RNF170 protein, an endoplasmic reticulum membrane ubiquitin ligase, mediates inositol 1,4,5-trisphosphate receptor ubiquitination and degradation

Abstract

Inositol 1,4,5-trisphosphate (IP(3)) receptors are endoplasmic reticulum membrane calcium channels that, upon activation, are degraded via the ubiquitin-proteasome pathway. While searching for novel mediators of IP(3) receptor processing, we discovered that RNF170, an uncharacterized RING domain-containing protein, associates rapidly with activated IP(3) receptors. RNF170 is predicted to have three membrane-spanning helices, is localized to the ER membrane, and possesses ubiquitin ligase activity. Depletion of endogenous RNF170 by RNA interference inhibited stimulus-induced IP(3) receptor ubiquitination, and degradation and overexpression of a catalytically inactive RNF170 mutant suppressed stimulus-induced IP(3) receptor processing. A substantial proportion of RNF170 is constitutively associated with the erlin1/2 (SPFH1/2) complex, which has been shown previously to bind to IP(3) receptors immediately after their activation. Depletion of RNF170 did not affect the binding of the erlin1/2 complex to stimulated IP(3) receptors, whereas erlin1/2 complex depletion inhibited RNF170 binding. These results suggest a model in which the erlin1/2 complex recruits RNF170 to activated IP(3) receptors where it mediates IP(3) receptor ubiquitination. Thus, RNF170 plays an essential role in IP(3) receptor processing via the ubiquitin-proteasome pathway.

FIGURE 1.

RNF170 is a RING domain-containing protein that associates with activated IP₃ receptors. A, αT3-1 cells were incubated without or with 100 nm GnRH for 7 min and harvested with 1% CHAPS lysis buffer, and then anti-IP₃R1 immunoprecipitates were subjected to SDS-PAGE and silver staining. The 20- to 25-kDa region of a representative gel is shown, and the 21.5-kDa band marked with an arrow was identified as RNF170. B, a multiple-sequence alignment of RNF170 homologs from selected vertebrates using ClustalW. Amino acid identity among all species shown is indicated with asterisks, whereas identity between at least rodents and humans is indicated with dots. Predicted are three putative transmembrane domains (yellow, TMHMM Server v 2.0), a RING-HC domain (pink, with zinc-coordinating residues in orange (9)), ER membrane localization (PSORTb v 3.0), and a molecular weight of ∼30 kDa (ExPASy Compute pI/MW). The peptide sequence boxed was used to raise anti-RNF170, and the individual residues marked with arrowheads were mutated in *RNF170^FLAG. C, topological model of RNF170 based on predictions of three transmembrane domains and the RING domain facing the cytosol. D, HeLa cells were transfected with either vector (lane 1) or with a construct containing the RNF170 encoding sequence isolated from αT3-1 cells (lane 2), and RNF170 expression was assessed in immunoblots (IB) with anti-RNF170. E, αT3-1 cells were treated for the indicated times with 100 nm GnRH and harvested with 1% CHAPS lysis buffer, and then anti-IP₃R1 immunoprecipitates were probed for ubiquitin, IP₃R1, p97, erlin2, and RNF170. F, the kinetics of GnRH-induced IP₃R1 ubiquitination and coimmunoprecipitation of p97, erlin2, and RNF170 were quantitated and graphed. G, SH-SY5Y cells were treated for the indicated times with 1 mm carbachol and harvested with 1% CHAPS lysis buffer, and then anti-IP₃R1 immunoprecipitates were probed for ubiquitin, IP₃R1, erlin2, and RNF170. In E and G, polyubiquitinated IP₃R1, unmodified IP₃R1, p97, erlin2, and RNF170 migrated at 275–380 kDa, 260 kDa, 97 kDa, 43 kDa, and 21.5 kDa, respectively.

FIGURE 2.

RNF170 possesses ubiquitin ligase activity. RNF170^FLAG (lanes 1–4) and *RNF170^FLAG (lane 5), immunopurified from transfected HeLa cells, were incubated with E1 (UBE1), E2 (UbcH5b) and HA-ubiquitin as indicated for 30 min at 30 °C. Samples were then subjected to SDS-PAGE and were probed in immunoblots (IB) with anti-HA epitope to assess ubiquitination (upper panel) or anti-FLAG epitope to assess RNF170^FLAG/*RNF170^FLAG levels (lower panel).

FIGURE 3.

RNF170 is an integral ER membrane protein. A, HeLa cells were cotransfected with cDNAs encoding the ER marker DsRed2-ER and wild-type RNF170^FLAG and were examined by confocal microscopy. B, HeLa cells transfected with cDNA encoding RNF170^FLAG were harvested in hypotonic buffer, sonicated, and fractionated by centrifugation (lanes 1 and 2). Pellets were then resuspended in hypotonic buffer, 0.1% SDS, or 0.1 m Na₂CO₃, and were recentrifuged (lanes 3–8). The supernatants (S) and pellets (P) from each centrifugation were then probed in immunoblots for the indicated proteins.

FIGURE 4.

Most RNF170 is constitutively associated with the erlin1/2 complex. Cells were harvested with 1% CHAPS lysis buffer. A, anti-erlin2 immunoprecipitates from αT3-1 cells (lane 3) plus controls (lanes 1 and 2) were subjected to SDS-PAGE and silver staining. The 15- to 50-kDa region of a representative gel is shown, and the 21.5-kDa band marked with an arrow was identified by mass spectrometry as RNF170. B, anti-erlin2 or anti-RNF170 immunoprecipitates from αT3-1 cells (lanes 3 and 6) plus controls (lanes 1, 2, 4 and 5) were subjected to SDS-PAGE and probed in immunoblots (IB) with anti-RNF170 or anti-erlin2 as indicated. The 15- to 50-kDa is shown, and the migration positions of RNF170 and erlin2 are indicated with arrows. C, αT3-1 cell lysates were incubated without antibody (lane 1) or with anti-erlin2 (lane 2), and the immunodepleted lysates were subjected to SDS-PAGE and probed in immunoblots with anti-erlin2 or with anti-RNF170. D, HeLa cells were transfected with vectors encoding RNF170^FLAG and/or erlin1^HA/erlin2^HA; cell lysates were incubated without antibody (lanes 1, 3, and 6), with anti-FLAG (F, lanes 2, 5, and 8), or with anti-HA (H, lanes 3, 6, and 9), and immunoprecipitates were probed in immunoblots with anti-HA or anti-FLAG.

FIGURE 5.

Depletion of endogenous RNF170 inhibits IP₃ receptor processing. A and B, αT3-1 cells were electroporated to express vectors encoding either Random or RNF170 siRNAs, and effects on 100 nm GnRH-induced IP₃ receptor polyubiquitination in anti-IP₃R1 immunoprecipitates (A) or down-regulation in cell lysates (B) were assessed. Erlin2 served as a loading control. The histograms and graph depict combined quantitated data. C and D, mHeLa cells were transfected to express either Random or RNF170 oligonucleotide siRNAs or vectors encoding erlin2 siRNA (6), and effects on 10 μm carbachol-induced IP₃ receptor ubiquitination in immunoprecipitates (C) or down-regulation in cell lysates (D) were assessed. Erlin2 and p97 served as loading controls. The histograms and graph depict combined quantitated data. †, p < 0.05 comparing polyubiquitination in Random versus RNF170 or erlin2 siRNA-expressing cells; *, p < 0.05 comparing IP₃R1 immunoreactivity in unstimulated Random versus RNF170 siRNA expressing cells; #, p < 0.05 comparing IP₃R1 down-regulation at each time point in Random versus RNF170 siRNA expressing cells. Polyubiquitinated IP₃R1, unmodified IP₃R1, p97, erlin2, and RNF170 migrated at 275–380 kDa, 260 kDa, 97 kDa, 43 kDa, and 21.5 kDa, respectively.

FIGURE 6.

Overexpression of RNF170^FLAG or *RNF170^FLAG modulates IP₃ receptor down-regulation. Rat1 cell clones stably expressing either RNF170 ^FLAG, *RNF170^FLAG, or empty vector were incubated without or with 10 nm ET1 for 1 h, and immunoreactivity of IP₃R1, erlin2 (loading control), or FLAG epitope (to monitor RNF170 ^FLAG or *RNF170^FLAG expression) was assessed. Data shown are representative of at least six vector-, RNF170 ^FLAG-, or *RNF170^FLAG-expressing clones. *, p < 0.05 comparing IP₃R1 immunoreactivity without or with ET1 for each clone.