Characterization of membrane steroid binding protein mRNA and protein in lens epithelial cells.
Epithelial cells of the ocular lens contain a 28 kDa membrane protein which is proposed to mediate high affinity binding of steroid hormones and rapid non-genomic actions of steroid hormones. It has been named membrane steroid binding protein (MSBP). Our purpose was to further characterize this protein from cultured bovine lens epithelial cells (BLEC) and compare it to similar forms of the protein present in other species and tissues. The size of the protein's mRNA was examined by Northern blot analysis using a digoxigenin-labelled antisense riboprobe. The sequence of the mRNA was obtained by RT-PCR amplification of poly A+ RNA recovered from cultured BLEC. PCR amplification was conducted using three sets of nested sense and antisense primers, one set at a time. The amino acid sequence of the lens protein was deduced from the revealed cDNA sequence. The hydropathy of the protein was examined by Kyte-Doolittle plots. The sequence of the lens protein's cDNA (about 1.7 kb total) described an open reading frame of 582 residues which coded for a protein of 194 amino acids. The presence of a C-terminal isoprenylation motif suggested by earlier work was not found in the coding region. The deduced amino acid sequence of the lens protein was extremely similar to those of other species and tissues, being 95-98% homologous with that of the other members. All of the MSBPs apparently contain a single membrane spanning domain in the amino terminal. The highly conserved nature of this protein implies a useful function to the cell. We speculate that the protein is a receptor which mediates rapid actions of steroids on lens epithelial cells, such as calcium mobilization, and that the protein plays a role in the mechanism of steroid induced cataracts.
Zhu
X L
XL
Department of Biochemistry, Kirksville College of Osteopathic Medicine, Kirksville, MO 63501, USA.
Sexton
P S
PS
Cenedella
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000737:chemistry*; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology*; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008861:Microsomes / Q000737:chemistry; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D017368:Protein Prenylation; D012333:RNA, Messenger / Q000648:ultrastructure*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013552:Swine
D008565:Membrane Proteins; D012333:RNA, Messenger; C433704:membrane steroid binding protein, lens