Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Oct 5;293(40):15706-15714.
doi: 10.1074/jbc.RA118.004547. Epub 2018 Aug 22.

The erlin2 T65I mutation inhibits erlin1/2 complex-mediated inositol 1,4,5-trisphosphate receptor ubiquitination and phosphatidylinositol 3-phosphate binding

Forrest A Wright  1 , Caden G Bonzerato  1 , Danielle A Sliter  2 , Richard J H Wojcikiewicz  3
Affiliations

The erlin2 T65I mutation inhibits erlin1/2 complex-mediated inositol 1,4,5-trisphosphate receptor ubiquitination and phosphatidylinositol 3-phosphate binding

Forrest A Wright et al. J Biol Chem. .

Abstract

The erlin1/2 complex is a ∼2-MDa endoplasmic reticulum membrane-located ensemble of the ∼40-kDa type II membrane proteins erlin1 and erlin2. The best defined function of this complex is to mediate the ubiquitination of activated inositol 1,4,5-trisphosphate receptors (IP3Rs) and their subsequent degradation. However, it remains unclear how mutations of the erlin1/2 complex affect its cellular function and cause cellular dysfunction and diseases such as hereditary spastic paraplegia. Here, we used gene editing to ablate erlin1 or erlin2 expression to better define their individual roles in the cell and examined the functional effects of a spastic paraplegia-linked mutation to erlin2 (threonine to isoleucine at position 65; T65I). Our results revealed that erlin2 is the dominant player in mediating the interaction between the erlin1/2 complex and IP3Rs and that the T65I mutation dramatically inhibits this interaction and the ability of the erlin1/2 complex to promote IP3R ubiquitination and degradation. Remarkably, we also discovered that the erlin1/2 complex specifically binds to phosphatidylinositol 3-phosphate, that erlin2 binds this phospholipid much more strongly than does erlin1, that the binding is inhibited by T65I mutation of erlin2, and that multiple determinants within the erlin2 polypeptide comprise the phosphatidylinositol 3-phosphate-binding site. Overall, these results indicate that erlin2 is the primary mediator of the cellular roles of the erlin1/2 complex and that disease-linked mutations of erlin2 can affect both IP3R processing and lipid binding.

Keywords: endoplasmic reticulum (ER); endoplasmic-reticulum-associated protein degradation (ERAD); erlin1; erlin2; inositol trisphosphate receptor (InsP3R); mutation; phosphatidylinositol phosphatase; phospholipid signaling; spastic paraplegia; ubiquitylation (ubiquitination).

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
The effects of erlin1 and erlin2 deletion on assembly. A, lysates were made from control, E1KO, and E2KO αT3 cells and probed in immunoblots for ubiquitin, IP3R1, p97, Hrd1, gp78, erlin1, erlin2, and RNF170. B, nondenaturing PAGE of cell lysates probed with anti-erlin1, anti-erlin2, or anti-p97 as a loading control. C, anti-erlin1 or anti-erlin2 immunoprecipitates (IP) from control, E1KO, and E2KO αT3 cells probed for the proteins indicated. D, membrane preparations from control, E1KO, and E2KO αT3 cells (lanes 1–3) and immunoprecipitates from control αT3 cells, incubated either without (lanes 4–6) or with 100 nm GnRH for 5 min (lane 7), were probed in immunoblots for the proteins indicated, including erlin1 and erlin2 with anti-erlinpan (bottom panels). Note that several erlin-unrelated background bands were recognized by anti-erlinpan even in the membrane preparations (lanes 1–3) but that these were not present in immunopurified material (lanes 4–7).
Figure 2.
Figure 2.
The effects of erlin1 and erlin2 deletion on IP3R1 ERAD. A, cells were treated with 100 nm GnRH for 0–20 min, and anti-IP3R1 immunoprecipitates and input lysates were probed in immunoblots for the proteins indicated. B, quantitated IP3R1-associated ubiquitin immunoreactivity graphed as a percentage of control cell maximum (n = 4). C, cells were treated with 100 nm GnRH for 0, 30, or 60 min, and cell lysates were probed for the proteins indicated. D, quantitated IP3R1 immunoreactivity graphed as a percentage of t = 0 values (n = 4). E, [Ca2+]c in control, E1KO, and E2KO αT3 cells exposed to 100 nm GnRH (n ≥ 9). Error bars represent S.E.
Figure 3.
Figure 3.
Effects of the T65I mutation on erlin2 function. A, ClustalO alignment of the N-terminal regions of erlin1 and erlin2 from various species (Homo sapiens, Mus musculus, and Rattus norvegicus). Amino acid identity is indicated by asterisks; colons and periods indicate strongly and weakly conservative differences, respectively. B, lysates from control and E2KO αT3 cells and E2KO cell lines stably expressing E2HAWT or E2HAT65I (two of each; lanes 3–6) were probed in immunoblots for ubiquitin, IP3R1, p97, erlin1, erlin2, HA, and RNF170. C, nondenaturing PAGE of cell lysates probed with anti-erlin1, anti-erlin2, or anti-p97 as a loading control. D, anti-erlin2 or anti-HA immunoprecipitates (IP) from cell lines stably expressing E2HAWT or E2HAT65I probed for the proteins indicated. E and F, cells were treated with 100 nm GnRH for 0, 5, or 10 min; anti-IP3R1 immunoprecipitates were probed in immunoblots for the proteins indicated; and ubiquitin immunoreactivity was quantitated and graphed (n = 2). G and H, cells were treated with 100 nm GnRH for 0, 30, or 60 min; lysates were probed for the proteins indicated; and IP3R1 immunoreactivity was quantitated and graphed (n = 7). I, [Ca2+]c in cell lines stably expressing E2HAWT or E2HAT65I exposed to 100 nm GnRH (n = 11). Error bars represent S.E.
Figure 4.
Figure 4.
The erlin1/2 complex binds specifically to PI(3)P. A and B, erlin1/2 complex was immunopurified from control αT3 cells, and binding to lipid arrays was assessed by probing with anti-erlin2. TG, triglyceride; DAG, diacylglycerol; PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol, PI(x)Py, various PI phosphates; LPA, lysophosphatidic acid; LPC, lysophosphocholine; S1P, sphingosine 1-phosphate. C, erlin1/2 complex was immunopurified from control and RNF170KO αT3 cells (11), and binding of equal amounts of complex to lipid arrays was assessed by probing with anti-erlin2. In the repeat experiment with similar results, RNF170 was depleted using siRNA (14). D, mouse E2FLAGHAWT and E1FLAGHAWT were expressed transiently in αT3 E1/E2KO cells and immunopurified with anti-HA, and binding of equal amounts to lipid arrays was assessed with anti-FLAG. The lowest panel shows an immunoblot of the material added to the lipid arrays probed with anti-FLAG, demonstrating that equal amounts of E1FLAGHAWT and E2FLAGHAWT (migrating at ∼ 47 and 48 kDa, respectively) were used. E, WT and chimeric erlin1 and erlin2 constructs either FLAGHA-tagged or HA-tagged. The switches were made between erlin2 and erlin1 at amino acid positions 31 and 33 and positions 299 and 301. Amino acid homology (identity) between mouse erlin2 and -1 in the N-terminal (NT), intervening, and C-terminal (CT) regions is 33, 86, and 25%, respectively. F, the constructs shown in E were expressed transiently in αT3 E1/E2KO cells and immunopurified with anti-HA, and binding of equal amounts of protein to PI(3)P (800 or 400 pmol) was assessed with anti-FLAG (lanes 1–4), anti-erlin2 (lanes 5 and 6), or anti-erlin1 (lanes 7 and 8). The lower panels show immunoblots of the material added to the PI(3)P arrays probed with the corresponding antibodies, demonstrating that matched amounts of the exogenous proteins were used. The epitopes for anti-erlin1 and anti-erlin2 are located in the C-terminal regions of erlin1 and -2 (13).
Figure 5.
Figure 5.
Effects of the T65I mutation on lipid binding. A, erlin complex was immunopurified with anti-HA from αT3 E2KO cell lines stably expressing E2HAWT or E2HAT65I, and binding to lipid arrays was assessed by probing with anti-erlin2. The lowest panels show immunoblots of material added to the lipid arrays probed with anti-erlin2 and anti-erlin1, demonstrating that approximately equal amounts of E1HAWT and E2HAT65I were used and that erlin1 coimmunoprecipitates with E2HAWT and E2HAT65I. B, E2HAWT and E2HAT65I were expressed transiently in αT3 E1/E2KO cells and immunopurified with anti-HA, and binding of equal amounts to lipid arrays was assessed with anti-erlin2. The lowest panel shows an immunoblot of material added to the lipid arrays probed with anti-erlin2, demonstrating that approximately equal amounts of E2HAWT and E2HAT65I were used.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Foskett J. K., White C., Cheung K. H., and Mak D. O. (2007) Inositol trisphosphate receptor Ca2+ release channels. Physiol. Rev. 87, 593–658 10.1152/physrev.00035.2006 - DOI - PMC - PubMed
    1. Fan G., Baker M. L., Wang Z., Baker M. R., Sinyagovskiy P. A., Chiu W., Ludtke S. J., and Serysheva I. I. (2015) Gating machinery of InsP3R channels revealed by electron microscopy. Nature 527, 336–341 10.1038/nature15249 - DOI - PMC - PubMed
    1. Berridge M. J. (2016) The inositol trisphosphate/calcium signaling pathway in health and disease. Physiol. Rev. 96, 1261–1296 10.1152/physrev.00006.2016 - DOI - PubMed
    1. Wojcikiewicz R. J. (1995) Type I, II and III inositol 1,4,5 trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J. Biol. Chem. 270, 11678–11683 10.1074/jbc.270.19.11678 - DOI - PubMed
    1. Monkawa T., Miyawaki A., Sugiyama T., Yoneshima H., Yamamoto-Hino M., Furuichi T., Saruta T., Hasegawa M., and Mikoshiba K. (1995) Heterotetrameric complex formation of inositol 1,4,5-trisphosphate receptor subunits. J. Biol. Chem. 270, 14700–14704 10.1074/jbc.270.24.14700 - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources