Structure and Intermolecular Interactions of the Luminal Dimerization Domain of Human IRE1α

Abstract

Accumulation of unfolded proteins in the lumen of the endoplasmic reticulum activates a signal transduction cascade that culminates in the transcriptional induction of genes encoding adaptive functions. One proximal sensor for this unfolded protein response is the protein kinase/endoribonuclease IRE1α. IRE1α is a type-I transmembrane glycoprotein for which the N-terminal luminal domain (NLD) senses the accumulation of unfolded proteins. Previously we demonstrated that the NLD forms a stable ligand-independent dimer linked by disulfide bridges. In this report we have identified the cysteine residues responsible for intermolecular disulfide bonding. However, this covalent interaction was not required for dimerization and/or signaling, suggesting that a cryptic dimer interface exists in the NLD that is independent of covalent disulfide interactions. Limited proteolysis of the NLD revealed characteristic fragments, all retaining the same N-terminal sequences as full-length NLD. Biochemical and functional studies using NLD truncation mutants indicated that the dimerization domain of the NLD is confined to the conserved motifs at the N-terminal regions where putative hydrophobic interactions exist. In addition, the peptide binding domain of the endoplasmic reticulum protein chaperone BiP interacted with the N-terminal region within the NLD. Our findings suggest that the NLD has at least two distinct types of interactions mediating dimerization and function in signaling,i.e. covalent interactions involving disulfide bond formation and hydrophobic interactions, with the hydrophobic interaction being the driving force for dimerization. Accumulation of unfolded proteins in the lumen of the endoplasmic reticulum activates a signal transduction cascade that culminates in the transcriptional induction of genes encoding adaptive functions. One proximal sensor for this unfolded protein response is the protein kinase/endoribonuclease IRE1α. IRE1α is a type-I transmembrane glycoprotein for which the N-terminal luminal domain (NLD) senses the accumulation of unfolded proteins. Previously we demonstrated that the NLD forms a stable ligand-independent dimer linked by disulfide bridges. In this report we have identified the cysteine residues responsible for intermolecular disulfide bonding. However, this covalent interaction was not required for dimerization and/or signaling, suggesting that a cryptic dimer interface exists in the NLD that is independent of covalent disulfide interactions. Limited proteolysis of the NLD revealed characteristic fragments, all retaining the same N-terminal sequences as full-length NLD. Biochemical and functional studies using NLD truncation mutants indicated that the dimerization domain of the NLD is confined to the conserved motifs at the N-terminal regions where putative hydrophobic interactions exist. In addition, the peptide binding domain of the endoplasmic reticulum protein chaperone BiP interacted with the N-terminal region within the NLD. Our findings suggest that the NLD has at least two distinct types of interactions mediating dimerization and function in signaling,i.e. covalent interactions involving disulfide bond formation and hydrophobic interactions, with the hydrophobic interaction being the driving force for dimerization. endoplasmic reticulum N-terminal luminal domain an S-tagged NLD protein a His6-tagged NLD protein unfolded protein response immunoglobulin heavy chain-binding protein peptide-binding domain of BiP nitrilotriacetic acid Caenorhabditis elegans Homo sapiens The endoplasmic reticulum (ER)1 monitors the folding status of newly synthesized secretory and transmembrane proteins and ensures that only properly folded proteins transit to the Golgi compartment. In response to accumulation of unfolded proteins in the ER, cells activate an intracellular signal transduction pathway called the unfolded protein response (UPR). The yeast UPR is a linear pathway in which the protein kinase/endoribonuclease Ire1p signaling mediates transcriptional activation of UPR target genes. When Ire1p inSaccharomyces cerevisiae is activated, it functions as a site-specific endoribonuclease (RNase) that splices HAC1mRNA encoding Hac1p, a basic-leucine zipper-containing transcription factor. Hac1p binds to the UPR element in the promoter region and induces transcription of target genes, includingKAR2, encoding the polypeptide-binding protein chaperone BiP/GRP78 that is a classical hallmark of UPR activation (1Patil C. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-355Crossref PubMed Scopus (676) Google Scholar). IRE1, PERK, and ATF6 are the three proximal ER stress transducers that regulate UPR signaling in metazoan species. IRE1 (yeast scIre1p homolog) and PERK are two type-I ER transmembrane serine/threonine protein kinase receptors, and ATF6 is a type-II ER transmembrane-activating transcription factor. The mammalian UPR includes three adaptive cellular responses that are activated to cope with the accumulation of unfolded proteins in the ER; 1) transcriptional induction of ER chaperones and folding catalysts; 2) transcriptional activation of genes encoding components of ER-associated protein degradation; and 3) general translational attenuation (1Patil C. Walter P. Curr. Opin. Cell Biol. 2001; 13: 349-355Crossref PubMed Scopus (676) Google Scholar, 2Mori K. Cell. 2000; 101: 451-454Abstract Full Text Full Text PDF PubMed Scopus (788) Google Scholar, 3Kaufman R.J. Scheuner D. Schroder M. Shen X. Lee K. Liu C.Y. Arnold S.M. Nat. Rev. Mol. Cell Biol. 2002; 3: 411-421Crossref PubMed Scopus (502) Google Scholar). Recent studies at the organismal level showed that IRE1 plays critical roles in normal embryogenesis in early development, and PERK function is required for glucose homeostasis in vivo (4Urano F. Wang X. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Science. 2000; 287: 664-666Crossref PubMed Scopus (2327) Google Scholar, 5Harding H.P. Zeng H. Zhang Y. Jungries R. Chung P. Plesken H. Sabatini D.D. Ron D. Mol. Cell. 2001; 7: 1153-1163Abstract Full Text Full Text PDF PubMed Scopus (1008) Google Scholar, 6Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Mol. Cell. 2001; 7: 1165-1176Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar, 7Lee K. Tirasophon W. Shen X. Michalak M. Prywes R. Okada T. Yoshida H. Mori K. Kaufman R.J. Genes Dev. 2002; 16: 452-466Crossref PubMed Scopus (833) Google Scholar). IRE1 and PERK are structurally similar to serine/threonine protein kinase receptors. Dimerization and trans-autophosphorylation is a universal mechanism for activation of this class of cell surface receptors (8Lemmon M.A. Schlessinger J. Trends Biochem. Sci. 1994; 19: 459-463Abstract Full Text PDF PubMed Scopus (433) Google Scholar, 9Weiss A. Schlessinger J. Cell. 1998; 94: 277-280Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). However, the biochemical and structural basis for this transmembrane signaling in response to conditions of ER stress is not understood. A biochemical and structural analysis of the NLD should provide insights into this novel transmembrane signaling event, which will in turn establish the foundation to understand the physiological functions of IRE1 and PERK. IRE1 and PERK contain a remarkably large N-terminal domain that resides in the ER lumen. The N-terminal luminal domains (NLDs) of IRE1 and PERK sense the accumulation of unfolded proteins by a common mechanism and transmit the signal across the ER membrane to induce receptor activation (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). To provide an experimental system amenable to study the biochemical and structural basis for transmembrane signaling mediated by the NLD, the entire IRE1α luminal region was produced in a soluble form by transient DNA transfection in COS-1 cells, termed the NLD (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). The soluble NLD formed homodimers in a ligand-independent manner. In addition, the NLD interacted with the membrane-bound full-length IRE1α receptor and the ER chaperone BiP. Interestingly, the NLD homodimer was stabilized by disulfide bridges. In this report we analyzed the biochemical and structural properties of the purified NLD homodimer. The cysteine residues responsible for intermolecular disulfide bond formation were identified, and their requirement for UPR signaling was examined. The core domain required for dimerization was defined by limited proteolysis and analysis of truncation mutants. Our studies demonstrated that sequences required for dimerization and signaling are confined to conserved motifs at the N terminus. The existence of a cryptic dimer interface and the significance of multiple types of intermolecular interactions within the NLD dimer are discussed. Proteases Glu-C, Lys-C, and trypsin,N-α-tosyl-l-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride, and other protease inhibitors were purchased from Roche Applied Science. Mouse α-His5 antibody and Ni-NTA agarose were from Qiagen. Mouse α-NLD antibody was previously described (12Tirasophon W. Welihinda A.A. Kaufman R.J. Genes Dev. 1998; 12: 1812-1824Crossref PubMed Scopus (746) Google Scholar). and were purchased from was and was from other were from The mammalian of and were described previously (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). DNA to were and into the of the The in an and of the N terminus. the an at the and residues to the of the by a and for ER The NLD protein acid residues The are and NLD proteins have a at the terminus. To N-terminal truncation was to by to encoding were into this at and and and were described previously (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). To a of encoding was The two were and into and in In the entire yeast was with the entire NLD of that was functional for UPR induction (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). The DNA to the of and were and into at to the full-length NLD. by was to cysteine mutants in mammalian and in for the mutants are and the NLD of the mutants are and yeast the mutants are and mutants of yeast were and acid mutants of were and BiP BiP was described previously Kaufman R.J. J. PubMed Scopus Google Scholar). A encoding the entire BiP was using as a The sense within the region a The was that it an at the and a acid was into the of BiP and acid a an and an The DNA was into the and of to To an DNA from J.A. Kaufman R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google was into the of to the In a was from the domain of binding To for the peptide binding domain of we in which was to to is the in the BiP The sense in the and the of the the contain at the and to the of the and by an and an were into of the full-length and was to proteolysis with Lys-C, at the of Glu-C, as at the of in the of In the of it at and The for and was To the of Glu-C, was in the In a of protein was in a The was to at for and by protease inhibitors by the was in a of with protease to protein of The was for Lys-C, for Glu-C, and for The were analyzed by acid proteins were membrane by The was with and with protein were and with and membrane were to N-terminal acid were by the of chloromethyl phenylmethylsulfonyl and protein were in the and at the of in the at of COS-1 cells and cell protein binding using and Ni-NTA protein and protein were as described previously (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). cell were and was as described previously (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). of was previously S. J. B. J. 16: PubMed Scopus Google Scholar, S. R.J. Cell. Full Text PDF PubMed Scopus Google Scholar). The for to was from at the of S. J. B. J. 16: PubMed Scopus Google Scholar). studies demonstrated that NLD dimer is linked by disulfide (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). acid showed that are two conserved IRE1 proteins and three PERK proteins Interestingly, the of the conserved IRE1 and PERK have are three in and To the roles of in the formation of linked to were to and mutants of the NLD. COS-1 cells were with COS-1 cells were with and cell were in the of is a The of of and NLD Cell were to and analyzed by conditions all the NLD mutants were as NLD and all the mutants were as not dimerization to intermolecular disulfide To that form intermolecular disulfide with two three from to were The is a functional truncation of the NLD not protein in COS-1 a of was as and forms by a of the mutants and were as forms In the and at a to the form showed that at and the intermolecular disulfide bonding. The in the forms by and in homodimers with and in a of two species. the forms were not in the mutants. and are for formation of intermolecular disulfide that two disulfide homodimer. One and the other this we that a disulfide and in we the for NLD to form were with to agarose and not the were by their with with In all the transfection and NLD were to a similar level as by of not The two NLD and formation in the of intermolecular disulfide that NLD dimer formation is independent of disulfide bonding. However, of the two disulfide within the NLD the in the dimer suggesting that disulfide interaction within the NLD dimer to and/or should that the intermolecular interactions NLD and NLD and and and were with only a of proteins in the that dimer formation is the two in the contain When of the homodimer and the other the within the dimer is The basis and significance for this is not and structural Previously we an in S. cerevisiae to the function of the NLD (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). In this yeast cells a of the of the UPR element of this the of a of When the NLD of yeast Ire1p was by the NLD of IRE1α retaining the signal peptide transmembrane and the domain of the the Ire1p receptor induction in of the luminal domain of PERK UPR signaling (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). in the NLD region into Ire1p receptors, and their function by cysteine in and were to sense ER stress in an to NLD The cysteine and the Ire1p receptor function in of to in and in yeast and not receptor signaling not receptors in the and were functional in the demonstrated that intermolecular disulfide are not required for receptor dimerization UPR signaling ER Our suggest that the driving force for NLD dimerization is from interactions other intermolecular disulfide To the interactions responsible for dimerization and to understand the structural of the NLD, we limited proteolysis of the Limited proteolysis by to and two and and two and in and other that all from the NLD of the three and two were from a and at the N-terminal by The N-terminal acid sequences of all the were to that of the full-length NLD (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). should that are and residues that are the entire NLD that the NLD to were to analysis by The were and their we to and The that all the protease are at the of the NLD indicated that the of the is the N-terminal In addition, analysis showed that the three by were not the N-terminal region is to contain the core for dimerization. To the that the core for NLD dimerization is at the N we truncation mutants of the and were in COS-1 cells by transient DNA The was to a level with full-length NLD, and of and were and were and the of and not not the mechanism for this protein is not in this it is that the regions to the of the soluble NLD. mutants were to for their to form with full-length NLD. was into COS-1 cells with the truncation and formation was by an binding the were by their interaction with In the of protein was and not in cells, their dimerization with the NLD was not examined. demonstrated that the N-terminal region of the NLD form the N-terminal region of the NLD are motifs that are conserved IRE1 from species. motifs are conserved in the NLD of PERK (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). we the N-terminal region of the NLD is to signal the of of the N-terminal region of the NLD the entire luminal domain from Ire1p receptor to induce the UPR (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). is that of the N-terminal mutants the conserved To the requirement for NLD Ire1p receptors from the of were and their function in UPR activation was using the full-length of and were all to ER However, was in UPR suggesting that of and NLD function and Ire1p signaling are only residues the of IRE1 and PERK and were to for yeast to signal the of the conserved residues are to the motifs at the N of the NLD (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). To the requirement of the conserved were to in of the receptors were to induction in cells (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). of the conserved residues of and not NLD function in the UPR with not demonstrated that the three conserved motifs at the N are and required for dimerization and UPR is to that of the conserved residues within this region of the NLD is critical for NLD and/or BiP with the NLD. BiP has two the N-terminal domain with peptide and the The of domain from and of of of the mechanism of BiP function C. PubMed Scopus Google Scholar, X. X. A. Science. PubMed Scopus Google Scholar). The domain has a with the in a The of a by and the peptide is in an in the The domain as a to regulate the peptide binding and in with the BiP is a acid of the domain of the binding is of binding and immunoglobulin heavy this BiP has J. Biol. Chem. Full Text PDF PubMed Google Scholar). is the full-length the domain all the and The and and only BiP interacted with and by a protein binding using and with and not all the three and to and the interaction BiP and was the interaction was with that of The of and proteins to Ni-NTA their in cell The interaction and BiP was not were to protein in COS-1 cells, and interaction with BiP was not examined. experimental all the NLD from to not to to a level by analysis and Our showed that the of BiP with the N-terminal regions of the NLD. However, the which is in in to BiP To within the NLD, we an peptide binding motifs for identified by an peptide S. J. B. J. 16: PubMed Scopus Google Scholar). for of with in proteins by The binding of a hydrophobic core of and two The of this is similar to the hydrophobic BiP binding previously described S. R.J. Cell. Full Text PDF PubMed Scopus Google Scholar). is to BiP the general of the peptide-binding of proteins are In all of the that the in are conserved in BiP X. X. A. Science. PubMed Scopus Google Scholar). the of acid in the is a of the for a peptide to BiP. The the for a the the for BiP. A by this system the peptide has a to to a of binding to BiP S. J. B. J. 16: PubMed Scopus Google Scholar). In the NLD of IRE1α and PERK, motifs at the N and three other regions at the were to contain with to with this are to hydrophobic in not However, it to of are in a in the folded NLD. that BiP binding regions are IRE1 and PERK The conserved motifs at the N contain and the of are A of are the region of the NLD. into regions and their are not conserved In the sequences of the of IRE1 and PERK have a common of and multiple which provide the biochemical basis for a common mechanism of dimerization and In this study we analyzed the by which the luminal domain of IRE1α forms full-length IRE1α receptor and the NLD form homodimers intermolecular disulfide (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). However, mutants not dimer formation and UPR suggesting that a cryptic dimer interface exists that is independent of the covalent To the of the NLD, we limited The indicated that all the to are at the region of the NLD. forms of the NLD were functional in their to with full-length NLD and to induce the UPR in we showed that of the conserved motifs from the N UPR In for the interactions responsible for we demonstrated that the NLD dimer is to of interactions as hydrophobic and interactions. showed that the N-terminal formed and was the level and/or analysis of dimer status of which the three conserved motifs at the N was functional in ER stress to signal the that the NLD three conserved motifs at the N terminus. Previously we demonstrated that a basic-leucine dimerization Ire1p receptor activation and UPR induction (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). that dimerization and the receptor activation hydrophobic interactions within the is to that the conserved motifs in the of IRE1 and PERK are by an of hydrophobic and hydrophobic we that hydrophobic interactions within the N of the NLD, intermolecular disulfide are the driving force for dimer The ER is a and from in and the of of this the of chaperones and folding that are at normal Our that the NLD is not to conditions that activate the NLD dimerization not covalent disulfide interactions, function will not by as IRE1α is a NLD is not for dimer formation UPR the NLD to function conditions that and/or the of the purified receptor not as as for which the ER is the (11Liu C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). the function of the NLD is not to in in the the NLD acid of conserved residues and from the terminus. The only region within IRE1 and PERK that is is at the N of the NLD. A common regions is the hydrophobic of that provide a hydrophobic interface for dimerization. for the for two domains to to the same ER stress their the NLD suggesting that a large hydrophobic dimer interface of identified hydrophobic regions in the conserved N-terminal motifs Our that the NLD a of the dimer ensures that the NLD structural and function in the of conditions that the of protein conditions that protein folding in the that the NLD has as a ER stress for to conditions that induce protein in the BiP is a of the UPR and with all three ER stress IRE1, PERK, and conditions (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, C.Y. Wong H.N. Schauerte J.A. Kaufman R.J. J. Biol. Chem. 2002; 277: 18346-18356Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Zhang Y. Harding H.P. Ron D. Nat. Cell Biol. 2000; PubMed Scopus Google Scholar, K. Y. H. A. K. Biochem. 2000; PubMed Scopus Google Scholar, J. X. Prywes R. Dev. Cell. 2002; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). accumulation of unfolded proteins in the ER, stress are from BiP to is that unfolded proteins and BiP that it is for interaction with IRE1, PERK, and from ATF6 to the Golgi where it is to form A. Zhang Y. Harding H.P. Ron D. Nat. Cell Biol. 2000; PubMed Scopus Google Scholar). The of IRE1 and PERK from BiP their for In this BiP senses unfolded proteins in the ER lumen and activates the BiP is the of UPR the of interaction with the luminal domains of the stress transducers to distinct regions within the ATF6 luminal domain were identified that have for BiP J. X. Prywes R. Dev. Cell. 2002; 3: Full Text Full Text PDF PubMed Scopus Google Scholar). A region in was identified as a K. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In the we analyzed interaction of with BiP to that BiP with the N-terminal region of the NLD, with to that contain residues Interestingly, the region of and the region of are in region region in is not required for signaling the UPR in yeast (10Liu C.Y. Schröder M. Kaufman R.J. J. Biol. Chem. 2000; 275: 24881-24885Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). to this region in PERK is required to ER UPR Our studies demonstrated that the NLD binds to the within the peptide binding domain of BiP we that the NLD binds to the peptide binding in BiP. Our that are multiple in the NLD. and BiP binding to the N-terminal region of the Our studies suggest that the binds BiP with Interestingly, and analysis showed that the is not in the dimer with BiP. that hydrophobic motifs within the N to form a dimer and the is an for BiP To this we N-terminal mutants of the NLD, and showed that all interacted with BiP not sequences in were not required for the to regions the are the in vivo for NLD dimerization. that the BiP interaction with the N-terminal with the homodimer for BiP to regulate IRE1 dimerization. The of BiP the level of IRE1 a for BiP to with the NLD. The of the NLD provide the driving force for receptor dimerization and activation conditions of ER In we have identified cysteine residues responsible for intermolecular disulfide bond showed and that of the intermolecular disulfide not receptor dimerization function in UPR The structural of the NLD was defined by limited proteolysis and functional showed that the functional dimerization domain is at the N of the NLD. The of the NLD the of functional that are proteins should in for of IRE1 to the accumulation of unfolded proteins in the ER to receptor