title: Phenylalkylamine Ca2+ antagonist binding protein. Molecular cloning, tissue distribution, and heterologous expression.
abstract: We recently characterized (Moebius, F. F., Burrows, G. G., Striessnig, J., and Glossmann H. (1993) Mol. Pharmacol. 43, 139-144) and purified (Moebius, F. F., Hanner, M., Knaus, H. G., Weber, F., Striessnig, J., and Glossmann, H. (1994) J. Biol. Chem. 269, 29314-29320) a binding protein for the phenylalkylamine Ca2+ antagonist emopamil. The emopamil-binding protein (EBP) acts as a high affinity acceptor for several antiischemic drugs and thus represents a potential common molecular target for antiischemic drug action. Degenerate oligonucleotides were synthesized according to the N-terminal amino acid sequence of purified EBP and used to amplify a guinea pig cDNA with reverse transcriptase-polymerase chain reaction and to clone full-length cDNAs from guinea pig and human liver cDNA libraries. The cDNAs coded for 229 (guinea pig) and 230 (human) amino acid 27-kDa polypeptides without significant sequence homology with any known protein. However, EBP shared structural features with pro- and eukaryotic drug transport proteins. The amino acid identity between human and guinea pig EBP was 73%. Hydrophobicity plots predicted four transmembrane segments. The C terminus contained a lysine-rich consensus sequence for the retrieval of type I integral membrane proteins to the endoplasmic reticulum. The heterologous expression of human and guinea pig EBP in Saccharomyces cerevisiae demonstrated that the expression of EBP alone is sufficient to form high affinity drug- and cation-binding domains identical to the [3H]-emopamil-binding site of guinea pig liver. Northern and Western blot analysis revealed high abundance of EBP in guinea pig epithelial tissues as liver, bowel, adrenal gland, testis, ovary, and uterus and low densities in brain, cerebellum, skeletal muscle, and heart. EBP is suggested to be the first structurally characterized member of a family of high affinity microsomal drug acceptor proteins carrying so called sigma-binding sites.
authors.affiliation: Institut für Biochemische Pharmakologie, Universität Innsbruck, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Hanner
authors.forename: F F
authors.initials: FF
authors.lastname: Moebius
authors.forename: F
authors.initials: F
authors.lastname: Weber
authors.forename: M
authors.initials: M
authors.lastname: Grabner
authors.forename: J
authors.initials: J
authors.lastname: Striessnig
authors.forename: H
authors.initials: H
authors.lastname: Glossmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D002121:Calcium Channel Blockers / Q000378:metabolism / Q000494:pharmacology; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers; D015870:Gene Expression; D015723:Gene Library; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D013251:Steroid Isomerases; D014700:Verapamil / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D002121:Calcium Channel Blockers; D002352:Carrier Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D014700:Verapamil; D013251:Steroid Isomerases; C090445:EBP protein, human; C052752:emopamil