The adhesion G protein–coupled receptor (GPCR) BAI subfamily was uncovered in 1997 by Dr. Tokino’s laboratory. They first discovered the hBAI1 gene as a target gene for the p53 tumor suppressor in a cDNA library of fetal human brain (Nishimori et al. 1997). They subsequently identified two homologous genes: hBAI2 and hBAI3 which are not regulated by p53 (Shiratsuchi et al. 1997). This adhesion GPCR subfamily was named human brain-specific Angiogenesis inhibitor because of the capacity of BAI1 to inhibit neovascularization in a rat cornea model, and its predominant expression in the human brain. In 2015, a new nomenclature has been proposed for all the adhesion GPCRs by the Adhesion GPCR Consortium and the International Union of Basic and Clinical Pharmacology Committee. For the BAI subfamily, this new nomenclature is: ADhesion G protein–coupled Receptor B or ADGRB1–3 (Hamann et al. 2015).
BAI3 Expression and Localization Pattern
hBAI3 was mapped on the chromosome 6q12 in human and on chromosome 1 in mice (Shiratsuchi et al. 1997). Analyses of tissues from mice show that mBai3 expression peaks at P1 and is restricted to the brain (Kee et al. 2004). In situ hybridizations confirm the high expression of mBai3 during the development; mBai3 spatial expression pattern shows notably a strong expression in the hippocampus and the dentate gyrus. It is also present in the cerebral cortex, except in layer I, in some posterior nuclei, with a strong expression observed in the inferior olive (IO) and in the deep nuclei and the Purkinje cells of the cerebellum. In the adult mouse, mBai3 expression becomes restricted to the pyramidal cells of the hippocampus, the Purkinje cells of the cerebellum, and a few neurons in the cerebral cortex (Sigoillot et al. 2015). At a subcellular level, BAI3 has been isolated by mass spectrometry in purified postsynaptic density preparations from the cortex and at the parallel fiber/Purkinje cell synapse in the cerebellum (Collins et al. 2006; Jordan et al. 2004). Most recently, immunolabeling revealed the subcellular localization of BAI3 in neurons. In transfected hippocampal neurons, BAI3 is first present in the actin-rich tips of the developing dendrites; later, it is found in dendritic spines and colocalizes with the postsynaptic marker PSD95 (Sigoillot et al. 2015; Lanoue et al. 2013). In the cerebellum, BAI3 is located adjacent to VGluT2 puncta, which labels the synaptic termination of climbing fibers on Purkinje cells (Kakegawa et al. 2015). Further electron microscopy analyses confirm the abundance of BAI3 at postsynaptic sites of climbing fibers synapses.
BAI3 Conservation and Structure
Although BAI3 has been identified for its homology with BAI1, the two receptors share only 48.1% identity in the mouse. On the contrary, BAI3 conservation during evolution is fairly high, with a homology in most of the vertebrates models greater than 90%, the zebrafish (Danio rerio) being the furthest with still more than 70% identity of its BAI3 protein with other species (Lanoue et al. 2013). The in silico study of the expressed sequences of BAI3 has put in evidence that during alternative splicing, some introns of the gene coding for BAI3 are transcribed, leading to the production of noncoding transcripts, also known as noncoding RNA (ncRNA). These ncRNA could be involved in the regulation of the proteic expression of the receptor (Bjarnadóttir et al. 2007).
BAI3 Up- and Downstream Partners
The only known ligands of the BAI3 receptors are the C1ql1–4 proteins. These molecules belong to the complement proteins family, proteins of the innate immune system. The C1ql proteins interact with the TSR domains of BAI3 (Bolliger et al. 2011), although C1ql1 seems to also bind to the CUB domain of the receptor (Kakegawa et al. 2015). Treatment of cultured hippocampal neurons with C1ql proteins reduces the density of excitatory synapses in a manner that is inhibited by the addition of a synthetic TSR-containing soluble fragment of BAI3 (Bolliger et al. 2011). The C1ql1 protein is highly expressed in inferior olivary neurons, and immunostaining reveals C1ql1 punctate labeling in the molecular layer of the cerebellum – where IO neurons extend their axon, the climbing fiber – colocalized with BAI3 labeling (Kakegawa et al. 2015).
BAI3 Downstream Partners
So far, the only downstream partners identified for BAI3 are the ELMO1–2 proteins (Hamoud et al. 2014; Lanoue et al. 2013). The short alpha helix formed by the RKR residues in the cytoplasmic tail of the BAI receptors, first identified in BAI1, is conserved among the BAI family and constitutes the binding motif for ELMO1 (Lanoue et al. 2013; Park et al. 2007). ELMO forms a complex with Dock to activate the RhoGTPase Rac1 and modulate the actin cytoskeleton (Park et al. 2007). The ELMO/Dock complex is conserved in the evolution as it is found in the Drosophila melanogaster where it has been involved in muscle formation (Hamoud et al. 2014), and in the mammalian brain where it plays important roles in neuronal development (Lanoue et al. 2013).
Roles of the BAI3 Receptor
Although BAI3 has been discovered almost 20 years ago, its major roles in the development have only begun to be described in the last 5 years.
Tumorigenesis and Angiogenesis
BAI3 expression is decreased in glioblastoma cell lines (Shiratsuchi et al. 1997). Moreover, it is significantly mutated in 13% of lung squamous cancers and 5% of lung adenocarcinomas (Kan et al. 2010). Thus, a study has recently identified BAI3 expression in small-cell lung cancer using immunohistochemistry (Bari et al. 2014). The protein is detected in the nucleus and not at the membrane, leading the authors to suggest a possible role of BAI3 in cell proliferation. Hence, BAI3 could be used as a potential marker to distinguish between different types of lung tumors (Bari et al. 2014). No biological function has been identified yet for a potential role of BAI3 in regulating angiogenesis or to play a role in the cardiovascular system. However, a genetic study searching for single nucleotide polymorphisms (SNP) candidates to determine venous thromboembolism identified a SNP in the proximity of the BAI3 promoter, suggesting its implication in the occurrence of the disease (Antoni et al. 2010).
In the mouse gastrointestinal tract, quantitative real-time PCR reveals that BAI3 is preferentially expressed in muscle-myenteric nerve layer (Ito et al. 2009). BAI3 has been identified in a myoblast differentiation and fusion model cell line (C2C12 cells) by semiquantitative RT-PCR (Hamoud et al. 2014). The receptor interacts with the ELMO protein, part of the ELMO/Dock complex that regulates the RhoGTPase Rac1 (Lanoue et al. 2013; Park et al. 2007), to regulate embryonic myogenesis. Myoblast fusion is impaired in cells depleted for the endogenous BAI3, establishing BAI3 as an essential effector upstream of ELMO in the regulation of myoblast fusion in vertebrates (Hamoud et al. 2014).
BAI3 in the Developing Central Nervous System
In 2008, a genetic study conducted on a Caucasian patient cohort presenting schizophrenic symptoms has put in evidence a link between SNPs in the gene coding for hBAI3 and disorganized symptoms of schizophrenia (DeRosse et al. 2008). Copy number variations, including microdeletion in the 6q12–13 region corresponding to hBAI3 allele, have also been found related to schizophrenic symptoms (Liao et al. 2012). These genetic studies, together with the identification of BAI3 in synaptic preparations, suggested an important role of the receptor in the regulation of neuronal development.
BAI3 has been shown to regulate dendrite morphogenesis partly via its interaction with ELMO1 and the regulation of the RhoGTPase Rac1 (Lanoue et al. 2013). ELMO1 is expressed in developing Purkinje cells, and downregulation of mBai3 both in vitro and in vivo in these neurons, as well as in cultured hippocampal neurons, leads to important defaults in the length and the branching of neuronal dendritic tree. Moreover, the expression of mutated forms of BAI3 or ELMO1 unable to bind to each other leads to the same phenotype which can be partly rescued by reexpression of wildtype forms of the proteins, confirming the important role of BAI3/ELMO1 interaction in the regulation of dendritogenesis (Lanoue et al. 2013).
BAI3 has been biochemically isolated in excitatory synapses, and immunocytochemistry on transfected hippocampal neurons shows its colocalization with the postsynaptic scaffolding protein PSD95 (Sigoillot et al. 2015), suggesting an important role of the BAI3 receptor in synaptogenesis. Indeed, two recent studies have shed light on the involvement of BAI3 in regulating the excitatory innervation of Purkinje cells in the cerebellum. Immunohistochemistry experiments show the expression of the BAI3 protein in the dendritic spines of Purkinje cells, notably the ones apposed to VGluT2 expressing axonal terminations, which are the synapses formed by the climbing fibers – the axons of the inferior olivary neurons – on Purkinje cells (Kakegawa et al. 2015). BAI3 knock down in the cerebellum impairs the connectivity between both climbing fiber and parallel fibers on Purkinje cells, with a reduced number of Purkinje dendritic spines and a decreased density of VGluT1 (parallel fiber marker) and VGluT2 puncta in the molecular layer of the cerebellar cortex (Kakegawa et al. 2015; Sigoillot et al. 2015). Thus, the C1ql1-BAI3 signaling regulates both the strengthening of a winner and the elimination of loser climbing fibers on a Purkinje cell, leading to a correct one-to-one climbing fiber-Purkinje cell innervation (Kakegawa et al. 2015).
BAI3 is an adhesion GPCR originally identified as a homologous of the p53 tumor suppressor target gene hBAI1. In the last 7 years, a ligand, C1ql, and a downstream effector, ELMO, have been uncovered, while its importance in regulating major developmental processes like muscle formation, dendritogenesis, and synaptogenesis have just begun to be described. C1ql molecules bind to either the CUB (C1ql1) or the TSRs (C1ql1–4) domain located on the N-terminus of the receptor. This binding leads to the correct formation and control of synapse, at least on the Purkinje cells of the cerebellum. Intracellularly, BAI3 interacts with ELMO, which is part of the ELMO/Dock complex that regulates the RhoGTPase Rac1, to regulate the correct development of muscle and the growth and branching of dendrites in neurons. Given the potential genetic link between BAI3 and some neurodevelopmental diseases, the aforementioned roles of BAI3 in neuronal development may be of clinical interest. The regulation of BAI3 expression is also linked to tumorigenesis, making it an interesting marker of cancer progression. However, numerous questions are still unanswered regarding its exact function in tumor formation, its downstream signaling pathways involved in the regulation of synaptogenesis, and the effect of ligand binding in muscle and dendrite development.