The mouse homolog of the bovine leukemia virus receptor is closely related to the delta subunit of adaptor-related protein complex AP-3, not associated with the cell surface

Abstract

A mouse cDNA (mBLVR1) which was highly homologous to the bovine cDNA of the bovine leukemia virus receptor (BLVR) gene was cloned. The mBLVR1 cDNA, of 4,730 bp, covered nearly the full length of the mRNA (about 5 kb) and included an open reading frame (ORF) encoding a protein of 1,199 amino acids. While the bovine BLVR protein was thought to be a type I transmembrane protein, the deduced protein coded by mBLVR1 did not appear to be a typical transmembrane protein. The ORF of mBLVR1 ended at a site 280 amino acids upstream of the termination codon of the bovine BLVR ORF, so the deduced mouse BLVR protein lacked the corresponding transmembrane and cytoplasmic regions of the predicted bovine BLVR protein. No significant hydrophobic region was found in the mouse protein. Recently, a human cDNA which was highly homologous (69.6% homology) to the mouse BLVR gene was reported. The cDNA encodes the delta subunit of the human adaptor-related protein complex AP-3, which aligned almost collinearly with the mouse BLVR protein. AP-3 and all other related adaptor protein complexes have been shown to be associated with intracellular vesicles but not with the cell surface. Thus, the mouse BLVR homolog appeared to be the mouse AP-3 delta subunit itself or closely related to it, but the bovine BLVR gene seemed slightly different from the adaptor subunit gene family.

FIG. 1

Nucleotide sequences of mouse BLVR (m) and bovine BLVR (b) (3, 4) cDNAs. Nucleotides identical to each other are indicated by dots. The start position of the mouse BLVR ORF is indicated by a bracket. The presumed ATG initiation codons and termination codons are indicated by boldface underlining. The fragments used for the probe in Southern blot analysis (Fig. 5A and B) are indicated by boldface letters. The predicted coding region of the bovine BLVR TM region (3) is indicated by lightface underlining.

FIG. 2

Deduced amino acid sequence of mouse BLVR (mBLVR) human AP-3 δ (hAP-3δ), and bovine BLVR (bBLVR). Dots in the upper and lower lines indicate identical amino acids between mouse BLVR and human AP-3 δ and between mouse BLVR and bovine BLVR, respectively. Two consensus N glycosylation sites of mBLVR are indicated by boldface letters. The predicted initiation codons and the TM region of bovine BLVR are indicated by boldface and lightface underlining, respectively.

FIG. 3

Schematic positions of ORFs in the mouse BLVR, human AP-3 δ, and bovine BLVR genes. The ORFs are indicated by open boxes, and putative methionine initiation codons are indicated by arrows. Probes used for the Southern hybridization analysis (Fig. 5) are indicated by shaded boxes. The putative TM region of bovine BLVR is indicated by a black box. Numbers are expressed as nucleotide positions relative to the mouse BLVR. The AP-3 δ gene lacks a region corresponding to nt 728 to 1000 of the mouse BLVR.

FIG. 4

Hydropathy plot of the deduced amino acid sequence of mBLVR. Average hydropathy values were calculated according to the algorithm of Kyte and Doolittle (18) using a 9-aa window. High values indicate hydrophobic regions, and low values indicate hydrophilic regions.

FIG. 5

Southern blot analysis of mouse and bovine genomic DNAs with mouse and bovine BLVR cDNA probes. Bovine (lanes 1 to 5) and mouse (lanes 6 to 10) genomic DNAs were digested with BamHI (lanes 1 and 6), HindIII (lanes 2 and 7), EcoRI (lanes 3 and 8), PstI (lanes 4 and 9), and KpnI (lanes 5 and 10). Five micrograms of DNAs were fractionated on an 0.8% agarose gel and blotted onto a nitrocellulose membrane. The same membrane was hybridized with the ³²P-labeled 0.6-kb mouse BLVR probe (nt 3159 to 3791) (A), the 0.6-kb bovine BLVR probe (nt 1486 to 2140) (B), and the entire 4.7-kb mBLVR1 cDNA probe (C) (Fig. 2).

FIG. 6

RNA expression of the mBLVR gene in mouse tissues. RNAs were isolated from various tissues of BALB/c mice. Five micrograms of total RNAs were fractionated on a 1% formaldehyde-agarose gel, blotted onto a nitrocellulose membrane, and hybridized with the entire ³²P-labeled 4.7-kb mBLVR1 cDNA probe. Left-side arrows indicate size markers of 28S (4.8 kb) and 18S (1.9 kb) rRNA. The major 5.0 kb RNA was detected in all organ samples. The minor 6.2- and 4.6-kb RNAs were detected in some organ samples. As a control, the same membrane was rehybridized with an 18S rDNA probe.