Research reportCharacterization of mouse brain-specific angiogenesis inhibitor 1 (BAI1) and phytanoyl-CoA alpha-hydroxylase-associated protein 1, a novel BAI1-binding protein
Introduction
We recently isolated a novel brain-specific protein (PAHX-AP1) associated with PAHX, a Refsum disease gene product, using the yeast-based two-hybrid assay [14]. Refsum disease (RfD) is an autosomal recessive disorder of the lipid metabolism. Its major clinical findings include retinitis pigmentosa, peripheral neuropathy, nerve deafness and cerebellar ataxia [32]. We showed a unique targeted expression of PAHX-AP1 in mouse and human brains, and highly conserved amino acid sequences between humans and mice (99%). In situ hybridization analyses of the rat brain showed that PAHX-AP1 is expressed in the most neurons of the cerebral cortex, dentate gyrus, hippocampus, the Purkinje cell layer and deep cerebellar nucleus, trigeminal, abducent, facial, and cochlear nuclei, and retina. It also showed that PAHX-AP1 is mainly expressed in the neuron but not in the oligodendrocyte, which is a major pathologic site of the polyneuropathy in RfD. It indicates that PAHX-AP1 is not involved in the pathogenesis of peripheral polyneuropathy. Interestingly, those subregions of the brain frequently show overt pathologic symptoms in RfD patients, and it is suggested that PAHX-AP1 is involved at least in the development of central neurologic deficits in RfD.
To characterize the function of PAHX-AP1 in the nervous system, we searched for cellular proteins that may associate with it by screening a mouse brain cDNA library with yeast two-hybrid system. Here we report the identification of the interaction between the cytoplasmic region (1254–1516 aa) of BAI1 and the whole encoding region of PAHX-AP1, and cloning of the mouse homolog (mBAI1, 5498 bp) of human BAI1. hBAI1 was reported to have a functional p53 binding sequence, and seven-span transmembrane region as well as extended extracellular and cytoplasmic domains [18]. hBAI1 possessed two presumably functional sites in its extracellular region, an Arg-Gly-Asp (RGD) motif and thrombospondin (TSP)-type 1 repeats. The RGD motif is a potential recognition sequence for binding integrins [4], and the TSP-type 1 repeats can inhibit angiogenesis in the rat cornea induced by bFGF [30]. To date, the functions of BAI1 in the brain are as yet unknown. In this study, we found that BAI1 also acts as an angiogenesis inhibitor in the rat cerebral cortex like the TSP in the cornea.
Three BAI-associated proteins (BAP1, 2, 3) were reported to interact with the cytoplasmic domain of hBAI1 by means of a yeast two-hybrid screening system [20], [22], [24]. However, structural analysis of the PAHX-AP1 revealed that there is no homology between the three BAPs and PAHX-AP1. We renamed PAHX-AP1 as BAP4. Considering the unique expression pattern and the same localization of BAI1 and BAP4 in the brain, interaction of BAI1 with BAP4 might be involved in several important neuronal functions, such as neuronal differentiation and angiogenesis inhibition. It suggests that interaction of PAHX with BAI1 through BAP4 is also related to the pathogenesis of the central neurologic symptoms of RfD.
Section snippets
Yeast two-hybrid assay, isolation and analysis of murine BAI1
The yeast two-hybrid assay was performed as described in the Matchmaker protocol (Clonetech, Palo Alto, CA). Briefly, the whole portion of murine PAHX-AP1 was amplified from the corresponding cDNA by PCR and cloned into the GAL4 DNA-binding domain vector, a bait vector, pGBT9 using the EcoRI and BamHI restriction sites. Y190 yeast cells were co-transformed with the bait vector harboring PAHX-AP1 cDNA and a prey vector containing mouse brain cDNA libraries (Clonetech), then cultured at 30°C in a
Cloning of the murine BAI1 cDNA
To identify new proteins that interact with and potentially regulate PAHX-AP1, a mouse brain cDNA library was screened by the yeast two-hybrid assay using the whole encoding region of murine PAHX-AP1 cDNA as bait. Several positive clones were obtained and data base surveys identified a high degree of the deduced amino acid sequence identity (90%, Fig. 2) between one of the positive clones and hBAI1. This cDNA insert corresponded to the cytoplasmic region (1254–1516 aa) of hBAI1. This murine
Discussion
In this study, we isolated the murine homolog of hBAI1 that interacts with PAHX-AP1, a Refsum disease gene product related protein, using the yeast two-hybrid assay. We revealed a unique expression of mBAI1 in the mouse brain, and highly conserved amino acid sequences between humans and mice (94%). In situ hybridization analyses showed that mBAI1 is co-localized with PAHX-AP1(BAP4) in the rat brain, such as in the most neurons of the cerebral cortex, olfactory bulb, dentate gyrus, hippocampus,
Acknowledgements
We thank S.M. Lee, S.R. Park and K.S. Lee (Chonnam University) for assistance with cloning and cell culture experiments. This study was supported by a grant (HMP-98-N-2-0024) of the 98 Good Health R and D Program, Ministry of Health and Welfare, Republic of Korea.
References (34)
- et al.
Endogenous plasminogen activator expression after embolic focal cerebral ischemia in mice
Brain Res.
(1999) - et al.
Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif
Trends Biochem. Sci.
(1991) - et al.
Tumor suppressor gene expression during normal and pathologic myocardial growth
J. Biol. Chem.
(1994) - et al.
F-spondin: a gene expressed at high levels in the floor plate encodes a secreted protein that promotes neural cell adhesion and neurite extension
Cell
(1992) - et al.
Identification of a brain specific protein that associates with a Refsum disease gene product. Phytanoyl-CoA alpha-hydroxylase
Mol. Brain Res.
(2000) - et al.
UNC-5, a transmembrane protein with immunoglobulin and thrombospondin type 1 domains, guides cell and pioneer axon migrations in C. elegans
Cell
(1992) - et al.
Cloning and characterization of BAI-associated protein 1: a PDZ domain-containing protein that interacts with BAI1
Biochem. Biophys. Res. Commun.
(1998) - et al.
Cloning and characterization of BAP3 (BAI-associated protein 3), a C2 domain-containing protein that interacts with BAI1
Biochem. Biophys. Res. Commun.
(1998) - et al.
Overexpression of thrombospondin-1 decreases angiogenesis and inhibits the growth of human cutaneous squamous cell carcinomas
Am. J. Pathol.
(1999) - et al.
Expression of thrombospondin-1 in ischemia-induced retinal neovascularization
Am. J. Pathol.
(1999)
Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase
J. Biol. Chem.
Activated microvessels express vascular endothelial growth factor and integrin alpha(v)beta3 during focal cerebral ischemia
J. Cereb. Blood Flow Metab.
A potential role for erythropoietin in focal permanent cerebral ischemia in mice
J. Cereb. Blood Flow Metab.
The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit
Genes Dev.
Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells
Cancer Res.
Expression of thrombospondin in the adult nervous system
J. Comp. Neurol.
Differential expression of thrombospondin 1,2, and 3 during murine development
Dev. Dyn.
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These authors contributed equally to this work.