BRET Analysis of GPCR Dimers in Neurons and Non-Neuronal Cells: Evidence for Inactive, Agonist, and Constitutive Conformations

G-protein-coupled receptors (GPCRs) are dimeric proteins, but the functional consequences of the process are still debated. Active GPCR conformations are promoted either by agonists or constitutive activity. Inverse agonists decrease constitutive activity by promoting inactive conformations. The histamine H3 receptor (H3R) is the target of choice for the study of GPCRs because it displays high constitutive activity. Here, we study the dimerization of recombinant and brain H3R and explore the effects of H3R ligands of different intrinsic efficacy on dimerization. Co-immunoprecipitations and Western blots showed that H3R dimers co-exist with monomers in transfected HEK 293 cells and in rodent brains. Bioluminescence energy transfer (BRET) analysis confirmed the existence of spontaneous H3R dimers, not only in living HEK 293 cells but also in transfected cortical neurons. In both cells, agonists and constitutive activity of the H3R decreased BRET signals, whereas inverse agonists and GTPγS, which promote inactive conformations, increased BRET signals. These findings show the existence of spontaneous H3R dimers not only in heterologous systems but also in native tissues, which are able to adopt a number of allosteric conformations, from more inactive to more active states.

Structural determinants and regulation of spontaneous activity in GABAA receptors

GABAA receptors are vital for controlling neuronal excitability and can display significant levels of constitutive activity that contributes to tonic inhibition. However, the mechanisms underlying spontaneity are poorly understood. Here we demonstrate a strict requirement for β3 subunit incorporation into receptors for spontaneous gating, facilitated by α4, α6 and δ subunits. The crucial molecular determinant involves four amino acids (GKER) in the β3 subunit’s extracellular domain, which interacts with adjacent receptor subunits to promote transition to activated, open channel conformations. Spontaneous activity is further regulated by β3 subunit phosphorylation and by allosteric modulators including neurosteroids and benzodiazepines. Promoting spontaneous activity reduced neuronal excitability, indicating that spontaneous currents will alter neural network activity. This study demonstrates how regional diversity in GABAA receptor isoform, protein kinase activity, and neurosteroid levels, can impact on tonic inhibition through the modulation of spontaneous GABAA receptor gating.

ZAC in GtoPdb v.2021.3

The zinc-activated channel (ZAC, nomenclature as agreed by the NC-IUPHAR Subcommittee for the Zinc Activated Channel) is a member of the Cys-loop family that includes the nicotinic ACh, 5-HT3, GABAA and strychnine-sensitive glycine receptors [2, 3, 4]. The channel is likely to exist as a homopentamer of 4TM subunits that form an intrinsic cation selective channel equipermeable to Na+, K+ and Cs+, but impermeable to Ca2+ and Mg2+ [4]. ZAC displays constitutive activity that can be blocked by tubocurarine and high concentrations of Ca2+ [4]. Although denoted ZAC, the channel is more potently activated by H+ and Cu2+, with greater and lesser efficacy than Zn2+, respectively [4]. ZAC is present in the human, chimpanzee, dog, cow and opossum genomes, but is functionally absent from mouse, or rat, genomes [2, 3].

Role of GPR39 in Neurovascular Homeostasis and Disease

GPR39, a member of the ghrelin family of G protein-coupled receptors, is zinc-responsive and contributes to the regulation of diverse neurovascular and neurologic functions. Accumulating evidence suggests a role as a homeostatic regulator of neuronal excitability, vascular tone, and the immune response. We review GPR39 structure, function, and signaling, including constitutive activity and biased signaling, and summarize its expression pattern in the central nervous system. We further discuss its recognized role in neurovascular, neurological, and neuropsychiatric disorders.

KRIBB11 Induces Apoptosis in A172 Glioblastoma Cells via MULE-Dependent Degradation of MCL-1

KRIBB11, an HSF1 inhibitor, was shown to sensitize various types of cancer cells to treatment with several anticancer drugs. However, the exclusive effects of KRIBB11 in preventing the growth of glioblastoma cells and the related mechanisms have not been elucidated yet. Herein, we aimed to examine the potential of KRIBB11 as an anticancer agent for glioblastoma. Using MTT and colony formation assays and Western blotting for c-PARP, we demonstrated that KRIBB11 substantially inhibits the growth of A172 glioma cells by inducing apoptosis. At the molecular level, KRIBB11 decreased anti-apoptotic protein MCL-1 levels, which was attributable to the increase in MULE ubiquitin ligase levels. However, the constitutive activity of HSF1 in A172 cells was not influenced by the exclusive treatment with KRIBB11. Additionally, based on cycloheximide chase assay, we found that KRIBB11 markedly retarded the degradation of MULE. In conclusion, stabilization of MULE upon KRIBB11 treatment is apparently an essential step for degradation of MCL-1 and the subsequent induction of apoptosis in A172 cells. Our results have expanded the knowledge on molecular pathways controlled by KRIBB11 and could be potentially effective for developing an inhibitory therapeutic strategy for glioblastoma.

Enrichment of SARM1 alleles encoding variants with constitutively hyperactive NADase in patients with ALS and other motor nerve disorders

SARM1, a protein with critical NADase activity, is a central executioner in a conserved programme of axon degeneration. We describe eight rare missense or in-frame microdeletion human SARM1 variant alleles, in patients with amyotrophic lateral sclerosis (ALS), hereditary spastic paraparesis (HSP), or other motor nerve disorders, that alter the SARM1 auto-inhibitory ARM domain and constitutively hyperactivate SARM1 NADase activity. The constitutive NADase activities of six of the variants are at least as high as that of SARM1 lacking the entire ARM domain and greatly exceed the basal activity of wild-type SARM1, even in the presence of nicotinamide mononucleotide (NMN), its physiological activator. This rise in constitutive activity alone is enough to promote neuronal degeneration in response to otherwise non-harmful mild stress. Importantly, we provide evidence that these gain-of-function alleles are enriched in ALS and HSP patients compared to matched individuals without these conditions within ALS and other motor nerve disorder databases. Together, these data suggest these are risk alleles for ALS and other motor nerve disorders. The broad phenotypic heterogeneity and variable age-of-onset in patients with these alleles raises intriguing questions about the pathogenic mechanism of hyperactive SARM1 variants.

RXRA DT448/9PP generates a dominant active variant capable of inducing maturation in acute myeloid leukemia cells

The retinoid receptors RARA and RXRA contribute to myeloid maturation in both mice and humans, and deletion of Rxra and Rxrb augments leukemic growth in mice. While defining the domains of RXRA that are required for anti-leukemic effects in mouse KMT2A-MLLT3 leukemia cells, we unexpectedly identified RXRA DT448/9PP as a constitutively active variant capable of inducing maturation and loss of their proliferative phenotype. RXRA DT448/9PP was associated with ligand-independent activity in reporter assays, with enhanced co-activator interactions, reduced engraftment in vivo, and activation of myeloid maturation transcriptional signatures that overlapped with cells treated with the potent RXRA agonist bexarotene, suggestive of constitutive activity that leads to leukemic maturation. Phenotypes of RXRA DT448/9PP appear to differ from two other RXRA mutations with forms of constitutive activity (F318A and S427F), in that DT448/9PP activity was resistant to mutations at critical ligand-interacting amino acids (R316A/L326A) and was resistant to pharmacologic antagonists, suggesting it may be ligand independent. These data provide further evidence that activated RXRs can regulate myeloid maturation and provide a novel constitutively active variant that may be germane for broader studies of RXR in other settings.

Analysis of Porcine RIG-I Like Receptors Revealed the Positive Regulation of RIG-I and MDA5 by LGP2

The RLRs play critical roles in sensing and fighting viral infections especially RNA virus infections. Despite the extensive studies on RLRs in humans and mice, there is a lack of systemic investigation of livestock animal RLRs. In this study, we characterized the porcine RLR members RIG-I, MDA5 and LGP2. Compared with their human counterparts, porcine RIG-I and MDA5 exhibited similar signaling activity to distinct dsRNA and viruses, via similar and cooperative recognitions. Porcine LGP2, without signaling activity, was found to positively regulate porcine RIG-I and MDA5 in transfected porcine alveolar macrophages (PAMs), gene knockout PAMs and PK-15 cells. Mechanistically, LGP2 interacts with RIG-I and MDA5 upon cell activation, and promotes the binding of dsRNA ligand by MDA5 as well as RIG-I. Accordingly, porcine LGP2 exerted broad antiviral functions. Intriguingly, we found that porcine LGP2 mutants with defects in ATPase and/or dsRNA binding present constitutive activity which are likely through RIG-I and MDA5. Our work provided significant insights into porcine innate immunity, species specificity and immune biology.