Identification of addicsin/GTRAP3-18 as a chronic morphine-augmented gene in amygdala

Abstract

Using subtractive cloning, we identified a 1.4 kb mRNA that was ubiquitously expressed in various tissues; this mRNA was highly up-regulated in amygdala nuclei in mice when morphine was repeatedly administered but not when an opiate-receptor antagonist was co-administered. The mRNA encodes a 23 kDa protein, designated 'addicsin'. This contains two putative PKC-phosphorylation motifs and several hydrophobic regions, and was recovered in a soluble protein fraction of brain lysate. Its primary structure showed 98% identity with that of rat glutamate-transporter-associated protein 3-18 (GTRAP3-18), a putative modulator of neural glutamate-transporter EAAC1. Up-regulation of addicsin expression by morphine may affect glutamate uptake in the amygdala, causing mice to develop morphine tolerance and dependence.

Modulation of EAAC1-Mediated Glutamate Uptake by Addicsin

“…Mouse addicsin is 98% identical to rat GTRAP3-18 and 95% similar to human JWA (Butchbach et al, 2002;Ikemoto et al, 2002). Moreover, addicsin has two putative PKC phosphorylation motifs (amino acids 18-20 and 138-140) as well as two putative cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II phosphorylation motifs (amino acids 27-31 and 35-39) (Butchbach et al, 2002;Ikemoto et al, 2002) (Fig. 1).…”

“…Addicsin, GTRAP3-18, and JWA have been independently identified by several research groups (Ikemoto et al, 2002;Lin et al, 2001;Zhou et al, GeneBank, AF070523, unpublished observations). We first identified addicsin as a novel mRNA encoding a 22-kDa hydrophobic protein that is highly expressed in the basomedial nucleus of the mouse amygdala following repeated morphine administration (Ikemoto et al, 2002). Meanwhile, GTRAP3-18 cDNA was identified as encoding an EAAC1 binding protein by yeast twohybrid screening of a rat brain cDNA library using the C-terminal intracellular domain of EAAC1 as bait (Lin et al, 2001).…”

“…The addicsin cDNA is approximately 1.4 kbp and consists of a 564-bp single open reading frame (Ikemoto et al, 2002). The addicsin gene contains three exons separated by two introns, and the sequence is highly conserved among vertebrates (Butchbach et al, 2002).…”

“…The addicsin gene contains three exons separated by two introns, and the sequence is highly conserved among vertebrates (Butchbach et al, 2002). Furthermore, addicsin is located on mouse chromosome 6, a location corresponding to human chromosome 3p (Butchbach et al, 2002;Ikemoto et al, 2002).…”

“…Mouse addicsin is a 22-kDa protein of 188 amino acids with putative transmembrane segments (Butchbach et al, 2002;Ikemoto et al, 2002). Mouse addicsin is 98% identical to rat GTRAP3-18 and 95% similar to human JWA (Butchbach et al, 2002;Ikemoto et al, 2002).…”