Characterization of single-stranded cAMP response element binding protein (ssCRE-BP) from mouse cerebellum.
It has been speculated that opiate tolerance and dependence may occur at the level of gene expression. Our previous studies have shown that the binding activity of a nuclear factor (ssCRE-BP) to single-stranded CRE of somatostatin gene is altered by long-term treatment with morphine in the mouse cerebellum. ssCRE-BP was purified from the mouse cerebellum by a combination of chromatography on DNA affinity agarose and Mono Q HR. The native protein exhibited a molecular size of 110-150 kDa by gel filtration, and two polypeptides of about 35-40 kDa were observed on SDS-PAGE. The cloning and sequencing of a cDNA encoding ssCRE-BP showed that the protein possesses a glycine-rich domain and a glutamine-rich domain in the amino terminus and the carboxyl terminus, respectively. To investigate the function of ssCRE-BP in the brain, recombinant glutathione-S-transferase (GST) fusion proteins containing ssCRE-BP were expressed in bacterial systems. Rabbit anti-ssCRE-BP antibodies were raised against a GST-ssCRE-BP fusion protein. Using the antibodies in western blot analysis, a polypeptide of approximately 66 kDa was detected in the brain. These findings indicate that ssCRE-BP is involved in opiate tolerance and dependence.
Osugi
T
T
Department of Pharmacology I, Osaka University School of Medicine, Japan.
Ding
Y
Y
Miki
N
N
D000818:Animals; D002531:Cerebellum / Q000378:metabolism*; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D004361:Drug Tolerance / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics
D017362:Cyclic AMP Response Element-Binding Protein; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase