Human GABA B G protein-coupled receptor (GPCR), a member of the class C family, mediates inhibitory neurotransmission and is implicated in epilepsy, pain, and addiction 1 . A unique GPCR known to require heterodimerization for function 2 – 6 , its two subunits, GABA B1 and GABA B2 , are structurally homologous but perform distinct and complementary functions. GABA B1 recognizes orthosteric ligand 7 , 8 , while GABA B2 couples with G protein 9 – 14 . Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane (TM) domain, and a cytoplasmic tail 15 . Whereas the VFT heterodimer structure has been resolved 16 , the structure of the full-length receptor and its transmembrane signaling mechanism remain unknown. Here we present a near full-length structure of the GABA B receptor, captured in an inactive state via cryo-electron microscopy (EM). Our structure reveals multiple ligands pre-associated with the receptor, including two large endogenous phospholipids embedded within the TM domains to maintain receptor integrity and modulate receptor function. We also identify a novel heterodimer interface between TM helices 5 and 3 of both subunits, which serves as a signature of the inactive conformation. A unique ′intersubunit latch′ within this TM interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

Metabotropic GABA receptor is a G protein-coupled receptor (GPCR) that mediates slow and prolonged inhibitory neurotransmission in the brain. It functions as a constitutive heterodimer composed of the GABA and GABA subunits. Each subunit contains three domains; the extracellular Venus flytrap module, seven-helix transmembrane region and cytoplasmic tail. In recent years, the three-dimensional structures of GABA receptor extracellular and intracellular domains have been elucidated. These structures reveal the molecular basis of ligand recognition, receptor heterodimerization and receptor activation. Here we provide a brief review of the GABA receptor structures, with an emphasis on describing the different ligand-bound states of the receptor. We will also compare these with the known structures of related GPCRs to shed light on the molecular mechanisms of activation and regulation in the GABA system, as well as GPCR dimers in general. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

Metabotropic GABA B receptors mediate a significant fraction of inhibitory neurotransmission in the brain. Native GABA B receptor complexes contain the principal subunits GABA B1 and GABA B2 , which form an obligate heterodimer, and auxiliary subunits, known as potassium channel tetramerization domain-containing proteins (KCTDs). KCTDs interact with GABA B receptors and modify the kinetics of GABA B receptor signaling. Little is known about the molecular mechanism governing the direct association and functional coupling of GABA B receptors with these auxiliary proteins. Here, we describe the high-resolution structure of the KCTD16 oligomerization domain in complex with part of the GABA B2 receptor. A single GABA B2 Cterminal peptide is bound to the interior of an open pentamer formed by the oligomerization domain of five KCTD16 subunits. Mutation of specific amino acids identified in the structure of the GABA B2 -KCTD16 interface disrupted both the biochemical association and functional modulation of GABA B receptors and G protein-activated inwardly rectifying K + channel (GIRK) channels. These interfacial residues are conserved among KCTDs, suggesting a common mode of KCTD interaction with GABA B receptors. Defining the binding interface of GABA B receptor and KCTD reveals a potential regulatory site for modulating GABA B -receptor function in the brain. GABA B receptor | KCTD | principal and auxiliary subunits | crystal structure M etabotropic γ-aminobutyric acid (GABA) type B (GABA B )receptors are implicated in various neurological and psychiatric disorders, including spasticity, epilepsy, depression, addiction, and anxiety (1-3). GABA B receptors provide a crucial component of inhibitory neurotransmission in the nervous system (1-3), via coupling to G i/o type G proteins that modulate three different downstream effectors: voltage-gated Ca 2+ channels, G proteinactivated inwardly rectifying K + (GIRK) channels, and adenylyl cyclase (1-3). The GABA B receptor functions as an obligatory heterodimer, consisting of the GABA B1 and GABA B2 subunits (4-9), whereby the GABA B1 subunit binds orthosteric ligand (10, 11) and the GABA B2 subunit couples to the G protein (12)(13)(14)(15)(16)(17)(18).Recent proteomic studies led to the discovery of a family of auxiliary proteins for the GABA B receptor, originally referred to as potassium channel tetramerization domain-containing (KCTD) proteins (19)(20)(21). A subset of KCTD proteins (numbered 8, 12, 12b, and 16) interact with the cytoplasmic tail of the GABA B2 subunit (3, 20, 21) and convey unique functional properties to the signaling of the GABA B receptor (3). For example, expression of each of the four KCTD proteins leads to acceleration of GABA B receptordependent activation of GIRK currents, albeit to different extents (20,22). KCTD12 and KCTD12b also promote rapid desensitization of the GABA B receptor-induced GIRK current, by uncoupling Gβγ from GIRK channels (20,22,23). By contrast, KCTD8 and KCTD16 generate primarily nondesensitizing receptor responses (20,22...

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near–full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.

Inducing long-term protective memory CD8+ T cells is a desirable goal for vaccines against intracellular pathogens. However, the mechanisms of differentiation of CD8+ T cells into long-lived memory cells capable of mediating protection of immunized hosts remain incompletely understood. We have developed an experimental system using mice immunized with WT or mutants of the intracellular bacterium Listeria monocytogenes (Lm) that either do or do not develop protective memory CD8+ T cells. We previously reported that mice immunized with Lm lacking functional SecA2, an auxiliary secretion system of gram-positive bacteria, did not differentiate functional memory CD8+ T cells that protected against a challenge infection with WT Lm. Herein we hypothesized that the p60 and NamA autolysins of Lm, which are major substrates of the SecA2 pathway, account for this phenotype. We generated Lm genetically deficient for genes encoding for the p60 and NamA proteins, ΔiapΔmurA Lm, and further characterized this mutant. Δp60ΔNamA Lm exhibited a strong filamentous phenotype, inefficiently colonized host tissues, and grew mostly outside cells. When Δp60ΔNamA Lm was made single unit (SU), cell invasion was restored to WT levels during vaccination, yet induced memory T cells still did not protect immunized hosts against recall infection. Recruitment of blood phagocytes and antigen-presenting cell activation was close to that of mice immunized with ΔActA Lm which develop protective memory. However, key inflammatory factors involved in optimal T cell-programming such as IL-12 and type I IFN (IFN-I) were lacking, suggesting that cytokine signals may largely account for the observed phenotype. Thus altogether, these results establish that p60 and NamA secreted by Lm promote primary host cell-invasion, the inflammatory response and the differentiation of functional memory CD8+ T cells, by preventing Lm filamentation during growth and subsequent triggering of innate sensing mechanisms.