Analysis of stimulatory effects of calmodulin on Purα binding to PUR.

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Purα, which binds to a single stranded (GGN) n sequence designated as a PUR, is discovered as a nuclear protein and is thought to be a regulator protein in replication and transcription processes.During the investigation of morphine tolerance and dependence, we found that 1) ssCRE (single stranded cyclic AMP response element) binding activity in the nuclear extract from morphine treated mouse brain and NG-108-15 cells, was decreased, 2) the ssCRE binding protein in the nuclear extract was identified as a Purα protein, and 3) the Purα binding acitivity is enhanced by an endogeneous activator, CaM.We have further investigated by using neuronal PC12 cells that 1) effects of morphine of CaM gene expression, and 2) effects of Purα on CRE-mediated gene expression.CaM expression estimated with RT-PCR was increase by morphine and DAMGO.It was a μ-opioid receptor mediated one and blocked by naloxone, antagonist. From northern blot analysis, we found that morphine stimulates CaM gene III express … More ion, but does't CaMI and CaMII.Purα expressed in PC12 cells suppresses somatostachin CRE-luciferase reporter gene activity and neurite outgrowth induced by forskolin. However, these inhibitory effects of Purα is disappeared, when a GGN consensus sequence for Purα is deleted from the somatostachin CRE.During the Purα expression study, we found that Purα localizes in cytoplasm mainly. Therefore, we examined EGFP- Purα distribution in the cell lines. EGFP- Purα expressed in PC12, NG-108-15, and COS-7 cells, localized in nuclear foci and mainly cytoplasm, a part of which seems to be a membrane bound form. Purα content in mouse tissues was also estimated. Among tested tissues, the brain shows the highest Purα content about 10-fold more compared to others. S3 and microsomal fractions from mouse brain exhibit higher Purα contents than nuclear fraction. When S3 fraction was incubated at 30℃ with Ca++ and subjected to western blot, a band of 42 kDa Purα was decreased and a novel 38 kDa Purα was concomitantly appeared with a time-dependent manner. It depends on Ca++ and a half-maximum concentration is about 0.3mM.We beliebe that Purα is a substrate for calpain in vitro. Less