Identification and Molecular Cloning of a Novel Brain-specific Receptor Protein That Binds to Brain Injury-derived Neurotrophic Peptide

Abstract

Brain injury-derived neurotrophic peptide (BINP) is a synthetic 13-mer peptide that supports neuronal survival and protects hippocampal neurons in primary cultures from cell death caused by glutamate. We have developed a monoclonal antibody named mAb 6A22 against the 40-kDa BINP-binding protein, p40BBP. mAb 6A22 inhibits binding between BINP and rat brain synaptosomes and abolishes the protective effect of BINP. The antigen of mAb 6A22 should be the BINP-binding protein that mediates the neuroprotective action of BINP. Using an expression cloning approach with mAb 6A22, we isolated a cDNA encoding a novel receptor protein that shows binding activity of BINP. COS7 cells transfected with the cloned cDNA show binding of BINP and cell surfaces that are stained by 6A22. The mRNA for p40BBP is specific for the rat brain and is increased after birth. From immunohistochemical studies using mAb 6A22, p40BBP increased after kainic acid treatment in rat hippocampal neurons.The normal functioning of the central nervous system presupposes well balanced interactions between different biochemically and structurally linked neuronal systems. Several proteins support neuronal survival and activities. Recently, some researchers have indicated that the central nervous system has the potential for partial recovery from injury. Neurotrophic factors play important roles in the maintenance of neuronal survival and normal brain function in the central nervous system (1), with much evidence indicating that a lack of neurotrophic factor(s) causes cell death. Furthermore, various studies have indicated that neurotrophic factor(s) may protect the nervous system from cell death initiated by disease or injury. Recently, many kinds of neurotrophic factors have been found and identified in the central nervous system, while some have been only newly discovered and are still to be characterized. Neurotrophic factors are produced by various kinds of cells, depending on the developmental stage, and some are increased at the sites of injury (2-8). It is therefore hoped that neurotrophic factors may have a possible therapeutic role in degenerative diseases of the nervous system (9). Understanding how neurons die and how neurotrophic factors prevent this cell death may be helpful for the development of clinically useful compounds. In the case of nerve growth factor, the receptors are TrkA and p75 (10). Nerve growth factor induces cell death in some cells that express only p75 (11-14); however, it supports the survival of neurons that express both TrkA and p75 (15-18). Receptor studies are necessary to elucidate the functions of these factors. Other kinds of receptors, such as G-protein-coupled receptor proteins, also exist in the brain. These receptor proteins are coupled to signal cascades and have biological functions (19,20).We have been studying neuronal survival using a 13-mer brain injury-derived neurotrophic peptide (BINP).1 BINP enhances the survival of cholinergic and mesencephalic dopaminergic neurons in cultures from P14 rats (21). It also ...

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“…As shown in Figure 1a, transcription derived from Ris1 was detected in most adult tissues, including the brain, lymph nodes, spleen, liver, kidney and the small and large intestines, and it was almost undetectable in thymus and testis. The major site of Ris1 expression was brain, supporting previous studies indicating a role for Ris1 in the nervous system (Hama et al, 2001).…”

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Applications of display technologies to proteomic analyses

“…In order to ef®ciently recover enough plasmids for each round of panning selection, the cell lines that support active replication of episomal plasmid DNA were often chosen for plasmid based expression cloning. Several genes encoding for cell receptor or transmembrane protein have been cloned by this way, including brain injury-derived neurotrophic peptide (BINP) binding receptor [Hama et al, 2001] and CD34 [Simmons et al, 1992]. Although the cell based plasmid expression cloning method has been mainly applied in cloning protein genes localized on the cell surface, with the application of direct¯uores-cence-activated cell sorting of permeabilized cells, this method can also be applied for cDNA cloning of the intracellular antigens [Horst et al, 1991].…”

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