We have cloned a σ receptor from rat brain and established its functional identity using a heterologous expression system. The cloned cDNA (1,582 bp long) codes for a protein of 223 amino acids that possesses a single putative transmembrane domain. The amino acid sequence of the rat brain σ receptor is highly homologous to that of the σ receptor recently cloned from guinea pig liver and a human placental cell line but is not related to any other known mammalian receptors. When expressed in HeLa cells, the rat brain σ receptor cDNA leads to a two‐ to threefold increase in haloperidol binding, and this cDNA‐induced binding is sensitive to inhibition by several σ receptor‐specific ligands. Kinetic analysis using the heterologous expression system has revealed that the rat brain σ receptor interacts with haloperidol with an apparent dissociation constant (KD) of 3 nM. Functional expression of the cloned rat brain σ receptor in HeLa cells also leads to an increase in the binding of two other σ ligands, namely, (+)‐pentazocine and (+)‐3‐(3‐hydroxyphenyl)‐N‐(1‐propyl)piperidine (PPP). Pharmacological characterization of the cloned rat brain σ receptor reveals that it exhibits severalfold higher affinity for clorgyline than for 1,3‐di(2‐tolyl)guanidine, it interacts with progesterone and testosterone, and its interaction with PPP is markedly enhanced by phenytoin. In addition, transfection of MCF‐7 cells, which do not express type 1 σ receptor mRNA or activity, with the cloned rat brain cDNA leads to the appearance of haloperidol‐sensitive binding of (+)‐pentazocine, a selective type 1 σ receptor ligand. These data show that the cloned rat brain cDNA codes for a functional type 1 σ receptor. Northern blot analysis with poly(A)+ RNA isolated from various rat tissues has indicated that the σ receptor‐specific transcript, 1.6 kb in size, is expressed abundantly in liver and moderately in intestine, kidney, brain, and lung.
a receptor is a protein that interacts with a variety of psychotomimetic drugs including cocaine and amphetamines and is believed to play an important role in the cellular functions of various tissues associated with the endocrine, immune, and nervous systems. Here we report on the structure and organization of the human gene coding for this receptor. The gene is -~7kbp long and contains four exons, interrupted by three introns. Exon 3 is the shortest (93 bp), and exon 4 is the longest (1,132 bp). Among the introns, intron 3 is the longest ('-~1250 bp). Exon 2 codes for the single transmembrane domain present in the receptor. 5' rapid amplification of cDNA end reactions with mRNA from the JAR human trophoblast cell line have identified 56 bp upstream of the translation start codon as the initiation site for transcription. This transcription start site has been confirmed by RNase protection analysis. Structural analysis of the 5' flanking region has revealed that the gene is TATAless. This region, however, contains a CCAATC box in the reverse complement and several GO boxes that are recognition sites for SP1. There are also consensus sequences for the liver-specific transcription factor nuclear factor-i IL, for a variety of cytokine responsive factors, and for the xenobiotic responsive factor called the arylhydrocarbon receptor. Southern blot analysis of the genomic DNA from Chinese hamster-human and mouse-human hybrid cell lines and fluorescent in situ hybridization with human metaphase chromosome spreads have shown that the gene is located on human chromosome 9, band p13, a region known to be associated with different psychiatric disorders.
We have cloned the Na(+)-dependent neutral amino acid transporter B0 (ATB0) from rabbit jejunum and from the human intestinal cell line Caco-2. Rabbit intestinal ATB0 (riATB0) cDNA codes for a protein of 541 amino acids with 10 potential transmembrane domains. When expressed in HeLa cells, riATB0 mediates the transport of several neutral amino acids, including glutamine, in a Na(+)-dependent manner. Anionic amino acids, cationic amino acids, and N-methylated amino acids are excluded by riATB0. When expressed in Xenopus laevis oocytes, riATB0 increases the transport of neutral amino acids severalfold. The induced transport activity is specific for neutral amino acids, with no noticeable interaction with anionic, cationic, and N-methylated amino acids. However, riATB0 does interact with anionic amino acids at acidic pH. In oocytes expressing riATB0, the neutral amino acid threonine evokes inward currents at a holding potential of -50 mV. The amino acid-evoked current is sensitive to membrane potential. The inward current increases as the membrane potential is hyperpolarized, but the current reverses at about -30 to -40 mV. Threonine evokes outward currents if the membrane potential is depolarized beyond this value. We have also cloned the ATB0 from the human intestinal cell line Caco-2. The Caco-2 ATB0 cDNA also codes for a protein of 541 amino acids that is essentially identical to the ATB0 expressed in the human choriocarcinoma cell line JAR. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction analysis of the RT-PCR products indicate that the human intestine and the human kidney proximal tubular cell line HKPT express an ATB0 identical to the ATB0 expressed in Caco-2 cells.
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