert. Des Weiteren mag ProF eine allgemeinere Rolle in einer Vielzahl von Vesikeltransport- Prozessen - in Adipozyten wie auch in anderen Zellsystemen - spielen und mit zahlreichen anderen Proteinen interagieren. Adaptor proteins are essential components of many signal transduction pathways. Although not enzymatically active themselves, they can recruit important signaling components such as protein kinases to protein complexes or to specific subcellular locations in response to an activating signal. To perform this task, adaptor proteins often contain several different domains for coordinated protein-protein, protein-lipid, or protein-nucleic acid interaction. In this thesis the identification and extensive characterization of one potential adaptor protein is described. The protein contains two domains, a WD-repeat propeller and a FYVE domain, designated as ProF. The FYVE domain allows the interaction of ProF with internal vesicles, whereas the WD-repeats provide a protein- protein interaction platform. ProF interacts with several proteins as demonstrated in adipocytes (fat cells) and other cellular systems. Among these interaction partners are two protein kinases, Akt and PKCζ, and VAMP2, a protein involved in fusion of vesicles with their target membranes. Overexpression and knock down of ProF showed that the protein regulates differentiation of pre-adipocytes and glucose uptake in adipocytes. Furthermore, ProF may be more generally involved in a variety of vesicular trafficking processes in adipocytes or other tissues and may bind to numerous other signaling proteins

Auxiliary GABAB Receptor Subunits Uncouple G Protein βγ Subunits from Effector Channels to Induce Desensitization

SummaryActivation of K+ channels by the G protein βγ 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 Gβγ subunits induces desensitization by uncoupling Gβγ 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 Gβγ signaling

Modular composition and dynamics of native GABAB receptors identified by high-resolution proteomics

GABAB receptors, the most abundant inhibitory G protein-coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABAB receptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-beta A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABAB receptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor