Cellular Signalling Vol. 4, No. 5, pp. 465--470, 1992. Printed in Great Britain.
0898-6568/92 $5.00 + 0,00 © 1992 Pergamon Press Ltd
MINI REVIEW
SIGNALLING ACROSS THE ENDOPLASMIC RETICULUM MEMBRANE: POTENTIAL MECHANISMS BRENDAN D. PRICE Stress Protein Group (.IF 205), Dana=Farber Cancer Institute, Harvard Medical School, 44 Binney St, Boston, MA 02115, U.S.A.
(Received 30 April 1992; and accepted 10 May 1992) AImraet--The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for the synthesis, assembly and post-translational modification of proteins destined for the lysosomes, Golgi and for secretion. The processes which occur in the lumen of the ER are vital to the correct functioning of the cell, and mechanisms must exist to enable the cell to monitor events within the lumen of the ER. How the cell is able to do this is not known, but it would apparently require the passage of signals from the lumen of the ER to the cytosoi, from where signals can be sent to, for example, the nucleus to effect changes in transcription. Here, it is suggested that the membrane of the ER may contain the components (i.e. receptors, kinases, etc.) required for transmembrane signalling in much the same way as the plasma membrane does. This hypothesis will be discussed in relation to known ER proteins which might act as signalling proteins.
Key words: Endoplasmic reticulum, GRPs, signalling, phosphorylation, G-proteins, protein transport, protein kinases. Tim ENDOPLAS~C RETICULUM (ER) is a membrane-bound structure consisting of cisternae that are spread throughout the cell. Proteins which are targeted to the lysosomes, Golgi apparatus, plasma membrane or for secretion, are all initially processed through the E R [1]. When proteins are translated on the m R N A - r i b o s o m e complex in the cytosol, the emergence o f a signal peptide at the N-terminal of the nascent protein directs the m R N A - r i b o some complex to attach to the ER via the signal peptide [2]. Protein synthesis then continues across the ER membrane and the protein emerges into the lumen o f the ER, or, for some membrane proteins (e.g. growth factor recep-
Abbreviations: BAPTA-- 1,2-bis(2-aminophenoxy)ethanN,N,N',N'-tetraacetic acid; EPO-R--erythropoietin receptor; ER--endoplasmic reticulum; GRPs---glucose-regnlated proteins; IL-3--interleukin-3; KDEL--Lys-Asp-GIu-Leu, ER retention signal; KKKK--Lys-Lys-Lys-Lys, nuclear localization sequence; NRK ceils---normalrat kidney cells; PDGF--platelet-derived growth factor; PTP-IB--protein tyrosine phosphatase IB. 465
tors), part of the protein may be inserted into the ER membrane. Once synthesis has been completed, a number of post-translational modifications may take place within the lumen o f the ER. These can include N-linked glycosylation, fatty acylation and assembly o f protein sub-units via disulphide bond formation into oligomeric structures [1]. Processed proteins are transported from the ER to the Golgi apparatus by the budding of transport vesicles from the face of the ER and the fusion o f these with the Golgi, where further processing may occur. Mature proteins are then directed to the lysosomes, plasma membrane or are targeted for secretion [1-3]. The biosynthetic and transport processes of the ER are highly complex and involve a wide range of biosynthetic events. The assembly of proteins within the lumen of the ER, and the transfer of these via the Golgi to the appropriate compartment must be tightly controlled to prevent, for instance, the delivery of lysosomal enzymes to the wrong organelle. Maintaining the structural integrity of the ER
466
B.D. PRICE
requires the continuous synthesis and incorporation of specific lipids and structural proteins into the membrane of the ER. The biosynthetic functions require various glycosyltransferases to carry out glycosylation, enzymes involved in fatty acylation, and a number of proteins involved in protein folding (see below), as well as controlling the levels of ATP, GTP, Ca 2+ (a major component of the ER) and cofactors required in the modification of secretory proteins. In addition, during mitosis, the secretory mechanism is blocked and the ER breaks down into small vesicles in preparation for cell division, reforming only after division has completed [24]. All of these processes must be closely monitored by the cell to ensure that the biochemical events occurring inside the ER proceed in an orderly and precise fashion. However, the ER is a membrane-bound organelle which is distinct from the cytosol. How, then, do the ER and cytosol (and nucleus) communicate with each other to control both the structure of the ER and the processing and secretion of various proteins? A family of 'stress proteins' (the glucose-regulated proteins or GRPs), which are found only in the ER, provide a good example of the requirement for communication across the ER membrane. If the folding and maturation of proteins within the lumen of the ER is disrupted, the cell can sense this and responds by increasing the transcription of the GRP genes. This process requires the transduction of a signal generated in the lumen of the ER to the cytosol and subsequently to the nucleus to alter GRP transcription. The GRPs (GRP78 and 94 [4]), and other proteins, including protein disulphide isomerase [5], ERp72 [6], calreticulin and many others [13] are resident proteins in the ER. GRP78 and 94 are major components of the ER. GRP78 functions as a molecular ehaperonin protein similar to hsp70 (with which it has 68% homology [7]) and has been found associated with immunogiobulin heavy chains [8], unfolded or incompletely glycosylated proteins [9] and may participate in the import and folding of nascent proteins targeted to the ER and Golgi systems
[10]. GRP78's function in the ER may be to bind to nascent proteins as they are delivered into the lumen and either help them to fold or to aid in the assembly of oligomeric proteins [3, 7, 10]. GRP78 has an intrinsic ATPase activity which may be utilized to modulate the binding and release of substrate proteins [7, 10]. Both GRP78 and 94 contain C-terminal Lys-Asp-Glu-Leu (KDEL) sequences which mark these proteins for retention within the ER [10]. Normally, the levels of the GRPs in the ER remain constant, but, under some conditions, the expression of these proteins is greatly increased. Cells exposed to low oxygen concentrations (hypoxia, e.g. poorly vascularized cells in the centre of tumours or during ischaemia), or low glucose levels (hypoglycemia), respond by greatly increasing the transcription of the GRPs, resulting in up to three- to five-fold increases in the levels of GRP78 and 94 in the ER [4, 11]. Increased GRP synthesis can also be induced by treating cells with A23187 (a Ca 2÷ ionophore which discharges ER Ca2÷), tunicamycin (which blocks N-linked glycosylation in the ER), 2-deoxyglucose (an inhibitor of glucose uptake and glycosylation), brefeldin A (which inhibits vesicle transport between the ER and the Golgi) and thapsigargin (an inhibitor of the ER Ca2+-ATPase [4, 12, 13]). All of these treatments result in the accumulation within the ER of underglycosylated or realfolded proteins which cannot be transported out of the ER to the Golgi apparatus. The cell responds to the accumulation of aberrant proteins by increasing the synthesis of GRP78, primarily through increased transcription of the GRP genes [12]. It is thought that the extra GRP78 and 94 are required to prevent aberrant proteins from precipitating in the ER and that GRPs carry out this function by binding to these accumulated proteins and maintaining their solubility [13]. In the presence of large quantities of defective proteins, GRP78 rapidly becomes bound up with aberrant proteins, and free (unbound) GRP levels fall dramatically [9, 16]. However, the mechanism by which the cell monitors the level of GRPs in the ER and the
Signal transductionfrom the ER lumento the cytosol nature of the signal it sends to the nucleus to increase their transcription is largely unknown. Whatever the signal transduction process, it requires a sensing mechanism in the ER to monitor the GRP levels, a means of communicating the signal across the ER membrane and the ability to pass this signal on to the nucleus to allow changes in transcription.
How do cells sense the level of GRP78? Recent data have shown that the free (unbound) GRP78 in the ER is both phosphorylated (on Ser/Thr residues) and ADP-ribosylated, but that the GRP78 bound to proteins contains neither modification [14, 15]. Further, when cells are induced to produce more GRP78 by starving the cells of glucose, the newly synthesized GRP78 is found in association with non-transported proteins and contains no modifications [16]. However, the amount of free GRP78 in glucose-starved cells is the same as in glucose-fed cells [15, 16]. This suggests that the levels of GRP78 are regulated to maintain the amount of free GRP78 at a constant level. When ER function is disrupted and non-transported proteins become trapped within the lumen, GRP78 binds to the accumulated proteins [8, 9] and is dephosphorylated and de-ribosylated. Normally, the association of GRP78 with target proteins would be transient, and GRP78 would dissociate after correct folding or glycosylation of the target protein, but when these processes are disrupted, GRP78 remains bound to the target protein for an extended period of time [16]. The decrease in free GRP78, monitored by the dephosphorylation and/or de-ribosylation of GRP78 then triggers an increase in transcription of the GRP genes. So it is likely that the cell monitors the levels of free, modified GRP78 and increases transcription in response to changes in post-translational modification. Whilst this may be the sensing mechanism within the ER, and suggests the presence of a GRP78 protein kinase and phosphatase, as well as ADP ribosylation factors (as
467
suggested in Ref. 16), it does not e~alain how the message is passed out of the ER.
Is Ca 2+ involved in signalling from the ER to the cytosol? The ER is a major cellular store for Ca 2+ and contains two important transmembrane proteins involved in regulating cellular Ca 2+ - - the inositol phosphate receptor [17] and the ER Ca2+-ATPase [18]. Both A23187 (a Ca 2+ ionophore) and thapsigargin (an inhibitor of the ER Ca2+-ATPase [18]) rapidly increase intracellular Ca 2+ levels by discharging ER Ca 2+ and both compounds can induce GRP transcription [13]. Elevated Ca 2+ might therefore be responsible for increasing transcription of the GRP genes in the same way that it affects the transcription of the closely related heat shock genes [19]. However, buffering the rise in intracellular Ca 2+ (with BAPTA or EGTA) does not affect the induction of the GRPs [20]. In addition, induction of GRP transcription by tunicamycin, 2deoxyglucose, brefeldin A or by the introduction of transport defective proteins does not alter the level of intracellular Ca 2+ [4, 12, 13, 20]. Changes in Ca 2+ are therefore not important for the induction of GRPs.
Are protein kinases/phosphatases involved in signalling? Recent work has identified a non-receptor, transmembrane tyrosine kinase, the Ltk kinase, which is localized to the ER and may play a role in signal transduction across the ER membrane [21]. The N-terminal domain contains a cysteine-rich region which faces the luminal side of the ER. The Ltk kinase activity is greatly increased by diamide, a thioloxidizing agent, resulting in the formation of active disulphide-linked Ltk dimers. This suggests that the Ltk kinase may be involved in signalling under conditions in which the redox state of the ER is altered, such as during hypoxia or glucose starvation (as noted in Ref. 21). Hypoxia and glucose starvation also result
468
B . D . PRICE
in elevat~n of GRPs. Given the properties displayed by the Ltk kinase, it is possible that it (or related kinases) may be involved in the regulation of GRP transcription and that the tyrosine kinase activity may result in the phosphorylation of target proteins in the cytosol which may be positive regulators of GRP transcription. However, since the Ltk kinase is expressed only in cells of lymphoid origin this should be treated with some caution. In addition to the Ltk kinase, a cAMP-dependent protein kinase has also been shown to be associated with the external face of the ER, and, on activation, may be released to the cytosol whilst the inhibitory subunits remain attached to the ER [25]. Protein tyrosine phosphatase 1B (PTP-1B), one of the major tyrosine phosphatases in cells, is associated with the ER membrane. PTP-1B is anchored to the cytosolic face of the ER by a 35-amino acid sequence and the active site is directed towards the cytosol [22]. The function of this ER localization is not clear, but it may be involved in regulating the normal function and transport properties of the ER. It has been speculated that PTP-1B may be required to ensure that tyrosine kinase receptors passing through the ER on their way to the plasma membrane are maintained in an inactive (dephosphorylated) form [22]. The 35-amino acid C-terminal fragment that attaches PTP-1B to the ER could be in contact with the interior of the ER and may participate in the passage of signals out of the ER, for instance by interacting with luminal ER proteins. PTP-1B activity may be controlled through phosphorylation (since the PTP-IB has been reported to be phosphorylated on serine residues [22]). The turning on or off of the PTP-1B activity may either terminate or enhance signals generated from the ER. Further evidence that phosphorylation is important in the induction of GRP transcription comes from the observation that genistein, a tyrosine kinase inhibitor, blocks the stimulation of GRP transcription, whereas okadaic acid, a phosphatase inhibitor, greatly enhances the stimulation of GRP transcription, indirectly
supporting a role for protein kinases in activation of GRP transcription [13]. G-proteins present on the E R
As well as protein kinases, the ER and Golgi contain a large number of trimeric and ras-like G-proteins which are involved in vesicle transport, fusion, targeting and release of secretory proteins (reviewed in Ref. 26). Two of these proteins are worthy of further investigation. An ER-specific trimeric G-protein has been detected on the outer face of the ER membrane which was released to the cytoplasm when GTP~S was added and could be ADP-ribosylated by pertussis toxin [27]. The second protein is yptl, a ras-related G-protein of 21,000 mol. wt which is required for vesicle transport between the Golgi and the ER [28]. A number of reports have shown that transport between the ER and the Golgi involves a trimeric G-protein whose function can be inhibited by GTPyS and AIFa- (which blocks trimeric G-proteins but not ras-like proteins [29]). Interestingly, cells treated with AIF4- also show enhanced synthesis of GRPs, although this effect might be due to inhibition of protein exit from the ER, causing proteins to accumulate in the lumen, rather than an effect on a G-protein involved in signal transduction [13]. So although the ER has a number of associated G-proteins [26-28], these seem to function mostly in control of vesicle budding and fusion rather than in signal transduction across the ER membrane. Further characterization of these proteins will be required, although the finding that at least one of these G-proteins [27] can bind reversibly to the ER may indicate a cytosolic function for ER G-proteins as well. What other processes require a trans-ER signalling system?
There are a number of other cellular processes apart from GRP transcription which require the transmission of a signal from the ER to the eytosoi. The budding of transport vesicles from the ER and their fusion to the
Signal transduction from the ER lumen to the cytosol Golgl apparatus involves the binding and cyclical release of a number of coat proteins (COPS) and G-proteins (including the ADPribosylation factor, ARF) on the cytosolic side of the ER (reviewed in Ref. 34). This process may be regulated by the presence of mature proteins inside the ER which require transport to the Golgi stacks, which suggests some form of trans-ER membrane signalling. Other examples include: (i) The gp55 protein of the Friend spleen focus-forming virus binds to the EPO-R and triggers cell growth, but the interaction of the EPO-R with the gp55 protein occurs within the lumen of the ER [30]. Neither the EPO-R or gp55 appear on the cell surface or in the medium. (ii) If N R K cells are transfected with the simian sarcoma virus derived v-sis gene [homologous to the platelet-derived growth factor (PDGF) B chain] to which an ER retention signal has been added, cells become morphologically transformed despite the failure to secrete v-sis [31]. (iii) A similar experiment in which interleukin-3 (IL-3) was given an ER retention signal and transfected into an IL-3 dependent cell line (murine 32D cells [32]) resulted in IL-3 independent growth. Again, no IL-3 was released into the medium. In each case, the ligand (either gp55, v-sis or IL-3) is apparently able to bind (and activate) its receptor within the ER and transmit a signal to the cell as if the receptor was at the plasma membrane. This is analogous to the way in which G R P transcription may be regulated. Most growth factor receptors are synthesized into the ER, with the extracellular domain (receptor site) on the luminal side and the kinase domain on the cytosolic side. This orientation is reversed when the receptors are delivered to the plasma membrane. The gp55, v-sis and IL-3 ligands may bind to the receptor binding sites on the luminal side of the ER, activating the receptor kinase activity on the cytosolic face of the ER. The interaction of receptor and ligand therefore results in the propagation of the signal into the cell. Whether the GRPs utilize such a system is unclear. It is possible that GRP78 (or one of its post-translationally modified forms) may interact with a
469
receptor on the luminal side of the F~R which can transduce a signal to the cytosolic side. For instance, ERD2, the protein which recognizes the ER retention signal (KDEL) is a 26,000 mol. wt integral membrane protein. Structural analysis of ERD2 from Saccharomyces cerevisiae, Kluveromyces lactis and humans reveals the presence of seven hydrophobic domains and suggests that all seven regions may span the membrane [33, 35]. This type of structure is common to many transmembrane receptors [3], and has the potential to be coupled to a G-protein. Since GRP78 also contains a K D E L signal, interactions with the K D E L receptor may result in signalling across the ER membrane. The presence of a variety of G-proteins on the outside of the ER may then serve to pass on the signal to the nucleus or other targets. This type of mechanism may work both ways, with receptors on the outside of the ER able to respond to cytoplasmic messages and pass the signal on to the lumen of the ER. Much more work needs to be done to elucidate the mechanism by which the ER and the cytosol can communicate with each other. The data presented here are speculative but outline an area of research which has not yet been fully explored. The ER contains all the 'classical' components of signalling systems - - receptors, G-proteins, kinases, phosphatases. Given the great interest of late in studying the structure and function of the ER, answers to these questions may soon be available to us. Acknowledgements--This work was supported by grant numbers R29 CA44940 and ROI 47407 from the NIH/NCI. Thanks to STUARTCALDERWOODfor discussions and comments on the manuscript.
REFERENCES !. Rose J. K. and Doms R. W. (1988) A. Rev. Cell Biol. 4, 257-288. 2. Lingappa V. R. (1991) Cell 65, 527-530. 3. Klausner R. O. (1989) Cell 57, 703-706. 4. Welch W. J., Garrels J. I., Thomas G. P., Lin J. J. and Feramisc~ J. R. (1983) J. biol. Chem. 258, 7102-711 !. 5. Freedman R. B. (1989) Cell 57, 1069-1072.
470
B.D. Palc~
6. McCauliffe D. P., Yang Y.-S., Wilson J., Sontheimer R. D. and Capra J. D. (1992) J. biol. Chem. 267, 2557-2562. 7. Munro S. and Pelham H. R. (1986) Cell 46, 291-300. 8. Hendershott L. M. (1990) J. Cell Biol. 111, 829-837. 9. Kasscnbrock C. K., Garcia P. D., Walter P. and Kelly R. B. (1988) Nature 333, 90-93. 10. Pelham H. B. (1989) A. Rev. Cell Biol. 5, 1-23. 11. Kang H. S. and Welch W. J. (1989) J. biol. Chem. 266, 5643-5649. 12. Chang S. C., Wooden S. K., Takafumi N., Kim Y. K., Lin A. Y., Kung L., Etteneilo J. W. and Lee A. S. (1987) Proc. hath. Acad. Sci. U.S.A. 84, 680-684. 13. Price B. D., Mannheim-Rodman L. and Calderwood S. K. (1992) J. Cell Physiol. (in press). 14. Lcno G. H. and Ledford B. E. (1989) Eur. J. Biochem. 186, 205-211. 15. Hendershot L. M., Ting J. and Lee A. S. (1988) Molec. Cell Biol. 8, 4250-4256. 16. Fr¢iden P. J., Gant J. R. and Hendershot L. M. (1992) Fur. Molec. Biol. Org. J. 11, 63-70. 17. Furuiehi T., Yoshikawa S., Miyawaki A., Wada K., Maeda N. and Mikoshiba K. (1989) Nature 342, 32-38. 18. Lytton J., Westlin M. and Hanley M. R. (1991) Proc. natn. Acad. Sci. U.S.A. 266, 17,067-17,071. 19. Price B. D. and Calderwood S. K. (1991) Molec. Cell Biol. 11, 3365-3368. 20. Drummand I. A., Lee A. S., Resendez E. and
21. 22.
23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
Steinhardt R. A. (1987) J. biol. Chem. 262, 12,801-12,805. Bauskin A. R., Alkalay I. and Bcn-Neriah Y. (1991) Cell 66, 685-696. Frangioni J. V., Beahm P. H., Shifrin V., Jost C. A. and Neel B. G. (1992) Cell 615, 545-560. Lucocq J. M., Berger E. G. and Warren G. (1988) J. Cell Biol. 109, 463-474. Toumikoski T., Felix M., Doree M. and Gruenberg J. (1989) Nature 342, 942-945. Nigam S. K. and Blobel G. (1989) J. biol. Chem. 264, 16,927-16,932. Hall A. (1990) Science 249, 635--640. Audigier Y., Nigam S. K. and Blobel G. (1988) J. biol. Chem. 263, 16,352-16,357. Baker D., Wuestehube L., Schekman R., Botstein D. and Segev N. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 355-359. Beckers C. J. and Balch W. E. (1989) J. Cell Biol. 108, 1245-1256. Yoshimura A., D'Andrea A. D. and Lodish H. F. (1990) Proc. hath. Acad. Sci. U.S.A. 87, 4139-4143. Bejeck B. E., Li D. Y. and Deuel T. F. (1989) Science 245, 1496--1499. Dunbar C. E., Browder T. M., Abrams J. S. and Nienhuis A. W. (1989) Science 245, 1493-1496. Lewis M. J. and Pelham H. R. (1992) Cell 611, 353-364. Rothman J. E. and Orci L. (1992) Nature 355, 409-4 15. Semenza J. C. and Pelham H. R. B. (1992) J. molec. Biol. 224, 1-5.
0898-6568/92 $5.00 + 0,00 © 1992 Pergamon Press Ltd
MINI REVIEW
SIGNALLING ACROSS THE ENDOPLASMIC RETICULUM MEMBRANE: POTENTIAL MECHANISMS BRENDAN D. PRICE Stress Protein Group (.IF 205), Dana=Farber Cancer Institute, Harvard Medical School, 44 Binney St, Boston, MA 02115, U.S.A.
(Received 30 April 1992; and accepted 10 May 1992) AImraet--The endoplasmic reticulum (ER) is a membrane-bound organelle responsible for the synthesis, assembly and post-translational modification of proteins destined for the lysosomes, Golgi and for secretion. The processes which occur in the lumen of the ER are vital to the correct functioning of the cell, and mechanisms must exist to enable the cell to monitor events within the lumen of the ER. How the cell is able to do this is not known, but it would apparently require the passage of signals from the lumen of the ER to the cytosoi, from where signals can be sent to, for example, the nucleus to effect changes in transcription. Here, it is suggested that the membrane of the ER may contain the components (i.e. receptors, kinases, etc.) required for transmembrane signalling in much the same way as the plasma membrane does. This hypothesis will be discussed in relation to known ER proteins which might act as signalling proteins.
Key words: Endoplasmic reticulum, GRPs, signalling, phosphorylation, G-proteins, protein transport, protein kinases. Tim ENDOPLAS~C RETICULUM (ER) is a membrane-bound structure consisting of cisternae that are spread throughout the cell. Proteins which are targeted to the lysosomes, Golgi apparatus, plasma membrane or for secretion, are all initially processed through the E R [1]. When proteins are translated on the m R N A - r i b o s o m e complex in the cytosol, the emergence o f a signal peptide at the N-terminal of the nascent protein directs the m R N A - r i b o some complex to attach to the ER via the signal peptide [2]. Protein synthesis then continues across the ER membrane and the protein emerges into the lumen o f the ER, or, for some membrane proteins (e.g. growth factor recep-
Abbreviations: BAPTA-- 1,2-bis(2-aminophenoxy)ethanN,N,N',N'-tetraacetic acid; EPO-R--erythropoietin receptor; ER--endoplasmic reticulum; GRPs---glucose-regnlated proteins; IL-3--interleukin-3; KDEL--Lys-Asp-GIu-Leu, ER retention signal; KKKK--Lys-Lys-Lys-Lys, nuclear localization sequence; NRK ceils---normalrat kidney cells; PDGF--platelet-derived growth factor; PTP-IB--protein tyrosine phosphatase IB. 465
tors), part of the protein may be inserted into the ER membrane. Once synthesis has been completed, a number of post-translational modifications may take place within the lumen o f the ER. These can include N-linked glycosylation, fatty acylation and assembly o f protein sub-units via disulphide bond formation into oligomeric structures [1]. Processed proteins are transported from the ER to the Golgi apparatus by the budding of transport vesicles from the face of the ER and the fusion o f these with the Golgi, where further processing may occur. Mature proteins are then directed to the lysosomes, plasma membrane or are targeted for secretion [1-3]. The biosynthetic and transport processes of the ER are highly complex and involve a wide range of biosynthetic events. The assembly of proteins within the lumen of the ER, and the transfer of these via the Golgi to the appropriate compartment must be tightly controlled to prevent, for instance, the delivery of lysosomal enzymes to the wrong organelle. Maintaining the structural integrity of the ER
466
B.D. PRICE
requires the continuous synthesis and incorporation of specific lipids and structural proteins into the membrane of the ER. The biosynthetic functions require various glycosyltransferases to carry out glycosylation, enzymes involved in fatty acylation, and a number of proteins involved in protein folding (see below), as well as controlling the levels of ATP, GTP, Ca 2+ (a major component of the ER) and cofactors required in the modification of secretory proteins. In addition, during mitosis, the secretory mechanism is blocked and the ER breaks down into small vesicles in preparation for cell division, reforming only after division has completed [24]. All of these processes must be closely monitored by the cell to ensure that the biochemical events occurring inside the ER proceed in an orderly and precise fashion. However, the ER is a membrane-bound organelle which is distinct from the cytosol. How, then, do the ER and cytosol (and nucleus) communicate with each other to control both the structure of the ER and the processing and secretion of various proteins? A family of 'stress proteins' (the glucose-regulated proteins or GRPs), which are found only in the ER, provide a good example of the requirement for communication across the ER membrane. If the folding and maturation of proteins within the lumen of the ER is disrupted, the cell can sense this and responds by increasing the transcription of the GRP genes. This process requires the transduction of a signal generated in the lumen of the ER to the cytosol and subsequently to the nucleus to alter GRP transcription. The GRPs (GRP78 and 94 [4]), and other proteins, including protein disulphide isomerase [5], ERp72 [6], calreticulin and many others [13] are resident proteins in the ER. GRP78 and 94 are major components of the ER. GRP78 functions as a molecular ehaperonin protein similar to hsp70 (with which it has 68% homology [7]) and has been found associated with immunogiobulin heavy chains [8], unfolded or incompletely glycosylated proteins [9] and may participate in the import and folding of nascent proteins targeted to the ER and Golgi systems
[10]. GRP78's function in the ER may be to bind to nascent proteins as they are delivered into the lumen and either help them to fold or to aid in the assembly of oligomeric proteins [3, 7, 10]. GRP78 has an intrinsic ATPase activity which may be utilized to modulate the binding and release of substrate proteins [7, 10]. Both GRP78 and 94 contain C-terminal Lys-Asp-Glu-Leu (KDEL) sequences which mark these proteins for retention within the ER [10]. Normally, the levels of the GRPs in the ER remain constant, but, under some conditions, the expression of these proteins is greatly increased. Cells exposed to low oxygen concentrations (hypoxia, e.g. poorly vascularized cells in the centre of tumours or during ischaemia), or low glucose levels (hypoglycemia), respond by greatly increasing the transcription of the GRPs, resulting in up to three- to five-fold increases in the levels of GRP78 and 94 in the ER [4, 11]. Increased GRP synthesis can also be induced by treating cells with A23187 (a Ca 2÷ ionophore which discharges ER Ca2÷), tunicamycin (which blocks N-linked glycosylation in the ER), 2-deoxyglucose (an inhibitor of glucose uptake and glycosylation), brefeldin A (which inhibits vesicle transport between the ER and the Golgi) and thapsigargin (an inhibitor of the ER Ca2+-ATPase [4, 12, 13]). All of these treatments result in the accumulation within the ER of underglycosylated or realfolded proteins which cannot be transported out of the ER to the Golgi apparatus. The cell responds to the accumulation of aberrant proteins by increasing the synthesis of GRP78, primarily through increased transcription of the GRP genes [12]. It is thought that the extra GRP78 and 94 are required to prevent aberrant proteins from precipitating in the ER and that GRPs carry out this function by binding to these accumulated proteins and maintaining their solubility [13]. In the presence of large quantities of defective proteins, GRP78 rapidly becomes bound up with aberrant proteins, and free (unbound) GRP levels fall dramatically [9, 16]. However, the mechanism by which the cell monitors the level of GRPs in the ER and the
Signal transductionfrom the ER lumento the cytosol nature of the signal it sends to the nucleus to increase their transcription is largely unknown. Whatever the signal transduction process, it requires a sensing mechanism in the ER to monitor the GRP levels, a means of communicating the signal across the ER membrane and the ability to pass this signal on to the nucleus to allow changes in transcription.
How do cells sense the level of GRP78? Recent data have shown that the free (unbound) GRP78 in the ER is both phosphorylated (on Ser/Thr residues) and ADP-ribosylated, but that the GRP78 bound to proteins contains neither modification [14, 15]. Further, when cells are induced to produce more GRP78 by starving the cells of glucose, the newly synthesized GRP78 is found in association with non-transported proteins and contains no modifications [16]. However, the amount of free GRP78 in glucose-starved cells is the same as in glucose-fed cells [15, 16]. This suggests that the levels of GRP78 are regulated to maintain the amount of free GRP78 at a constant level. When ER function is disrupted and non-transported proteins become trapped within the lumen, GRP78 binds to the accumulated proteins [8, 9] and is dephosphorylated and de-ribosylated. Normally, the association of GRP78 with target proteins would be transient, and GRP78 would dissociate after correct folding or glycosylation of the target protein, but when these processes are disrupted, GRP78 remains bound to the target protein for an extended period of time [16]. The decrease in free GRP78, monitored by the dephosphorylation and/or de-ribosylation of GRP78 then triggers an increase in transcription of the GRP genes. So it is likely that the cell monitors the levels of free, modified GRP78 and increases transcription in response to changes in post-translational modification. Whilst this may be the sensing mechanism within the ER, and suggests the presence of a GRP78 protein kinase and phosphatase, as well as ADP ribosylation factors (as
467
suggested in Ref. 16), it does not e~alain how the message is passed out of the ER.
Is Ca 2+ involved in signalling from the ER to the cytosol? The ER is a major cellular store for Ca 2+ and contains two important transmembrane proteins involved in regulating cellular Ca 2+ - - the inositol phosphate receptor [17] and the ER Ca2+-ATPase [18]. Both A23187 (a Ca 2+ ionophore) and thapsigargin (an inhibitor of the ER Ca2+-ATPase [18]) rapidly increase intracellular Ca 2+ levels by discharging ER Ca 2+ and both compounds can induce GRP transcription [13]. Elevated Ca 2+ might therefore be responsible for increasing transcription of the GRP genes in the same way that it affects the transcription of the closely related heat shock genes [19]. However, buffering the rise in intracellular Ca 2+ (with BAPTA or EGTA) does not affect the induction of the GRPs [20]. In addition, induction of GRP transcription by tunicamycin, 2deoxyglucose, brefeldin A or by the introduction of transport defective proteins does not alter the level of intracellular Ca 2+ [4, 12, 13, 20]. Changes in Ca 2+ are therefore not important for the induction of GRPs.
Are protein kinases/phosphatases involved in signalling? Recent work has identified a non-receptor, transmembrane tyrosine kinase, the Ltk kinase, which is localized to the ER and may play a role in signal transduction across the ER membrane [21]. The N-terminal domain contains a cysteine-rich region which faces the luminal side of the ER. The Ltk kinase activity is greatly increased by diamide, a thioloxidizing agent, resulting in the formation of active disulphide-linked Ltk dimers. This suggests that the Ltk kinase may be involved in signalling under conditions in which the redox state of the ER is altered, such as during hypoxia or glucose starvation (as noted in Ref. 21). Hypoxia and glucose starvation also result
468
B . D . PRICE
in elevat~n of GRPs. Given the properties displayed by the Ltk kinase, it is possible that it (or related kinases) may be involved in the regulation of GRP transcription and that the tyrosine kinase activity may result in the phosphorylation of target proteins in the cytosol which may be positive regulators of GRP transcription. However, since the Ltk kinase is expressed only in cells of lymphoid origin this should be treated with some caution. In addition to the Ltk kinase, a cAMP-dependent protein kinase has also been shown to be associated with the external face of the ER, and, on activation, may be released to the cytosol whilst the inhibitory subunits remain attached to the ER [25]. Protein tyrosine phosphatase 1B (PTP-1B), one of the major tyrosine phosphatases in cells, is associated with the ER membrane. PTP-1B is anchored to the cytosolic face of the ER by a 35-amino acid sequence and the active site is directed towards the cytosol [22]. The function of this ER localization is not clear, but it may be involved in regulating the normal function and transport properties of the ER. It has been speculated that PTP-1B may be required to ensure that tyrosine kinase receptors passing through the ER on their way to the plasma membrane are maintained in an inactive (dephosphorylated) form [22]. The 35-amino acid C-terminal fragment that attaches PTP-1B to the ER could be in contact with the interior of the ER and may participate in the passage of signals out of the ER, for instance by interacting with luminal ER proteins. PTP-1B activity may be controlled through phosphorylation (since the PTP-IB has been reported to be phosphorylated on serine residues [22]). The turning on or off of the PTP-1B activity may either terminate or enhance signals generated from the ER. Further evidence that phosphorylation is important in the induction of GRP transcription comes from the observation that genistein, a tyrosine kinase inhibitor, blocks the stimulation of GRP transcription, whereas okadaic acid, a phosphatase inhibitor, greatly enhances the stimulation of GRP transcription, indirectly
supporting a role for protein kinases in activation of GRP transcription [13]. G-proteins present on the E R
As well as protein kinases, the ER and Golgi contain a large number of trimeric and ras-like G-proteins which are involved in vesicle transport, fusion, targeting and release of secretory proteins (reviewed in Ref. 26). Two of these proteins are worthy of further investigation. An ER-specific trimeric G-protein has been detected on the outer face of the ER membrane which was released to the cytoplasm when GTP~S was added and could be ADP-ribosylated by pertussis toxin [27]. The second protein is yptl, a ras-related G-protein of 21,000 mol. wt which is required for vesicle transport between the Golgi and the ER [28]. A number of reports have shown that transport between the ER and the Golgi involves a trimeric G-protein whose function can be inhibited by GTPyS and AIFa- (which blocks trimeric G-proteins but not ras-like proteins [29]). Interestingly, cells treated with AIF4- also show enhanced synthesis of GRPs, although this effect might be due to inhibition of protein exit from the ER, causing proteins to accumulate in the lumen, rather than an effect on a G-protein involved in signal transduction [13]. So although the ER has a number of associated G-proteins [26-28], these seem to function mostly in control of vesicle budding and fusion rather than in signal transduction across the ER membrane. Further characterization of these proteins will be required, although the finding that at least one of these G-proteins [27] can bind reversibly to the ER may indicate a cytosolic function for ER G-proteins as well. What other processes require a trans-ER signalling system?
There are a number of other cellular processes apart from GRP transcription which require the transmission of a signal from the ER to the eytosoi. The budding of transport vesicles from the ER and their fusion to the
Signal transduction from the ER lumen to the cytosol Golgl apparatus involves the binding and cyclical release of a number of coat proteins (COPS) and G-proteins (including the ADPribosylation factor, ARF) on the cytosolic side of the ER (reviewed in Ref. 34). This process may be regulated by the presence of mature proteins inside the ER which require transport to the Golgi stacks, which suggests some form of trans-ER membrane signalling. Other examples include: (i) The gp55 protein of the Friend spleen focus-forming virus binds to the EPO-R and triggers cell growth, but the interaction of the EPO-R with the gp55 protein occurs within the lumen of the ER [30]. Neither the EPO-R or gp55 appear on the cell surface or in the medium. (ii) If N R K cells are transfected with the simian sarcoma virus derived v-sis gene [homologous to the platelet-derived growth factor (PDGF) B chain] to which an ER retention signal has been added, cells become morphologically transformed despite the failure to secrete v-sis [31]. (iii) A similar experiment in which interleukin-3 (IL-3) was given an ER retention signal and transfected into an IL-3 dependent cell line (murine 32D cells [32]) resulted in IL-3 independent growth. Again, no IL-3 was released into the medium. In each case, the ligand (either gp55, v-sis or IL-3) is apparently able to bind (and activate) its receptor within the ER and transmit a signal to the cell as if the receptor was at the plasma membrane. This is analogous to the way in which G R P transcription may be regulated. Most growth factor receptors are synthesized into the ER, with the extracellular domain (receptor site) on the luminal side and the kinase domain on the cytosolic side. This orientation is reversed when the receptors are delivered to the plasma membrane. The gp55, v-sis and IL-3 ligands may bind to the receptor binding sites on the luminal side of the ER, activating the receptor kinase activity on the cytosolic face of the ER. The interaction of receptor and ligand therefore results in the propagation of the signal into the cell. Whether the GRPs utilize such a system is unclear. It is possible that GRP78 (or one of its post-translationally modified forms) may interact with a
469
receptor on the luminal side of the F~R which can transduce a signal to the cytosolic side. For instance, ERD2, the protein which recognizes the ER retention signal (KDEL) is a 26,000 mol. wt integral membrane protein. Structural analysis of ERD2 from Saccharomyces cerevisiae, Kluveromyces lactis and humans reveals the presence of seven hydrophobic domains and suggests that all seven regions may span the membrane [33, 35]. This type of structure is common to many transmembrane receptors [3], and has the potential to be coupled to a G-protein. Since GRP78 also contains a K D E L signal, interactions with the K D E L receptor may result in signalling across the ER membrane. The presence of a variety of G-proteins on the outside of the ER may then serve to pass on the signal to the nucleus or other targets. This type of mechanism may work both ways, with receptors on the outside of the ER able to respond to cytoplasmic messages and pass the signal on to the lumen of the ER. Much more work needs to be done to elucidate the mechanism by which the ER and the cytosol can communicate with each other. The data presented here are speculative but outline an area of research which has not yet been fully explored. The ER contains all the 'classical' components of signalling systems - - receptors, G-proteins, kinases, phosphatases. Given the great interest of late in studying the structure and function of the ER, answers to these questions may soon be available to us. Acknowledgements--This work was supported by grant numbers R29 CA44940 and ROI 47407 from the NIH/NCI. Thanks to STUARTCALDERWOODfor discussions and comments on the manuscript.
REFERENCES !. Rose J. K. and Doms R. W. (1988) A. Rev. Cell Biol. 4, 257-288. 2. Lingappa V. R. (1991) Cell 65, 527-530. 3. Klausner R. O. (1989) Cell 57, 703-706. 4. Welch W. J., Garrels J. I., Thomas G. P., Lin J. J. and Feramisc~ J. R. (1983) J. biol. Chem. 258, 7102-711 !. 5. Freedman R. B. (1989) Cell 57, 1069-1072.
470
B.D. Palc~
6. McCauliffe D. P., Yang Y.-S., Wilson J., Sontheimer R. D. and Capra J. D. (1992) J. biol. Chem. 267, 2557-2562. 7. Munro S. and Pelham H. R. (1986) Cell 46, 291-300. 8. Hendershott L. M. (1990) J. Cell Biol. 111, 829-837. 9. Kasscnbrock C. K., Garcia P. D., Walter P. and Kelly R. B. (1988) Nature 333, 90-93. 10. Pelham H. B. (1989) A. Rev. Cell Biol. 5, 1-23. 11. Kang H. S. and Welch W. J. (1989) J. biol. Chem. 266, 5643-5649. 12. Chang S. C., Wooden S. K., Takafumi N., Kim Y. K., Lin A. Y., Kung L., Etteneilo J. W. and Lee A. S. (1987) Proc. hath. Acad. Sci. U.S.A. 84, 680-684. 13. Price B. D., Mannheim-Rodman L. and Calderwood S. K. (1992) J. Cell Physiol. (in press). 14. Lcno G. H. and Ledford B. E. (1989) Eur. J. Biochem. 186, 205-211. 15. Hendershot L. M., Ting J. and Lee A. S. (1988) Molec. Cell Biol. 8, 4250-4256. 16. Fr¢iden P. J., Gant J. R. and Hendershot L. M. (1992) Fur. Molec. Biol. Org. J. 11, 63-70. 17. Furuiehi T., Yoshikawa S., Miyawaki A., Wada K., Maeda N. and Mikoshiba K. (1989) Nature 342, 32-38. 18. Lytton J., Westlin M. and Hanley M. R. (1991) Proc. natn. Acad. Sci. U.S.A. 266, 17,067-17,071. 19. Price B. D. and Calderwood S. K. (1991) Molec. Cell Biol. 11, 3365-3368. 20. Drummand I. A., Lee A. S., Resendez E. and
21. 22.
23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.
Steinhardt R. A. (1987) J. biol. Chem. 262, 12,801-12,805. Bauskin A. R., Alkalay I. and Bcn-Neriah Y. (1991) Cell 66, 685-696. Frangioni J. V., Beahm P. H., Shifrin V., Jost C. A. and Neel B. G. (1992) Cell 615, 545-560. Lucocq J. M., Berger E. G. and Warren G. (1988) J. Cell Biol. 109, 463-474. Toumikoski T., Felix M., Doree M. and Gruenberg J. (1989) Nature 342, 942-945. Nigam S. K. and Blobel G. (1989) J. biol. Chem. 264, 16,927-16,932. Hall A. (1990) Science 249, 635--640. Audigier Y., Nigam S. K. and Blobel G. (1988) J. biol. Chem. 263, 16,352-16,357. Baker D., Wuestehube L., Schekman R., Botstein D. and Segev N. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 355-359. Beckers C. J. and Balch W. E. (1989) J. Cell Biol. 108, 1245-1256. Yoshimura A., D'Andrea A. D. and Lodish H. F. (1990) Proc. hath. Acad. Sci. U.S.A. 87, 4139-4143. Bejeck B. E., Li D. Y. and Deuel T. F. (1989) Science 245, 1496--1499. Dunbar C. E., Browder T. M., Abrams J. S. and Nienhuis A. W. (1989) Science 245, 1493-1496. Lewis M. J. and Pelham H. R. (1992) Cell 611, 353-364. Rothman J. E. and Orci L. (1992) Nature 355, 409-4 15. Semenza J. C. and Pelham H. R. B. (1992) J. molec. Biol. 224, 1-5.
