- Email: [email protected]
Calreticulin-Integrin Bidirectional Signaling Complex
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
232, 354–358 (1997)
RC976195
Calreticulin-Integrin Bidirectional Signaling Complex Qiang Zhu, Peter Zelinka, Tracy White, and Marvin L. Tanzer1 Department of BioStructure and Function, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-3705
Received January 22, 1997
Calreticulin has mutiple functions, diverse cellular locations, and putative isoforms. It likely maintains integrin avidity by binding a integrin cytoplasmic tails and is a surface lectin which triggers cell spreading. In the present study, we have immunocaptured a cell surface complex from B16 mouse melanoma cells which contains a6b1 integrin, two molecular forms of calreticulin, and KDEL docking protein (KDEL-R). One of the calreticulins, ‘‘endocalreticulin’’, a 52 kDa protein, does not become surface biotinylated, and is probably bound to a integrin cytoplasmic tails; it disappears when B16 cells adhere to laminin, and two ubiquitinated calreticulins appear. One ubiquitinated species, a 125 kDa protein, is restricted to focal contacts whereas a second species, a 75 kDa protein, is in focal contacts and surrounding plasma membrane; it also arises when cells bind non-specific surfaces. The other calreticulin, ‘‘ectocalreticulin’’, a 62 kDa protein, becomes surface biotinylated, is probably anchored to surface KDEL-R, and cooperates with a6b1 integrin, triggering cell spreading. The present results suggest a model in which calreticulin-integrin surface complex functions as a symbiotic unit, transmitting information in both directions across the plasma membrane. q 1997 Academic Press
Cell surface calreticulins have been implicated in cell behavior. Among such calreticulins is the human neutrophil collectin receptor which binds the plasma opsonins: complement C1q, mannose binding protein, lung surfactant protein A and conglutinin (1, 2). Although the collectin receptor displays antibody cross-reactivity, similar peptidic sequences, and similar migration on SDS/PAGE to intracellular calreticulin, it has a different overall charge. Surface calreticulin is shed when 1 Correspondent. Fax: 860-679-2910. E-mail: [email protected]. Abbreviations used are: CHAPS, [{3-[(3-cholamidopropyl)-dimetylammonio]-1-propanesulfonate}]; PMSF, phenylmethylsufonyl fluoride; PAGE, polyacrylamide gel electrophoresis; KDEL-R, KDEL receptor; WGA, wheat germ agglutinin; HRP, horseradish peroxidase.
neutrophils are stimulated with the peptide FMLP and differs from intracellular calreticulin in these cells (3). Murine B16 melanoma cell surface protein, B50, a prominent immunogen, shows antibody cross-reactivity and identical N-terminal amino acid sequences to murine calreticulin, and is also shed (4-6). Recently, human fetal fibroblast surface calreticulin has been implicated as a fibrinogen receptor which, upon occupancy, stimulates cell proliferation (7). A putative isoform of calreticulin binds the motif KxGFFKR in the cytoplasmic tail of a integrins and co-localizes with integrins in focal contacts (8); that isoform maintains b1 integrins primed for ligand engagement (9). The isoform itself, endocalreticulin, has not been directly detected, perhaps because of low concentration and lability. Ectocalreticulin also cooperates with b1 integrins but plays a different role from endocalreticulin. Several cell lines, including mouse B16 melanoma cells, efficiently attach to but fail to spread on laminin which had been synthesized without its customary repertoire of Nlinked carbohydrates (10). Spreading of the spherical laminin-adherent cells begins, within minutes, when laminin carbohydrates are restored (11). This receptormediated response is due to ectocalreticulin which recognizes laminin oligomannosides (12-14). Ectocalreticulin cannot, by itself, initiate cell adhesion and spreading but must act simultaneously with, or sequential to integrin engagement (11). Prior to cell adhesion, ectocalreticulin and integrin diffusely cover the outer cell surface (12, 13); following cell adhesion to laminin, podosomal focal contacts appear and contain concentrated ectocalreticulin (and endocalreticulin) and b1 integrin (12-14). Subsequent studies, described here, indicate that in pre-adherent cells ecto- and endocalreticulin and integrin heterodimer are affiliated in a functional complex which spans the plasma membrane. MATERIALS AND METHODS Surface biotinylation and immunocapture. B16 mouse melanoma cells were surface biotinylated as previously described (13), followed by cell lysis in 0.02M Tris, pH 7.4, 0.1 M NaCl, 1% CHAPS, 5 mM
354
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
Vol. 232, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CaCl2 , 2 mg/ml leupeptin, 0.4 mg/ml antipain, 2 mg/ml benzamidine, 2 mg/ml aprotinin, 1 mg/ml chymostatin, 1 mg/ml pepstatin, and 1 mM PMSF. The lysate was cleared by centrifugation at 10,000 1 g for 30 min. Immunocapture was carried out by standard protocols. Briefly, cell lysates were initally incubated with pre-immune sera and protein A beads to remove non-specific proteins; after removal of the beads by pelleting, anti-calreticulin or anti-b1 integrin antiserum and protein A beads were added to the lysate which was rotated overnight in the cold. The beads were harvested and exhaustively washed to remove non-specific proteins. The immunocaptured proteins were solubilized and separated by SDS/PAGE in 5-15% linear gradient gels, using a standard buffer system and then transferred to a membrane for immunoblotting. Immunoreactive proteins were visualized using ECL. Antibodies used were: anti-calreticulin, Affinity BioReagents, Golden, CO; anti-calreticulin peptides, amino acids 7-24 and amino acids 383-400, R. Sontheimer; anti-KDEL docking protein (KDEL-R), H.-D. So¨ling; anti-a6 integrin, American Research Products, Belmont, MA, and V. Quaranta; anti-b1 integrin, Pharmingen, San Diego, CA ; anti-ubiquitin, Sigma, St. Louis, MO. Cell membrane fractionation and focal contact formation. Cells in suspension were disrupted by mechanical means after osmotic swelling and cellular membranes were partitioned in a discontinuous sucrose density gradient (15). Membranes in each density phase were pelleted, washed three times with isotonic buffer, then subjected to SDS/PAGE and immunoblotting. Focal contacts were induced in suspended cells using laminin-coated paramagnetic microspheres followed by disruption of the cells and isolation of focal contacts adherent to harvested microspheres (16). Focal contact proteins on harvested microspheres were solubilized in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM PMSF), separated by SDS/PAGE and detected by immunblotting. Plasma membranes, from a wash fraction, were similarly analyzed. Cell adhesion, without focal contact formation, was induced in suspended cells using wheat germ agglutinin (WGA)-coated paramagentic microspheres followed by disruption of the cells and isolation of plasma membranes adherent to harvested microspheres (17).
RESULTS Complex immunocapture and calreticulin isoforms. The calreticulin-integrin complex is immunocaptured by antibody to either calreticulin or integrin; the complex contains ecto- and endocalreticulins, a6b1 integrin, KDEL docking protein (KDEL-R), plus two unidentified proteins (Fig. 1). Each of these proteins, except for endocalreticulin, is accessible to cell surface biotinylation; the KDEL docking protein signal is faint because most of the protein is intramembranous and unavailable for biotinylation; other experiments (Fig. 2A) confirm that ecto- and endocalreticulins are associated with plasma membranes, ER membranes and Golgi membranes; the relative abundance of both calreticulins varies in those cellular locations. Importantly, although both calreticulins are associated with plasma membranes, only ectocalreticulin is accessible to cell surface labeling. Concerning this point, we had previously optimized B16 cell biotinylation and shown that a consistent population of surface proteins is biotinylated, and that biotinylation did not label intracellular proteins (13). As for antibody specificity, we had shown that Affinity BioReagents anti-calreticulin antiserum
FIG. 1. Proteins of the calreticulin-integrin complex obtained from suspended cells. SDS/PAGE gels in panel A were performed in the presence of DTT. (A) Immunocaptured complex, obtained using anti-b1 integrin antibody. Lane 1, detection of surface-biotinylated proteins in the complex (streptavidin-HRP probe); the location of the integrin subunits was determined by parallel immunoblots. Lane 2a, detection of calreticulins in the complex by anti-calreticulin immunoblotting. Note that 52 kDa calreticulin did not become cell surfacebiotinylated. Lane 2b, detection of KDEL-R in the complex, immunoblotted with anti-KDEL-R antibody. (B) Detection of a6 integrin in the complex, immunocaptured with anti-calreticulin antibody and immunoblotted using anti-a6 antibody.
does not recognize proteins other than calreticulins in total cell lysates (13). Cells quickly release ectocalreticulin but do not release endocalreticulin (Fig. 2B); cells which have been surface biotinylated prior to incubation release biotinylated ectocalreticulin (data not shown). The composite results imply that surface shedding accounts for ectocalreticulin release, probably due to detachment from KDEL-R, perhaps by KDEL proteolysis. Prior studies, using metabolic labeling of calreticulin, support surface shedding as the release mechanism in B16 cells (5). Endocalreticulin becomes ubiquitinated when cells adhere. When B16 cells adhere to laminin they form podosomal focal contacts (13). When focal contacts are induced on suspended cells by laminin-coated microspheres, plasma membrane endocalreticulin disappears and two new immunoreactive species, 75 kDa and 125 kDa, appear in the focal contacts (Fig. 3A). The 75 kDa species, by itself, is also present in plasma membrane surrounding the focal contacts (data not shown). When suspended cells adhere to WGA-coated microspheres, a circumstance in which cells do not form focal contacts, endocalreticulin disappears and only the 75 kDa species appears in the plasma membrane (Fig. 3A). Separate experiments show that the 75 and 125 kDa species do not become surface-labeled, whether cells are biotinylated before or after adhesion (data not shown). Both species are ubiquitinated (Fig. 3B); additional studies show that the 75 kDa species is slow to react with C-terminal anti-calreticulin antibodies (Fig.
355
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
Vol. 232, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Relative abundance of endo- and ectocalreticulin in cell compartments obtained from suspended cells. (A) B16 cells were disrupted and cell membranes were partitioned by density gradient centrifugation, followed by membrane pelleting and sequential washes, then SDS/ PAGE (/DTT) of pelleted membranes, and anti-calreticulin immunoblotting. (B) Release of calreticulin into the culture medium by B16 cells during a 20 min incubation period. Spent medium was concentrated, followed by SDS/PAGE (/DTT) and immunblotting with anticalreticulin antibody.
3C), probably as a consequence of epitope masking by attached ubiquitin molecules. Ectocalreticulin appears as a doublet in these experiments, perhaps due to variable N-glycosylation (18). DISCUSSION The composite results, plus extant information, suggest a model for the calreticulin-integrin complex which accounts for cooperative activities between endocalreticulin, ectocalreticulin, and integrin (Fig. 4). Endocalreticulin’s putative role is to keep integrins primed for ligand engagement (8, 9). Integrins must be activated by cells in order to bind to the appropriate ligands; by itself, expression of an integrin on the cell surface is inadequate for adhesion (19, 20). The path-
ways by which cells activate integrins remain inadequately defined and one attractive model postulates that cell-type-specific energy-dependent cytoplasmic signals reach integrin cytoplasmic domains (21) - and are then propagated to an integrin’s ligand engagement site, increasing its avidity. Integrin-bound endocalreticulin presumably sustains that activated state. In contrast, ectocalreticulin’s role is that of a lectin receptor, acting in concert with a6b1 integrin to initiate spreading of laminin-adherent cells (11-14). The apparent conversion of endocalreticulin into ubiquitinated species when cells adhere implies that integrin engagement sends a transmembrane signal, triggering the conversion. Two different ubiquitinated endocalreticulins are formed, one of which is restricted to focal contacts. These observations imply high speci-
FIG. 3. Effects of microbead adhesion to plasma membranes on cell surface calreticulins. (A) Left frame, immunoblotted calreticulins of pre-adherent plasma membranes of suspended cells; Upper frame, immunoblotted calreticulins harvested from WGA-coated microspheres adherent to plasma membranes of suspended cells; Lower frame, immunoblotted calreticulins harvested from laminin-coated microspheres adherent to plasma membranes of suspended cells. (B) Immunobloted focal contact proteins harvested from laminin-coated microspheres adherent to plasma membranes of suspended cells, using anti-ubiquitin antibody. (C) Immunoblotted calreticulins harvested from WGAcoated microspheres adherent to plasma membranes of suspended cells. Lane 1, antibody vs. calreticulin peptide, amino acids 383-400; Lane 2, antibody vs. calreticulin peptide, amino acids 7-24. 356
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
Vol. 232, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Postulated calreticulin-integrin bi-directional signaling complex on the cell surface. Endocalreticulin binds the a6 integrin subunit cytoplasmic tail at KxGFFKR (8), keeping integrin heterodimer primed for ligand engagement (9). Ectocalreticulin putatively binds integrin heterodimer, is most likely anchored to KDEL docking protein (31), engages laminin oligomannosides (11-14) and probably signals to a6b1 integrin which transmits the signal to the cytoskeleton, triggering cell spreading.
ficity - consistent with current views of ubiquitination processes (22-24). Although ubiquitinated proteins are generally thought to be marked for destruction (22, 23), accumulating evidence suggests that selective removal of ubiquitin may occur, regenerating the original protein (24). A regulatory cycle, analogous to protein phosphorylation and de-phosphorylation, can be envisioned in which ubiquitinated endocalreticulins may have diminished avidity for the consensus motif in the cytoplasmic tail of integrin a subunits. If cells detach from laminin, selective de-ubiquitination could replenish endocalreticulin in order for it to sustain another round of integrin activation and subsequent cell attachment. This concept is consistent with the observations that ubiquitinated endocalreticulins remain associated with plasma membranes of adherent cells and do not become surface biotinylated, suggesting that ubiquitinated endocalreticulins continue to be bound at the cytoplasmic face of the membranes. Ectocalreticulin and endocalreticulin must be different molecules, given their opposite trans-membrane locations and individual molecular sizes. Endocalreticulin is probably translated from an alternative mRNA transcript of the single calreticulin gene, the mRNA arising either from a unique start site or from alternative splicing. At the very least, endocalreticulin must lack a signal sequence since it does not seem to enter the ER lumen; its smaller size, compared to ectocalreticulin, also implies that it may lack additional domains; in this regard, more than one calreticulin mRNA has been detected (25, 26). Most likely, the two calreticulins independently com-
bine with newly synthesized nascent a6b1 integrin, endocalreticulin binding the a6 cytoplasmic tail, and intralumenal ectocalreticulin, as an endoplasmic reticulum molecular chaperone (27-30), binding nascent a6b1 integrin hydrophobic domains. The composite complex, preserving its transmembrane topography, could then be translocated to the cell surface. Conceivably, the signal for translocation may be endocalreticulin attachment, which may ‘‘mark’’ the complex, enabling it to avoid Golgi recycling of intralumenal KDEL-R anchored ectocalreticulin (31). Smalheiser (32) has proposed that an initial ‘‘accidental’’ transit of proteins to cell locations other than their primary location may sometimes have imparted an evolutionary advantage to cells. Thus, the inaugural ‘‘leakage’’ of intracellular calreticulin-integrin complex to the cell surface may have conferred new properties to certain cells, enhancing their survival. Evolutionary pressure could then ensure that such cells have a sustained selective advantage. ACKNOWLEDGMENTS Research funding provided by National Institutes of Health Research Grant AR-17720, American Cancer Society Institutional Grant 152J-129, and a UCONN Faculty Research Grant. We are grateful to Drs. H.-D. So¨ling, R. Sontheimer, and V. Quaranta for antibodies to KDEL docking protein, calreticulin, and a6 integrin, respectively. We thank Drs. William Upholt and Stephen Helfand for critically reading the manuscript. A portion of this work was presented in poster form (Zhu, Q., Zelinka, P., White, T., and Tanzer, M. L. (1996) Mol. Biol. Cell 7, 66a).
REFERENCES 1. Malhotra, R. (1993) Behring Inst. Mitt. 93, 254–261. 2. Miyamura, K., Reid, K. B. M., and Holmskov, U. (1994) Trends Glycosci. Glycotech. 6, 286–309. 3. Eggleton, P., Lieu, T. S., Zappi, E. G., Sastry, K., Coburn, J., Zaner, K. S., Sontheimer, R. D., Capra, J. D., Ghebrehiwet, B., and Tauber, A. I. (1994) Clin. Immunol. Immunopath. 72, 405– 409. 4. Gersten, D. M., Bijwaard, K. E., Law, L. W., and Hearing, V. J. (1990) Biochim. Biophys. Acta 1096, 20–25. 5. Gersten, D. M., Moody, D., Vieira, W. D., Law, L. W., and Hearing, V. J. (1992) Biochim. Biophys. Acta 1138, 109–114. 6. Bijwaard, K., Hearing, V. J., Sontheimer, R., Lieu, T. S., Vieira, W. D., and Gersten, D. M. (1995) Melanoma Res. 5, 327–336. 7. Gray, A. J., Park, P. W., Broekelmann, T. J., Laurent, G. J., Reeves, J. T., Stenmark, K. R., and Mecham, R. P. (1995) J. Biol. Chem. 270, 26602–26606. 8. Leung-Hagesteijn, C. Y., Milankov, K., Michalak, M., Wilkins, J., and Dedhar, S. (1994) J. Cell Sci. 107, 589–600. 9. Coppolino, M., Leung-Hagesteijn, C., Dedhar, S., and Wilkins, J. (1995) J. Biol. Chem. 270, 23132–23138. 10. Dean, J. W., Chandrasekaran, S., and Tanzer, M. L. (1990) J. Biol. Chem. 265, 12553–12562. 11. Chandrasekaran, S., Tanzer, M. L., and Giniger, M. S. (1994) J. Biol. Chem. 269, 3356–3366. 12. Chandrasekaran, S., Tanzer, M. L., and Giniger, M. S. (1994) J. Biol. Chem. 269, 3367–3373.
357
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
Vol. 232, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
13. White, T. K., Zhu, Q., and Tanzer, M. L. (1995) J. Biol. Chem. 270, 15926–15929. 14. McDonnell, J., Jones, G. E., White, T. K., and Tanzer, M. L. (1996) J. Biol. Chem. 271, 7891–7894. 15. Vidugiriene, J., and Menoe, A. K. (1994) J. Cell Biol. 127, 333– 341. 16. Plopper, G., McNamee, H. P., Dike, L. E., Bojanowski, K., and Ingber, D. E. (1995) Mol. Biol. Cell 6, 1349–1365. 17. Warnock, D. E., Roberts, C., Lutz, M. S., Blackburn, W. A., Jr, W. W. Y., and Baenzinger, J. U. (1993) J. Biol. Chem. 268, 10145–10153. 18. Jethmalani, S. M., and Henle, K. J. (1994) Biochem. Biophys. Res. Commun. 205, 780–787. 19. Hynes, R. O. (1992) Cell 69, 11–25. 20. Diamond, M. S., and Springer, T. A. (1994) Current Biology 4, 506–517. 21. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Ann. Rev. Cell Dev. Biol. 11, 549–599.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
Ciechanover, A. (1994) Cell 79, 13–21. Hochstrasser, M. (1995) Current Opinion Cell Biol. 7, 215–223. Jennissen, H. P. (1995) European J. Biochem. 231, 1–30. Treves, S., Zorzato, F., and Pozzan, T. (1992) Biochem. J. 287, 579–581. Liu, N., Fine, R. E., and Johnson, R. J. (1993) Biochim. Biophys. Acta 1202, 70–76. Nauseef, W. M., McCormick, S. J., and Clark, R. A. (1995) J. Biol. Chem. 270, 4741–4747. Peterson, J. R., Ora, A., Van, P. N., and Helenius, A. (1995) Mol. Biol. Cell 6, 1173–1184. Spiro, R. G., Zhu, Q., Bhoyroo, V., and So¨ling, H.-D. (1996) J. Biol. Chem. 271, 11588–11594. Wada, I., Imai, S., Kai, M., Sakane, F., and Kanoh, H. (1995) J. Biol. Chem. 270, 20298–20304. Nilsson, T., and Warren, G. (1994) Current Opinion Cell Biol. 6, 517–521. Smalheiser, N. R. (1996) Mol. Biol. Cell 7, 1003–1014.
358
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
232, 354–358 (1997)
RC976195
Calreticulin-Integrin Bidirectional Signaling Complex Qiang Zhu, Peter Zelinka, Tracy White, and Marvin L. Tanzer1 Department of BioStructure and Function, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-3705
Received January 22, 1997
Calreticulin has mutiple functions, diverse cellular locations, and putative isoforms. It likely maintains integrin avidity by binding a integrin cytoplasmic tails and is a surface lectin which triggers cell spreading. In the present study, we have immunocaptured a cell surface complex from B16 mouse melanoma cells which contains a6b1 integrin, two molecular forms of calreticulin, and KDEL docking protein (KDEL-R). One of the calreticulins, ‘‘endocalreticulin’’, a 52 kDa protein, does not become surface biotinylated, and is probably bound to a integrin cytoplasmic tails; it disappears when B16 cells adhere to laminin, and two ubiquitinated calreticulins appear. One ubiquitinated species, a 125 kDa protein, is restricted to focal contacts whereas a second species, a 75 kDa protein, is in focal contacts and surrounding plasma membrane; it also arises when cells bind non-specific surfaces. The other calreticulin, ‘‘ectocalreticulin’’, a 62 kDa protein, becomes surface biotinylated, is probably anchored to surface KDEL-R, and cooperates with a6b1 integrin, triggering cell spreading. The present results suggest a model in which calreticulin-integrin surface complex functions as a symbiotic unit, transmitting information in both directions across the plasma membrane. q 1997 Academic Press
Cell surface calreticulins have been implicated in cell behavior. Among such calreticulins is the human neutrophil collectin receptor which binds the plasma opsonins: complement C1q, mannose binding protein, lung surfactant protein A and conglutinin (1, 2). Although the collectin receptor displays antibody cross-reactivity, similar peptidic sequences, and similar migration on SDS/PAGE to intracellular calreticulin, it has a different overall charge. Surface calreticulin is shed when 1 Correspondent. Fax: 860-679-2910. E-mail: [email protected]. Abbreviations used are: CHAPS, [{3-[(3-cholamidopropyl)-dimetylammonio]-1-propanesulfonate}]; PMSF, phenylmethylsufonyl fluoride; PAGE, polyacrylamide gel electrophoresis; KDEL-R, KDEL receptor; WGA, wheat germ agglutinin; HRP, horseradish peroxidase.
neutrophils are stimulated with the peptide FMLP and differs from intracellular calreticulin in these cells (3). Murine B16 melanoma cell surface protein, B50, a prominent immunogen, shows antibody cross-reactivity and identical N-terminal amino acid sequences to murine calreticulin, and is also shed (4-6). Recently, human fetal fibroblast surface calreticulin has been implicated as a fibrinogen receptor which, upon occupancy, stimulates cell proliferation (7). A putative isoform of calreticulin binds the motif KxGFFKR in the cytoplasmic tail of a integrins and co-localizes with integrins in focal contacts (8); that isoform maintains b1 integrins primed for ligand engagement (9). The isoform itself, endocalreticulin, has not been directly detected, perhaps because of low concentration and lability. Ectocalreticulin also cooperates with b1 integrins but plays a different role from endocalreticulin. Several cell lines, including mouse B16 melanoma cells, efficiently attach to but fail to spread on laminin which had been synthesized without its customary repertoire of Nlinked carbohydrates (10). Spreading of the spherical laminin-adherent cells begins, within minutes, when laminin carbohydrates are restored (11). This receptormediated response is due to ectocalreticulin which recognizes laminin oligomannosides (12-14). Ectocalreticulin cannot, by itself, initiate cell adhesion and spreading but must act simultaneously with, or sequential to integrin engagement (11). Prior to cell adhesion, ectocalreticulin and integrin diffusely cover the outer cell surface (12, 13); following cell adhesion to laminin, podosomal focal contacts appear and contain concentrated ectocalreticulin (and endocalreticulin) and b1 integrin (12-14). Subsequent studies, described here, indicate that in pre-adherent cells ecto- and endocalreticulin and integrin heterodimer are affiliated in a functional complex which spans the plasma membrane. MATERIALS AND METHODS Surface biotinylation and immunocapture. B16 mouse melanoma cells were surface biotinylated as previously described (13), followed by cell lysis in 0.02M Tris, pH 7.4, 0.1 M NaCl, 1% CHAPS, 5 mM
354
0006-291X/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
Vol. 232, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CaCl2 , 2 mg/ml leupeptin, 0.4 mg/ml antipain, 2 mg/ml benzamidine, 2 mg/ml aprotinin, 1 mg/ml chymostatin, 1 mg/ml pepstatin, and 1 mM PMSF. The lysate was cleared by centrifugation at 10,000 1 g for 30 min. Immunocapture was carried out by standard protocols. Briefly, cell lysates were initally incubated with pre-immune sera and protein A beads to remove non-specific proteins; after removal of the beads by pelleting, anti-calreticulin or anti-b1 integrin antiserum and protein A beads were added to the lysate which was rotated overnight in the cold. The beads were harvested and exhaustively washed to remove non-specific proteins. The immunocaptured proteins were solubilized and separated by SDS/PAGE in 5-15% linear gradient gels, using a standard buffer system and then transferred to a membrane for immunoblotting. Immunoreactive proteins were visualized using ECL. Antibodies used were: anti-calreticulin, Affinity BioReagents, Golden, CO; anti-calreticulin peptides, amino acids 7-24 and amino acids 383-400, R. Sontheimer; anti-KDEL docking protein (KDEL-R), H.-D. So¨ling; anti-a6 integrin, American Research Products, Belmont, MA, and V. Quaranta; anti-b1 integrin, Pharmingen, San Diego, CA ; anti-ubiquitin, Sigma, St. Louis, MO. Cell membrane fractionation and focal contact formation. Cells in suspension were disrupted by mechanical means after osmotic swelling and cellular membranes were partitioned in a discontinuous sucrose density gradient (15). Membranes in each density phase were pelleted, washed three times with isotonic buffer, then subjected to SDS/PAGE and immunoblotting. Focal contacts were induced in suspended cells using laminin-coated paramagnetic microspheres followed by disruption of the cells and isolation of focal contacts adherent to harvested microspheres (16). Focal contact proteins on harvested microspheres were solubilized in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM PMSF), separated by SDS/PAGE and detected by immunblotting. Plasma membranes, from a wash fraction, were similarly analyzed. Cell adhesion, without focal contact formation, was induced in suspended cells using wheat germ agglutinin (WGA)-coated paramagentic microspheres followed by disruption of the cells and isolation of plasma membranes adherent to harvested microspheres (17).
RESULTS Complex immunocapture and calreticulin isoforms. The calreticulin-integrin complex is immunocaptured by antibody to either calreticulin or integrin; the complex contains ecto- and endocalreticulins, a6b1 integrin, KDEL docking protein (KDEL-R), plus two unidentified proteins (Fig. 1). Each of these proteins, except for endocalreticulin, is accessible to cell surface biotinylation; the KDEL docking protein signal is faint because most of the protein is intramembranous and unavailable for biotinylation; other experiments (Fig. 2A) confirm that ecto- and endocalreticulins are associated with plasma membranes, ER membranes and Golgi membranes; the relative abundance of both calreticulins varies in those cellular locations. Importantly, although both calreticulins are associated with plasma membranes, only ectocalreticulin is accessible to cell surface labeling. Concerning this point, we had previously optimized B16 cell biotinylation and shown that a consistent population of surface proteins is biotinylated, and that biotinylation did not label intracellular proteins (13). As for antibody specificity, we had shown that Affinity BioReagents anti-calreticulin antiserum
FIG. 1. Proteins of the calreticulin-integrin complex obtained from suspended cells. SDS/PAGE gels in panel A were performed in the presence of DTT. (A) Immunocaptured complex, obtained using anti-b1 integrin antibody. Lane 1, detection of surface-biotinylated proteins in the complex (streptavidin-HRP probe); the location of the integrin subunits was determined by parallel immunoblots. Lane 2a, detection of calreticulins in the complex by anti-calreticulin immunoblotting. Note that 52 kDa calreticulin did not become cell surfacebiotinylated. Lane 2b, detection of KDEL-R in the complex, immunoblotted with anti-KDEL-R antibody. (B) Detection of a6 integrin in the complex, immunocaptured with anti-calreticulin antibody and immunoblotted using anti-a6 antibody.
does not recognize proteins other than calreticulins in total cell lysates (13). Cells quickly release ectocalreticulin but do not release endocalreticulin (Fig. 2B); cells which have been surface biotinylated prior to incubation release biotinylated ectocalreticulin (data not shown). The composite results imply that surface shedding accounts for ectocalreticulin release, probably due to detachment from KDEL-R, perhaps by KDEL proteolysis. Prior studies, using metabolic labeling of calreticulin, support surface shedding as the release mechanism in B16 cells (5). Endocalreticulin becomes ubiquitinated when cells adhere. When B16 cells adhere to laminin they form podosomal focal contacts (13). When focal contacts are induced on suspended cells by laminin-coated microspheres, plasma membrane endocalreticulin disappears and two new immunoreactive species, 75 kDa and 125 kDa, appear in the focal contacts (Fig. 3A). The 75 kDa species, by itself, is also present in plasma membrane surrounding the focal contacts (data not shown). When suspended cells adhere to WGA-coated microspheres, a circumstance in which cells do not form focal contacts, endocalreticulin disappears and only the 75 kDa species appears in the plasma membrane (Fig. 3A). Separate experiments show that the 75 and 125 kDa species do not become surface-labeled, whether cells are biotinylated before or after adhesion (data not shown). Both species are ubiquitinated (Fig. 3B); additional studies show that the 75 kDa species is slow to react with C-terminal anti-calreticulin antibodies (Fig.
355
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
Vol. 232, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Relative abundance of endo- and ectocalreticulin in cell compartments obtained from suspended cells. (A) B16 cells were disrupted and cell membranes were partitioned by density gradient centrifugation, followed by membrane pelleting and sequential washes, then SDS/ PAGE (/DTT) of pelleted membranes, and anti-calreticulin immunoblotting. (B) Release of calreticulin into the culture medium by B16 cells during a 20 min incubation period. Spent medium was concentrated, followed by SDS/PAGE (/DTT) and immunblotting with anticalreticulin antibody.
3C), probably as a consequence of epitope masking by attached ubiquitin molecules. Ectocalreticulin appears as a doublet in these experiments, perhaps due to variable N-glycosylation (18). DISCUSSION The composite results, plus extant information, suggest a model for the calreticulin-integrin complex which accounts for cooperative activities between endocalreticulin, ectocalreticulin, and integrin (Fig. 4). Endocalreticulin’s putative role is to keep integrins primed for ligand engagement (8, 9). Integrins must be activated by cells in order to bind to the appropriate ligands; by itself, expression of an integrin on the cell surface is inadequate for adhesion (19, 20). The path-
ways by which cells activate integrins remain inadequately defined and one attractive model postulates that cell-type-specific energy-dependent cytoplasmic signals reach integrin cytoplasmic domains (21) - and are then propagated to an integrin’s ligand engagement site, increasing its avidity. Integrin-bound endocalreticulin presumably sustains that activated state. In contrast, ectocalreticulin’s role is that of a lectin receptor, acting in concert with a6b1 integrin to initiate spreading of laminin-adherent cells (11-14). The apparent conversion of endocalreticulin into ubiquitinated species when cells adhere implies that integrin engagement sends a transmembrane signal, triggering the conversion. Two different ubiquitinated endocalreticulins are formed, one of which is restricted to focal contacts. These observations imply high speci-
FIG. 3. Effects of microbead adhesion to plasma membranes on cell surface calreticulins. (A) Left frame, immunoblotted calreticulins of pre-adherent plasma membranes of suspended cells; Upper frame, immunoblotted calreticulins harvested from WGA-coated microspheres adherent to plasma membranes of suspended cells; Lower frame, immunoblotted calreticulins harvested from laminin-coated microspheres adherent to plasma membranes of suspended cells. (B) Immunobloted focal contact proteins harvested from laminin-coated microspheres adherent to plasma membranes of suspended cells, using anti-ubiquitin antibody. (C) Immunoblotted calreticulins harvested from WGAcoated microspheres adherent to plasma membranes of suspended cells. Lane 1, antibody vs. calreticulin peptide, amino acids 383-400; Lane 2, antibody vs. calreticulin peptide, amino acids 7-24. 356
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
Vol. 232, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 4. Postulated calreticulin-integrin bi-directional signaling complex on the cell surface. Endocalreticulin binds the a6 integrin subunit cytoplasmic tail at KxGFFKR (8), keeping integrin heterodimer primed for ligand engagement (9). Ectocalreticulin putatively binds integrin heterodimer, is most likely anchored to KDEL docking protein (31), engages laminin oligomannosides (11-14) and probably signals to a6b1 integrin which transmits the signal to the cytoskeleton, triggering cell spreading.
ficity - consistent with current views of ubiquitination processes (22-24). Although ubiquitinated proteins are generally thought to be marked for destruction (22, 23), accumulating evidence suggests that selective removal of ubiquitin may occur, regenerating the original protein (24). A regulatory cycle, analogous to protein phosphorylation and de-phosphorylation, can be envisioned in which ubiquitinated endocalreticulins may have diminished avidity for the consensus motif in the cytoplasmic tail of integrin a subunits. If cells detach from laminin, selective de-ubiquitination could replenish endocalreticulin in order for it to sustain another round of integrin activation and subsequent cell attachment. This concept is consistent with the observations that ubiquitinated endocalreticulins remain associated with plasma membranes of adherent cells and do not become surface biotinylated, suggesting that ubiquitinated endocalreticulins continue to be bound at the cytoplasmic face of the membranes. Ectocalreticulin and endocalreticulin must be different molecules, given their opposite trans-membrane locations and individual molecular sizes. Endocalreticulin is probably translated from an alternative mRNA transcript of the single calreticulin gene, the mRNA arising either from a unique start site or from alternative splicing. At the very least, endocalreticulin must lack a signal sequence since it does not seem to enter the ER lumen; its smaller size, compared to ectocalreticulin, also implies that it may lack additional domains; in this regard, more than one calreticulin mRNA has been detected (25, 26). Most likely, the two calreticulins independently com-
bine with newly synthesized nascent a6b1 integrin, endocalreticulin binding the a6 cytoplasmic tail, and intralumenal ectocalreticulin, as an endoplasmic reticulum molecular chaperone (27-30), binding nascent a6b1 integrin hydrophobic domains. The composite complex, preserving its transmembrane topography, could then be translocated to the cell surface. Conceivably, the signal for translocation may be endocalreticulin attachment, which may ‘‘mark’’ the complex, enabling it to avoid Golgi recycling of intralumenal KDEL-R anchored ectocalreticulin (31). Smalheiser (32) has proposed that an initial ‘‘accidental’’ transit of proteins to cell locations other than their primary location may sometimes have imparted an evolutionary advantage to cells. Thus, the inaugural ‘‘leakage’’ of intracellular calreticulin-integrin complex to the cell surface may have conferred new properties to certain cells, enhancing their survival. Evolutionary pressure could then ensure that such cells have a sustained selective advantage. ACKNOWLEDGMENTS Research funding provided by National Institutes of Health Research Grant AR-17720, American Cancer Society Institutional Grant 152J-129, and a UCONN Faculty Research Grant. We are grateful to Drs. H.-D. So¨ling, R. Sontheimer, and V. Quaranta for antibodies to KDEL docking protein, calreticulin, and a6 integrin, respectively. We thank Drs. William Upholt and Stephen Helfand for critically reading the manuscript. A portion of this work was presented in poster form (Zhu, Q., Zelinka, P., White, T., and Tanzer, M. L. (1996) Mol. Biol. Cell 7, 66a).
REFERENCES 1. Malhotra, R. (1993) Behring Inst. Mitt. 93, 254–261. 2. Miyamura, K., Reid, K. B. M., and Holmskov, U. (1994) Trends Glycosci. Glycotech. 6, 286–309. 3. Eggleton, P., Lieu, T. S., Zappi, E. G., Sastry, K., Coburn, J., Zaner, K. S., Sontheimer, R. D., Capra, J. D., Ghebrehiwet, B., and Tauber, A. I. (1994) Clin. Immunol. Immunopath. 72, 405– 409. 4. Gersten, D. M., Bijwaard, K. E., Law, L. W., and Hearing, V. J. (1990) Biochim. Biophys. Acta 1096, 20–25. 5. Gersten, D. M., Moody, D., Vieira, W. D., Law, L. W., and Hearing, V. J. (1992) Biochim. Biophys. Acta 1138, 109–114. 6. Bijwaard, K., Hearing, V. J., Sontheimer, R., Lieu, T. S., Vieira, W. D., and Gersten, D. M. (1995) Melanoma Res. 5, 327–336. 7. Gray, A. J., Park, P. W., Broekelmann, T. J., Laurent, G. J., Reeves, J. T., Stenmark, K. R., and Mecham, R. P. (1995) J. Biol. Chem. 270, 26602–26606. 8. Leung-Hagesteijn, C. Y., Milankov, K., Michalak, M., Wilkins, J., and Dedhar, S. (1994) J. Cell Sci. 107, 589–600. 9. Coppolino, M., Leung-Hagesteijn, C., Dedhar, S., and Wilkins, J. (1995) J. Biol. Chem. 270, 23132–23138. 10. Dean, J. W., Chandrasekaran, S., and Tanzer, M. L. (1990) J. Biol. Chem. 265, 12553–12562. 11. Chandrasekaran, S., Tanzer, M. L., and Giniger, M. S. (1994) J. Biol. Chem. 269, 3356–3366. 12. Chandrasekaran, S., Tanzer, M. L., and Giniger, M. S. (1994) J. Biol. Chem. 269, 3367–3373.
357
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC
Vol. 232, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
13. White, T. K., Zhu, Q., and Tanzer, M. L. (1995) J. Biol. Chem. 270, 15926–15929. 14. McDonnell, J., Jones, G. E., White, T. K., and Tanzer, M. L. (1996) J. Biol. Chem. 271, 7891–7894. 15. Vidugiriene, J., and Menoe, A. K. (1994) J. Cell Biol. 127, 333– 341. 16. Plopper, G., McNamee, H. P., Dike, L. E., Bojanowski, K., and Ingber, D. E. (1995) Mol. Biol. Cell 6, 1349–1365. 17. Warnock, D. E., Roberts, C., Lutz, M. S., Blackburn, W. A., Jr, W. W. Y., and Baenzinger, J. U. (1993) J. Biol. Chem. 268, 10145–10153. 18. Jethmalani, S. M., and Henle, K. J. (1994) Biochem. Biophys. Res. Commun. 205, 780–787. 19. Hynes, R. O. (1992) Cell 69, 11–25. 20. Diamond, M. S., and Springer, T. A. (1994) Current Biology 4, 506–517. 21. Schwartz, M. A., Schaller, M. D., and Ginsberg, M. H. (1995) Ann. Rev. Cell Dev. Biol. 11, 549–599.
22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.
Ciechanover, A. (1994) Cell 79, 13–21. Hochstrasser, M. (1995) Current Opinion Cell Biol. 7, 215–223. Jennissen, H. P. (1995) European J. Biochem. 231, 1–30. Treves, S., Zorzato, F., and Pozzan, T. (1992) Biochem. J. 287, 579–581. Liu, N., Fine, R. E., and Johnson, R. J. (1993) Biochim. Biophys. Acta 1202, 70–76. Nauseef, W. M., McCormick, S. J., and Clark, R. A. (1995) J. Biol. Chem. 270, 4741–4747. Peterson, J. R., Ora, A., Van, P. N., and Helenius, A. (1995) Mol. Biol. Cell 6, 1173–1184. Spiro, R. G., Zhu, Q., Bhoyroo, V., and So¨ling, H.-D. (1996) J. Biol. Chem. 271, 11588–11594. Wada, I., Imai, S., Kai, M., Sakane, F., and Kanoh, H. (1995) J. Biol. Chem. 270, 20298–20304. Nilsson, T., and Warren, G. (1994) Current Opinion Cell Biol. 6, 517–521. Smalheiser, N. R. (1996) Mol. Biol. Cell 7, 1003–1014.
358
AID
BBRC 6195
/
6922$$$401
02-20-97 08:22:58
bbrcg
AP: BBRC