Unveiling Mechanical Activation: GAIN Domain Unfolding and Dissociation in Adhesion GPCRs.

Adhesion G protein-coupled receptors (aGPCRs) have extracellular regions (ECRs) containing GPCR autoproteolysis-inducing (GAIN) domains. The GAIN domain enables the ECR to self-cleave into N- and C-terminal fragments. However, the impact of force on the GAIN domain's conformation, critical for mechanosensitive aGPCR activation, remains unclear. Our study investigated the mechanical stability of GAIN domains in three aGPCRs (B, G, and L subfamilies) at a loading rate of 1 pN/s. We discovered that forces of a few piconewtons can destabilize the GAIN domains. In autocleaved aGPCRs ADGRG1/GPR56 and ADGRL1/LPHN1, these forces cause the GAIN domain detachment from the membrane-proximal Stachel sequence, preceded by partial unfolding. In noncleavable aGPCR ADGRB3/BAI3 and cleavage-deficient mutant ADGRG1/GPR56-T383G, complex mechanical unfolding of the GAIN domain occurs. Additionally, GAIN domain detachment happens during cell migration. Our findings support the mechanical activation hypothesis of aGPCRs, emphasizing the sensitivity of the GAIN domain structure and detachment to physiological force ranges.

Multifaceted genome-wide study identifies novel regulatory loci in SLC22A11 and ZNF45 for body mass index in Indians.

Obesity, a risk factor for multiple diseases (e.g. diabetes, hypertension, cancers) originates through complex interactions between genes and prevailing environment (food habit and lifestyle) that varies across populations. Indians exhibit a unique obesity phenotype with high abdominal adiposity for a given body weight compared to matched white populations suggesting presence of population-specific genetic and environmental factors influencing obesity. However, Indian population-specific genetic contributors for obesity have not been explored yet. Therefore, to identify potential genetic contributors, we performed a two-staged genome-wide association study (GWAS) for body mass index (BMI), a common measure to evaluate obesity in 5973 Indian adults and the lead findings were further replicated in 1286 Indian adolescents. Our study revealed novel association of variants-rs6913677 in BAI3 gene (p = 1.08 × 10-8) and rs2078267 in SLC22A11 gene (p = 4.62 × 10-8) at GWAS significance, and of rs8100011 in ZNF45 gene (p = 1.04 × 10-7) with near GWAS significance. As genetic loci may dictate the phenotype through modulation of epigenetic processes, we overlapped genetic data of identified signals with their DNA methylation patterns in 236 Indian individuals and performed methylation quantitative trait loci (meth-QTL) analysis. Further, functional roles of discovered variants and underlying genes were speculated using publicly available gene regulatory databases (ENCODE, JASPAR, GeneHancer, GTEx). The identified variants in BAI3 and SLC22A11 genes were found to dictate methylation patterns at unique CpGs harboring critical cis-regulatory elements. Further, BAI3, SLC22A11 and ZNF45 variants were located in repressive chromatin, active enhancer, and active chromatin regions, respectively, in human subcutaneous adipose tissue in ENCODE database. Additionally, these genomic regions represented potential binding sites for key transcription factors implicated in obesity and/or metabolic disorders. Interestingly, GTEx portal identify rs8100011 as a robust cis-expression quantitative trait locus (cis-eQTL) in subcutaneous adipose tissue (p = 1.6 × 10-7), and ZNF45 gene expression in skeletal muscle of Indian subjects showed an inverse correlation with BMI indicating its possible role in obesity. In conclusion, our study discovered 3 novel population-specific functional genetic variants (rs6913677, rs2078267, rs8100011) in 2 novel (SLC22A11 and ZNF45) and 1 earlier reported gene (BAI3) for BMI in Indians. Our study decodes key genomic loci underlying obesity phenotype in Indians that may serve as prospective drug targets in future.

An investigation into the potential role of brain angiogenesis inhibitor protein 3 (BAI3) in the tumorigenesis of small-cell carcinoma: a review of the surrounding literature.

Brain angiogenesis inhibitor protein 3 (BAI3) is from the adhesion group of seven-transmembrane spanning G protein-coupled receptors (GPCRs) and has been identified via gene expression profiling as being upregulated in small-cell lung cancer (SCLC) tumors. It has subsequently been validated as a sensitive and specific immunohistochemical marker for SCLC, helping to differentiate these tumors from morphologically similar large-cell neuroendocrine (LCNEC) malignancies. It is, however, still unclear as to the role BAI3 proteins might play in SCLC and indeed how they might contribute to tumorigenesis. Interestingly, the pattern of staining observed on immunohistochemistry was in fact nuclear as opposed to the membranous staining pattern expected of transmembrane-bound molecules. This fact has lead the authors to believe that the protein receptor is structurally altered in SCLC and that this modification may confer different behavioral properties that contribute toward tumorigenesis. Nuclear localization is not unique to BAI3 and has been reported in a number of GPCRs and frequently correlates with survival outcomes. BAI3 has the potential to act as target for pharmaceutical intervention inline with developing trends in molecular pathology aiming to provide personalized, treatment regimes based on tumor-specific mutation profiles. The adhesion group of the GPCR superfamily is still poorly understood. We present a review of the existing literature regarding the role they play in both physiological and disease states and the mechanisms by which they influence a range of cellular processes.

BAI3, CDX2 and VIL1: a panel of three antibodies to distinguish small cell from large cell neuroendocrine lung carcinomas.

AIMS: Discriminating small-cell lung carcinoma (SCLC) from large-cell neuroendocrine carcinoma (LCNEC) rests on morphological criteria, and reproducibility has been shown to be poor. We aimed to identify immunohistochemical markers to assist this diagnosis. METHODS AND RESULTS: Gene expression profiling on laser captured frozen tumour samples from eight SCLC and eight LCNEC tumours identified a total of 888 differentially expressed genes (DEGs), 23 of which were validated by qRT-PCR. Antibodies to four selected gene products were then evaluated as immunohistochemical markers on a cohort of 173 formalin-fixed paraffin-embedded (FFPE) SCLC/LCNEC tumour samples, including 26 indeterminate tumours without a consensus diagnosis. Three markers, CDX2, VIL1 and BAI3, gave significantly different results in the two tumour types (P < 0.0001): CDX2 and VIL1 in combination (either marker positive) showed sensitivity and specificity of 81% for LCNEC while BAI3 showed 89% sensitivity and 75% specificity for SCLC. Of the 26 indeterminate tumours 15 (58%) showed an immunophenotype suggesting either SCLC or LCNEC, eight (31%) showed staining of both tumour types, and three (11%) were negative for all markers. CONCLUSION: A panel of three markers, BAI3, CDX2 and VIL1, is a useful adjunct in the diagnosis of these tumour types.

Creation of a novel CRISPR-generated allele to express HA epitope-tagged C1QL1 and improved methods for its detection at synapses.

C1QL1 is expressed in a subset of cells in the brain and likely has pleiotropic functions, including the regulation of neuron-to-neuron synapses. Research progress on C1QL proteins has been slowed by a dearth of available antibodies. Therefore, we created a novel knock-in mouse line in which an HA-tag is inserted into the endogenous C1ql1 locus. We examined the entire brain, identifying previously unappreciated nuclei expressing C1QL1, presumably in neurons. By total numbers, however, the large majority of C1QL1-expressing cells are of the oligodendrocyte lineage. Subcellular immunolocalization of synaptic cleft proteins is challenging, so we developed a new protocol to improve signal at synapses. Lastly, we compared various anti-HA antibodies to assist future investigations using this and likely other HA epitope-tagged alleles.

Generation and initial characterization of mice lacking full-length BAI3 (ADGRB3) expression.

Brain-specific angiogenesis inhibitor 3 (ADGRB3/BAI3) belongs to the family of adhesion G protein-coupled receptors. It is most highly expressed in the brain where it plays a role in synaptogenesis and synapse maintenance. Genome-wide association studies have implicated ADGRB3 in disorders such as schizophrenia and epilepsy. Somatic mutations in ADGRB3 have also been identified in cancer. To better understand the in vivo physiological role of ADGRB3, we used CRISPR/Cas9 editing to generate a mouse line with a 7-base pair deletion in Adgrb3 exon 10. Western blot analysis confirmed that homozygous mutants (Adgrb3∆7/∆7 ) lack full-length ADGRB3 expression. The mutant mice were viable and reproduced in Mendelian ratios but demonstrated reduced brain and body weights and deficits in social interaction. Measurements of locomotor function, olfaction, anxiety levels and prepulse inhibition were comparable between heterozygous and homozygous mutants and wild-type littermates. Since ADGRB3 is also expressed in organs such as lung and pancreas, this new mouse model will facilitate elucidation of ADGRB3's role in non-central nervous system-related functions. Finally, since somatic mutations in ADGRB3 were identified in patients with several cancer types, these mice can be used to determine whether loss of ADGRB3 function contributes to tumour development.

C1ql1-Bai3 signaling is necessary for climbing fiber synapse formation in mature Purkinje cells in coordination with neuronal activity.

Changes in neural activity induced by learning and novel environments have been reported to lead to the formation of new synapses in the adult brain. However, the underlying molecular mechanism is not well understood. Here, we show that Purkinje cells (PCs), which have established adult-type monosynaptic innervation by climbing fibers (CFs) after elimination of weak CFs during development, can be reinnervated by multiple CFs by increased expression of the synaptic organizer C1ql1 in CFs or Bai3, a receptor for C1ql1, in PCs. In the adult cerebellum, CFs are known to have transverse branches that run in a mediolateral direction without forming synapses with PCs. Electrophysiological, Ca2+-imaging and immunohistochemical studies showed that overexpression of C1ql1 or Bai3 caused these CF transverse branches to elongate and synapse on the distal dendrites of mature PCs. Mature PCs were also reinnervated by multiple CFs when the glutamate receptor GluD2, which is essential for the maintenance of synapses between granule cells and PCs, was deleted. Interestingly, the effect of GluD2 knockout was not observed in Bai3 knockout PCs. In addition, C1ql1 levels were significantly upregulated in CFs of GluD2 knockout mice, suggesting that endogenous, not overexpressed, C1ql1-Bai3 signaling could regulate the reinnervation of mature PCs by CFs. Furthermore, the effects of C1ql1 and Bai3 overexpression required neuronal activity in the PC and CF, respectively. C1ql1 immunoreactivity at CF-PC synapses was reduced when the neuronal activity of CFs was suppressed. These results suggest that C1ql1-Bai3 signaling may mediate CF synaptogenesis in mature PCs, potentially in concert with neuronal activity.

Loss of Brain Angiogenesis Inhibitor-3 (BAI3) G-Protein Coupled Receptor in Mice Regulates Adaptive Thermogenesis by Enhancing Energy Expenditure.

Effective energy expenditure is critical for maintaining body weight (BW). However, underlying mechanisms contributing to increased BW remain unknown. We characterized the role of brain angiogenesis inhibitor-3 (BAI3/ADGRB3), an adhesion G-protein coupled receptor (aGPCR), in regulating BW. A CRISPR/Cas9 gene editing approach was utilized to generate a whole-body deletion of the BAI3 gene (BAI3-/-). In both BAI3-/- male and female mice, a significant reduction in BW was observed compared to BAI3+/+ control mice. Quantitative magnetic imaging analysis showed that lean and fat masses were reduced in male and female mice with BAI3 deficiency. Total activity, food intake, energy expenditure (EE), and respiratory exchange ratio (RER) were assessed in mice housed at room temperature using a Comprehensive Lab Animal Monitoring System (CLAMS). While no differences were observed in the activity between the two genotypes in male or female mice, energy expenditure was increased in both sexes with BAI3 deficiency. However, at thermoneutrality (30 °C), no differences in energy expenditure were observed between the two genotypes for either sex, suggesting a role for BAI3 in adaptive thermogenesis. Notably, in male BAI3-/- mice, food intake was reduced, and RER was increased, but these attributes remained unchanged in the female mice upon BAI3 loss. Gene expression analysis showed increased mRNA abundance of thermogenic genes Ucp1, Pgc1α, Prdm16, and Elov3 in brown adipose tissue (BAT). These outcomes suggest that adaptive thermogenesis due to enhanced BAT activity contributes to increased energy expenditure and reduced BW with BAI3 deficiency. Additionally, sex-dependent differences were observed in food intake and RER. These studies identify BAI3 as a novel regulator of BW that can be potentially targeted to improve whole-body energy expenditure.

miR-142 downregulation alleviates the impairment of spatial learning and memory, reduces the level of apoptosis, and upregulates the expression of pCaMKII and BAI3 in the hippocampus of APP/PS1 transgenic mice.

MicroRNA-142-5p (miR-142-5p) has been found to be dysregulated in several neurodegenerative disorders. However, little is known about the involvement of miR-142-5p in Alzheimer's disease (AD). Brain angiogenesis inhibitor 3 (BAI3), which belongs to the adhesion-G protein-coupled receptor subgroup, contributes to a variety of neuropsychiatric disorders. Despite its very high expression in neurons, the role of BAI3 in AD remains elusive, and its mechanism at the cellular and molecular levels needs to be further elucidated. The current study sought to investigate whether miR-142-5p influenced BAI3 expression and neuronal synaptotoxicity induced by Aß, both in APP/PS1 transgenic mice and a cellular model of Alzheimer's disease. Altered expression of miR-142-5p was found in the hippocampus of AD mice. Inhibition of miR-142 could upregulate BAI3 expression, enhance neuronal viability and prevent neurons from undergoing apoptosis. In addition, the reduction of phosphorylation of Synapsin I and calcium/calmodulin-dependent protein kinase II (CaMKII), as well as the expression of PSD-95 in the hippocampus of APP/PS1 transgenic mice, were significantly restored by inhibiting miR-142. Meanwhile, the levels of Aß1-42, ß-APP, BACE-1 and PS-1 in cultured neurons were detected, and the effects of inhibiting miR-142 on spatial learning and memory were also observed. Interestingly, we found that BAI3, an important regulator of excitatory synapses, was a potential target gene of miR-142-5p. Collectively, our findings suggest that miR-142 inhibition can alleviate the impairment of spatial learning and memory, reduce the level of apoptosis, and upregulate the expression of pCaMKII and BAI3 in the hippocampus of APP/PS1 transgenic mice; thus, appropriate interference of miR-142 may provide a potential therapeutic approach to rescue cognitive dysfunction in AD patients.

A Synaptic Circuit Required for Acquisition but Not Recall of Social Transmission of Food Preference.

During social transmission of food preference (STFP), the combination of an olfactory sensory input with a social cue induces long-term memory of a food odor. How a social cue produces long-term learning of an olfactory input, however, remains unknown. Here we show that the neurons of the anterior olfactory nucleus (AON), which form abundant synaptic projections onto granule cells in the olfactory bulb (OB), express the synaptogenic molecule C1ql3. Deletion of C1ql3 in the dorsolateral AON impaired synaptic AON→OB connections and abolished acquisition, but not recall, of STFP memory without significantly affecting basal olfaction. Moreover, deletion in granule cells of the OB of Bai3, a postsynaptic GPCR that binds C1ql3, similarly suppressed synaptic transmission at AON→OB projections and abolished acquisition, but not recall, of STFP memory. Thus, synaptic AON→OB connections are selectively required for STFP memory acquisition and are formed by an essential interaction of presynaptic C1ql3 with postsynaptic Bai3.

Analysis of changes in transcription start site distribution by a classification approach.

Change in transcription start site (TSS) usage is an important mechanism for the control of transcription process, and has a significant effect on the isoforms being transcribed. One of the goals in the study of TSS is the understanding of how and why their usage differs in different tissues or under different conditions. In light of recent efforts in the mapping of transcription start site landscape using high-throughput sequencing approaches, a quantitative and automated method is needed to process all the data that are being produced. In this work we propose a statistical approach that will classify changes in TSS distribution between different samples into several categories of changes that may have biological significance. Genes selected by the classifiers can then be analyzed together with additional supporting data to determine their biological significance. We use a set of time-course TSS data from mouse dendritic cells stimulated with lipopolysaccharide (LPS) to demonstrate the usefulness of our method.