Expression and pharmacological characterization of the human peripheral-type benzodiazepine receptor in yeast.
Recently we cloned the cDNA coding for the putative human peripheral-type benzodiazepine receptor (hPBR). This report describes the expression of this cDNA in Saccharomyces cerevisiae and the characterization of the recombinant protein. The expression was achieved by placing the receptor cDNA under the control of a galactose-regulated artificial promoter. After galactose induction, the transformed cells expressed a functional hPBR which displayed a Kd for the specific peripheral-type ligand [3H]PK11195 of 9.9 +/- 1.3 nM and a maximal binding capacity of 249,300 +/- 50,400 sites/cell. The pharmacological characterization of the recombinant receptor, determined in competitive ligand binding experiments, agrees closely with that described for the natural receptor expressed by human cells. Furthermore, the binding was stereospecific as shown by the displacement of the [3H]PK11195 binding by PK14067 (-Q1) and not by PK14068 (+Q1). Photolabeling experiments showed that transformed cells expressed a 18 kDa protein which was specifically labeled with [3H]PK14105. Altogether these results show that the cDNA transfected in yeast encodes a 18 kDa protein with the expected characteristics of the hPBR.
Riond
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J
Laboratoire de Biochimie des Protéines, Sanofi Elf-BioRecherches, Labège Innopole, France.
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D000345:Affinity Labels / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D007546:Isoquinolines / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011963:Receptors, GABA-A / Q000235:genetics* / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology / Q000648:ultrastructure; D014316:Tritium
D000345:Affinity Labels; D007546:Isoquinolines; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D014316:Tritium; D004247:DNA; C051238:PK 14105