A putative G protein-coupled transmembrane polypeptide, named Pael receptor, was identified as an interacting protein with Parkin, a gene product responsible for autosomal recessive juvenile Parkinsonism (AR-JP). When overexpressed in cells, this receptor tends to become unfolded, insoluble, and ubiquitinated in vivo. The insoluble Pael receptor leads to unfolded protein-induced cell death. Parkin specifically ubiquitinates this receptor in the presence of ubiquitin-conjugating enzymes resident in the endoplasmic reticulum and promotes the degradation of insoluble Pael receptor, resulting in suppression of the cell death induced by Pael receptor overexpression. Moreover, the insoluble form of Pael receptor accumulates in the brains of AR-JP patients. Here, we show that the unfolded Pael receptor is a substrate of Parkin, the accumulation of which may cause selective neuronal death in AR-JP.

Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations in the parkin gene. Parkin protein is characterized by a ubiquitin-like domain at its NH 2 -terminus and two RING finger motifs and an IBR (in between RING fingers) at its COOH terminus (RING-IBR-RING).Here, we show that Parkin is a RING-type E3 ubiquitin-protein ligase which binds to E2 ubiquitinconjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif. Moreover, we found that unfolded protein stress induces up-regulation of both the mRNA and protein level of Parkin. Furthermore, overexpression of Parkin, but not a set of mutants without the E3 activity, specifically suppressed unfolded protein stress-induced cell death. These findings demonstrate that Parkin is an E3 enzyme and suggest that it is involved in the ubiquitination pathway for misfolded proteins derived from endoplasmic reticulum and contributes to protection from neurotoxicity induced by unfolded protein stresses. AR-JP1 is one of the most common forms of the familial Parkinson's diseases and is characterized by juvenile onset, a recessive mode of inheritance and selective loss of the dopaminergic neurons in the substantia nigra without Lewy bodies (intraneuronal accumulations of aggregated proteins) (1). In 1998, the gene responsible for AR-JP was identified and designated parkin (2).Recently, several proteins with RING finger motifs have been identified as E3 ubiquitin ligases, which are responsible for substrate recognition and for promotion of substrate ubiquitination in conjunction with ubiquitin-conjugating enzymes (E2s) (3-10). In RING-type E3s, RING finger motifs serve as recruiting motifs for specific E2 ubiquitin-conjugating enzymes. These facts suggest that Parkin, which contains a RING-IBR-RING motif, is a new member of E3 ubiquitin ligases.On the other hand, the fact that the deletion of the parkin gene causes the neuronal death of the substantia nigra in AR-JP patients suggests the cell-protective function of Parkin. Given that Parkin is involved in both the ubiquitin-proteasome pathway and cell death protection, an interesting possibility is that Parkin may inhibit a certain type of cell death through proteasome-mediated protein degradation. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) would constitute an unfolded protein stress or ER stress, which may lead to cell death. Normal cells deal with unfolded protein stress by several mechanisms, including transcriptional induction of genes that facilitate protein folding or removal of misfolded proteins and degradation that is dependent on the cytosolic ubiquitin-proteasome pathway (11).Here, we provide evidence that Parkin is a RING-type E3 ubiquitin-protein ligase. Moreover, we show that Parkin is up-regulated in response to unfolded protein stress and suppresses unfolded protein stress-induced cell death via its E3 activity, suggesting that the physiological role of Parkin involves dealing with unfolded protein stress. EXPERIMENTAL PROCEDUREScDNAs and Antibodies (Abs)...

Unfolded Pael receptor (Pael-R) is a substrate of the E3 ubiquitin ligase Parkin. Accumulation of Pael-R in the endoplasmic reticulum (ER) of dopaminergic neurons induces ER stress leading to neurodegeneration. Here, we show that CHIP, Hsp70, Parkin, and Pael-R formed a complex in vitro and in vivo. The amount of CHIP in the complex was increased during ER stress. CHIP promoted the dissociation of Hsp70 from Parkin and Pael-R, thus facilitating Parkin-mediated Pael-R ubiquitination. Moreover, CHIP enhanced Parkin-mediated in vitro ubiquitination of Pael-R in the absence of Hsp70. Furthermore, CHIP enhanced the ability of Parkin to inhibit cell death induced by Pael-R. Taken together, these results indicate that CHIP is a mammalian E4-like molecule that positively regulates Parkin E3 activity.

Selective loss of dopaminergic neurons is the final common pathway in Parkinson's disease. Expression of Parkin associated endothelin-receptor like receptor (Pael-R) in mouse brain was achieved by injecting adenoviral vectors carrying a modified neuron-specific promoter and Cre recombinase into the striatum. Upregulation of Pael-R in the substantia nigra pars compacta of mice by retrograde infection induced endoplasmic reticulum (ER) stress leads to death of dopaminergic neurons. The role of ER stress in dopaminergic neuronal vulnerability was highlighted by their decreased survival in mice deficient in the ubiquitin-protein ligase Parkin and the ER chaperone ORP150 (150 kDa oxygen-regulated protein). Dopamine-related toxicity was also a key factor, as a dopamine synthesis inhibitor blocked neuronal death in parkin null mice. These data suggest a model in which ER- and dopamine-related stress are major contributors to decreased viability of dopaminergic neurons in a setting relevant to Parkinson's disease.

Parkinson's disease (PD)1 is a movement disorder characterized pathologically as a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Most cases of PD are sporadic, and the etiology of common PD remains unknown. However, recent identification of gene mutations in familial cases of PD has advanced our understanding of the molecular mechanisms behind the neurodegeneration associated with this disease.