Divalent cations regulate the folding and activation status of integrins during their intracellular trafficking

Integrins are divalent cation-dependent, αβ heterodimeric adhesion receptors that control many fundamental aspects of cell behaviour by bi-directional signalling between the extracellular matrix and intracellular cytoskeleton. The activation state of cell surface integrins is tightly regulated by divalent cation occupancy of the ligand-binding pocket and by interaction with cytoplasmic adaptor proteins, such as talin. These agents elicit gross conformational changes across the entire molecule, which specify the activation state. Much less is known about the activation state of newly synthesised integrins or the role of cations during the early folding and trafficking of integrins. Here we use a number of well-characterised, conformation-specific antibodies to demonstrate that β1-integrins adopt the bent, inactive conformation after assembly with α-integrins in the endoplasmic reticulum. Folding and assembly are totally dependent on the binding of Ca2+ ions. In addition, Ca2+ binding prevents integrin activation before its arrival at the cell surface. Activation at the cell surface occurs only following displacement of Ca2+ with Mg2+ or Mn2+. These results demonstrate the essential roles played by divalent cations to facilitate folding of the β-integrin subunit, to prevent inappropriate intracellular integrin signalling, and to activate ligand binding and signalling at the cell surface.


Introduction
The expression of functional integrin molecules is a highly orchestrated process that begins with the assembly of integrin heterodimers in the endoplasmic reticulum (ER), and ends with priming and ligand-induced activation of the molecule at the cell surface (Hynes, 2002). Despite the structural heterogeneity exhibited by the 24 different mammalian integrin dimers, the processes of priming and activation are thought to be conserved. Throughout these processes, changes in the conformation of the molecule have a key role in both facilitating intracellular trafficking and in converting a passive receptor at the cell surface into a highaffinity, highly specific adhesion molecule. These changes in conformation involve transition from an inactive form with low affinity for ligand, to a primed form with high affinity for ligand, to a fully activated ligand-bound cell adhesion receptor (Luo and Springer, 2006). Each stage in the activation process is characterised by gross conformational changes in the integrin structure. The inactive form is thought to exist as a hairpin or bent structure with the globular ligand-binding domains facing the membrane. Upon priming, there is a dramatic straightening of the molecule, which forms a more extended structure, with the binding domains now protruding from the membrane. Evidence to support such a switchblade movement of the integrin ectodomain comes from the crystal structure of the V3 (Xiong et al., 2001) and  IIb  3 (Zhu et al., 2008) integrins, which have a bent conformation that is considered to be the inactive form. Furthermore, electron microscopy, hydrodynamic volume and antibody epitope-mapping studies have revealed the transition between a bent and extended form upon priming and ligand binding (Beglova et al., 2002;Takagi et al., 2002). Although our understanding of the events that underlie affinity regulation has significantly advanced in the last few years, there are still crucial aspects of the process that remain unclear. During inside-out signalling, integrin activation is regulated by binding of intracellular proteins such as talin to the -integrin cytoplasmic tail (Tadokoro et al., 2003), which leads to the separation of the and -integrin legs (Vinogradova et al., 2002;Luo et al., 2004;Anthis et al., 2009) and increased affinity for ligand (Luo and Springer, 2006). Because intracellular signalling molecules could bind to integrins and activate them inside the cells, it is still not known how cells avoid unwanted intracellular signalling. Above all, we do not know the conformational state of newly synthesised integrin molecules and whether or not they become primed or activated inside the cell.
Integrins contain several cation-binding sites that regulate the ligand-binding affinity of the receptor. Different cations have markedly different effects on ligand affinity: in general, Mn 2+ supports ligand binding, Mg 2+ does so to a lesser extent, and Ca 2+ does not support ligand binding at all (Gailit and Ruoslahti, 1988). It has also been proposed that divalent cations can themselves cause pronounced conformational changes that result in a shift in the equilibrium between the active and inactive forms (Mould et al., 1995). It is clear that ligand binding and cation binding are intimately linked because all of the regions implicated in ligand recognition lie at, or close to, cation-binding sites (Xiong et al., 2001;Xiong et al., 2002). Ca 2+ and Mg 2+ are the predominant physiological cations present in the cells in mM concentrations (Montero et al., 1995;Laurant and Touyz, 2000), with Mn 2+ present at much lower levels (1-14 M) (Schramm and Brandt, 1986;

Summary
Integrins are divalent cation-dependent,  heterodimeric adhesion receptors that control many fundamental aspects of cell behaviour by bi-directional signalling between the extracellular matrix and intracellular cytoskeleton. The activation state of cell surface integrins is tightly regulated by divalent cation occupancy of the ligand-binding pocket and by interaction with cytoplasmic adaptor proteins, such as talin. These agents elicit gross conformational changes across the entire molecule, which specify the activation state. Much less is known about the activation state of newly synthesised integrins or the role of cations during the early folding and trafficking of integrins. Here we use a number of well-characterised, conformation-specific antibodies to demonstrate that 1-integrins adopt the bent, inactive conformation after assembly with -integrins in the endoplasmic reticulum. Folding and assembly are totally dependent on the binding of Ca 2+ ions. In addition, Ca 2+ binding prevents integrin activation before its arrival at the cell surface. Activation at the cell surface occurs only following displacement of Ca 2+ with Mg 2+ or Mn 2+ . These results demonstrate the essential roles played by divalent cations to facilitate folding of the -integrin subunit, to prevent inappropriate intracellular integrin signalling, and to activate ligand binding and signalling at the cell surface. Smith et al., 1994). Although the binding of cations to integrin at the cell surface is well established, very little is known about the timing of cation association during biosynthesis and trafficking through the secretory pathway.
In humans, the biosynthesis of the integrin heterodimers begins with the selective assembly of one of the 18 -integrin subunits with one of the 8 -integrin subunits in the ER. Both subunits require binding to their partner to ensure correct folding Lu et al., 1998). To ensure a level of quality control of cell surface receptors, integrin heterodimers cannot be transported from the ER to the plasma membrane unless they have attained their native structure (Ho and Springer, 1983;Kishimoto et al., 1987). However, constitutively active integrins can be expressed at the cell surface (Larson et al., 1990), demonstrating that the quality control does not discriminate between inactive and primed conformations. The ability of cells to allow only heterodimers with the ability to become primed to exit the ER is a crucial aspect of integrin biosynthesis.
In this study, we characterised various conformational states of 1-integrins during biosynthesis and trafficking to the cell surface using a variety of conformation-specific antibodies. We found that, after assembly of the and -integrins in the ER, integrin heterodimers adopt the bent, inactive conformation, which is dependent on the binding of Ca 2+ . We show that Ca 2+ binding in the ER is essential for correct folding and assembly, and to maintain intracellular integrin in a bent conformation until it reaches the cell surface. Taken together, these results demonstrate that Ca 2+ has a crucial role in integrin folding, assembly and trafficking, and maintains the receptors in an inactive form until they reach the cell surface.

Probing the folding and assembly of integrins using monoclonal antibodies
Many monoclonal antibodies have been raised that are reactive towards a variety of conformations of 1-integrins (Mould, 1996;Humphries, 2000;Byron et al., 2009). Our initial aim was to use a selection of these antibodies to determine the folding, assembly and trafficking of integrins. Antibodies were first characterised in terms of their conformation specificity. Radiolabelled 1-subunits were generated by expressing the protein in an in vitro translation system supplemented with semi-permeabilised (SP) HT1080 cells (Wilson et al., 1995). The translation was carried out under reducing conditions to generate material that was translocated into the ER of SP-cells but which failed to fold correctly because of a lack of disulphide bond formation (Jessop et al., 2007). In addition, translation was carried out in the absence of a reductant to allow disulphide formation and correct folding. Finally, correctly folded 1-subunit was denatured with SDS in the presence or absence of reductant to generate denatured 1-subunits with intact or reduced disulphide residues. The various forms of 1-subunit were then immunoisolated with four different monoclonal antibodies, 8E3, 9EG7, TS2/16 and JB1A, which were previously reported to exhibit a wide range of functional activities.
A translation product synthesised in the absence of reducing agent with an approximate molecular weight of 100 kDa was immunoisolated by all the antibodies tested (Fig. 1A,lanes 1,5,9,13). This protein has been shown previously to be fully translated, glycosylated 1-subunit (Jessop et al., 2007). In addition, JB1A, but none of the other antibodies, recognised a translation product with an approximate molecular weight of 80 kDa that 1673 Divalent cations regulate integrin trafficking corresponds to unglycosylated, non-translocated 1-subunit (Fig.  1A, lanes 13-16). When translation was performed in the presence of reductant, only JB1A recognised the synthesised translation product (Fig. 1A, lane 14). Denatured 1-subunit with and without the reduced disulphides was immunoisolated by 8E3 and JB1A (Fig. 1A,lanes 3,4,15,16), whereas 9EG7 only immunoisolated the denatured protein if the disulphides were still intact (Fig. 1A, lanes 7 and 8). TS2/16 did not recognise any translation products that had been reduced or denatured. These results demonstrate that JB1A recognises all forms of the protein tested and that TS2/16 only recognises non-denatured, folded 1-subunit. The epitope recognised by 8E3 forms during correct folding of the protein and cannot be destroyed by denaturation in SDS, either with or without reducing agent. However, if the protein is prevented from folding following synthesis, the epitope for 8E3 does not form. (A)Human 1-integrin was translated in a reticulocyte lysate containing radiolabelled methionine and SP cells for 2 hours. Translations were carried out in the absence (lanes 1,3,4,5,7,8,9,11,12,13,15,16) or presence (lanes 2,6,10,14) of reducing agent. Translation products were treated with the indicated antibodies under native (lanes 1,2,5,6,9,10,15,16) or denaturing conditions in the absence (lanes 3,7,11,15) or presence (lanes 4,8,12,16) of added DTT. Isolated translation products were separated by reducing SDS-PAGE and visualised by exposure to film. Radiolabelled ER form of 1integrin (1Ј) is indicated. (B)HT1080 cells were radiolabelled for 30 minutes and chased for a further 2 hours. Cell lysates were treated with the indicated antibodies under native (lanes 1,4,7,10) or denaturing conditions in the absence (lanes 2,5,8,11) or presence (lanes 3,6,9,12) of added DTT. Isolated proteins were separated by reducing SDS-PAGE and visualised by exposure to film. The identity of radiolabelled proteins is as indicated: , -integrin subunits; 1Ј, ER form of 1-integrin subunit; 1, Golgi form of 1-integrin subunit. Molecular size markers are shown in kDa.
To extend these findings to endogenous newly synthesised 1subunits, HT1080 cells were pulse-labelled for 30 minutes followed by a chase for 2 hours. These conditions are known to allow the assembly of -integrin dimers and their transport to the Golgi complex . Two distinct forms of 1-subunit can be recognised under these conditions: an ER form (1Ј) that migrates in the same position as the fully translocated translation product and a Golgi form (1) that has a slower mobility as a result of modification of the oligosaccharide side chains . Following labelling, 1-subunits were immunoisolated from cell lysates with antibodies as indicated (Fig. 1B). JB1A recognised both the 1 and 1Ј forms of the protein under all conditions tested, confirming that this antibody is not conformation specific. In addition, under native conditions, some slower-migrating radiolabelled proteins were co-immunoisolated with mobility that was similar to that of the -subunits (Fig. 1B, lane 10). HT1080 cells make several -chains, any of which might assemble with 1-subunits. TS2/16 recognised the 1Ј-subunit and -subunit forms and co-immunoisolated assembled -subunits, but 1-subunit recognition was lost after denaturation ( Fig. 1B, lanes 7-9). By contrast, 9EG7 recognised only the 1Ј-subunit under native conditions, but could recognise both the 1Ј and 1 forms following denaturation in the absence, but not presence of reductant ( Fig. 1B, lanes 4-6). Finally, 8E3 recognised the 1Ј-subunit only under native conditions, and weakly recognised both the 1Ј-and subunits under denaturing conditions, irrespective of whether the disulphides had been reduced (Fig. 1B, lanes 1-3). These results demonstrate that the panel of antibodies recognised different conformations of the 1-subunit. Most interestingly, under native conditions, 8E3 and 9EG7 recognised the monomeric ER form; however, the epitopes only become exposed in the Golgi form after denaturation. This observation suggests that the epitopes are present in monomeric 1-integrins, but become lost following assembly with -integrin subunits. The results for 9EG7 extend those published previously which demonstrate that this antibody can recognise an -integrin heterodimer, but only following binding to Mn 2+ ions (Bazzoni et al., 1995). In addition, these two antibodies have been shown to report unbending of 1-integrin molecules during their activation Askari et al., 2010). It is, therefore, highly likely that 9EG7 recognises an epitope in the heterodimer that only becomes exposed following chain unbending or denaturation.

Early folding events during the biosynthesis of 1-integrin
After characterisation of the various forms of 1-integrins recognised by the panel of antibodies, the folding, assembly and trafficking of endogenous protein was then studied in HT1080 cells. HT1080 cells were pulsed with 35 S-labelled amino acids and chased for varying lengths of time (Fig. 2). In most of the time courses, two labelled proteins at approximately 66 kDa and 220 kDa were observed, which bound non-specifically to Protein-G-Sepharose ( Fig. 2A). All the antibodies recognised and isolated the 1Ј-integrin ER form, which diminished in intensity following 60 minutes of chase (Fig. 2B,D-F). The 1-integrin Golgi form was only recognised by TS2/16 and JB1A and appeared after 45 minutes. In addition, -integrins were co-immunoisolated by TS2/16 and JB1A, presumably following assembly with the 1integrin. A precursor-product relationship was seen between the 1Ј and the 1-integrins, as demonstrated by quantification of the TS2/16 autoradiograph (Fig. 2C). As expected, no Golgi form of 1-integrin was recognised by 9EG7 or 8E3 (Fig. 2E,F).
Together, these results indicate that newly synthesised 1integrins acquire their native conformation early, following synthesis in the ER. Exit from the ER and transport to the Golgi results in loss of the 9EG7 and 8E3 epitopes. The co-isolation of -integrins with the ER form of 1-integrin (Fig. 2B, 15 and 30 minute time points) indicates that assembly occurs within the ER and is a prerequisite for exit from the ER. No Golgi-modified chains were recognised and no -integrins were co-immunoisolated by 9EG7 or 8E3 indicating that assembly with the -integrin prevents their reactivity towards these antibodies. Hence newly synthesised, Golgi-localised 1-integrin molecules exhibit an inactive, bent conformation.

Endogenous 1-integrin is mainly in the inactive conformation
To extend studies with radiolabelled 1-integrin subunits that report the conformation of newly synthesised protein, the activation status of the total cellular integrin complement was determined. 1integrin receptors were immunoisolated from cell lysates using 9EG7 and isolated 1-integrins detected by western blotting with JB1A (Fig. 3A). As the 9EG7 epitope has been shown to be cation sensitive (Bazzoni et al., 1995;Mould et al., 1998), immunoisolation was also carried out following treatment of the lysate with Ca 2+ , Mn 2+ and Mn 2+ in combination with a 50 kDa ligand-binding fragment of fibronectin. No 1-integrins were isolated either when no cations were added, or following the addition of Ca 2+ (Fig. 3A, lanes 1 and 2). However, in the presence of added Mn 2+ ions alone, or in combination with the 50 kDa fragment of fibronectin, 1-integrins were isolated (Fig. 3A, lanes  3 and 4). Both the ER and Golgi forms were identified, which further demonstrates that assembly with the -integrin occurs within the ER, and that even the ER-localised integrin heterodimer adopts a bent conformation. In addition, the majority of 1-integrins in HT1080 cells appeared to be in an inactive conformation because they are not recognised by 9EG7 in the absence of added Mn 2+ .
To differentiate between intracellular and cell surface integrins, cell surface proteins were biotinylated and 1-integrins or Mn 2+ (lane 3). 1-integrin was immunoisolated from cell lysate with 9EG7 and subjected to SDS-PAGE and western blotting with avidin conjugated to peroxidase. Black arrowhead indicates the mature 1-integrin band. 9EG7 binding to cell surface 1 heterodimers in HT1080 cells was analysed by flow cytometry; in presence of (C) Ca 2+ , (D) Ca 2+ and 50K fragment of fibronectin, (E) Mn 2+ and (F) Mn 2+ with 50K fragment of fibronectin. Solid line, 9EG7; dotted line, rat IgG control. (G)HT1080 cells were radiolabelled for 30 minutes and chased for varying lengths of time as indicated. Cell lysis was performed in lysis buffer with 2 mM EGTA to remove Ca 2+ ions and lysates were immunoisolated with 9EG7. Immunoisolated material was separated by SDS-PAGE under reducing conditions and radiolabelled proteins were visualised by autoradiography. Black arrowheads indicate precursor 1-integrin of ~100 kDa (1Ј) and ~125 kDa mature 1-integrin (1). (H)HT1080 cells were radiolabelled for 30 minutes and chased for 2 hours. Cell lysis was performed in lysis buffer containing added EGTA (lane 1), EGTA and Mn 2+ (lane 2) and EGTA and Ca 2+ (lane 3) and lysates were immunoisolated with 9EG7. Immunoisolated material was separated by SDS-PAGE under reducing conditions and radiolabelled proteins were visualised by autoradiography. The identity of radiolabelled proteins is as follows: , -integrin chains immunoisolated with 1-integrin antibody as a result of assembly; 1Ј, precursor 1-integrin subunit; 1, mature 1integrin subunit.
immunoisolated. Isolated, biotinylated proteins were detected by blotting with peroxidase-conjugated avidin (Fig. 3B). Cell lysis was performed either in the absence or presence of Ca 2+ or Mn 2+ . As was the case with total cellular integrins, no 1-integrin subunits were isolated in the absence of added cations or following the addition of Ca 2+ (Fig. 3B, lanes 1 and 2), but in the presence of Mn 2+ , a 125 kDa mature 1-integrin was observed (Fig. 3B, lane  3). In addition, a slightly slower-migrating protein was also seen, which is likely to represent biotinylated -integrins co-isolated with the 1-integrins. This result indicates that most cell surface 1-integrins are in an inactive conformation in HT1080 cells. To confirm these biotinylation results, we also performed flow cytometry on HT1080 cells to analyse 9EG7 binding to cell surface integrins (Fig. 3C-F). Binding of 9EG7 was only observed in the presence of Mn 2+ , with or without protein ligand (Fig. 3E,F). No binding was seen in the presence of Ca 2+ even after the addition of protein ligand (Fig. 3C,D).

Ca 2+ prevents activation of 1-integrins
The lack of activation of 1-integrins suggests that a mechanism exists to maintain the molecule in a bent conformation during trafficking through the cell. The bent inactive conformation could be maintained by binding of an adaptor protein or cation. Integrins contain several cation-binding sites, and binding of distinct divalent cations can regulate the ligand-binding affinity of the receptor at the cell surface. To determine whether cation binding maintains newly synthesised integrins in an inactive state, the time course of integrin biosynthesis was repeated in the presence of EGTA in the lysis buffer to chelate cations such as Ca 2+ . In the presence of EGTA, 9EG7 immunoisolated the ER form of 1-integrin (Fig.  3G). In contrast to the previous time course, 9EG7 also immunoisolated the Golgi form (from 60 minute chase). This result implies that in the absence of cations, the 9EG7 epitope becomes accessible on mature 1-integrin, indicating a role for cations in the prevention of unbending and activation. To determine which cation stabilises the inactive, bent conformation, excess Mn 2+ or Ca 2+ was added to the lysis buffer before immunoisolation with 9EG7 (Fig. 3H). Both Golgi and ER forms of 1-integrins were immunoisolated in the absence and presence of Mn 2+ , together with co-assembled -integrins (Fig. 3H, lanes 1 and 2). The gel was exposed for longer than the time course to reveal the integrins, resulting in an increased background. In the presence of Ca 2+ , only the ER-unassembled form of 1-integrin was immunoisolated (Fig. 3H, lane 3). These results demonstrate that it is the binding of Ca 2+ to the integrin chains that maintains any resulting integrin molecules in a bent conformation before and during trafficking of the receptor to the cell surface.

Ca 2+ is required for folding and intracellular trafficking of 1-integrin
Given the role of Ca 2+ in the maintenance of inactive receptors, it was important to establish when Ca 2+ became bound to 1-integrins and whether it was required for correct folding. To address the role of intracellular Ca 2+ , free Ca 2+ was depleted from the ER by treating cells with low concentrations of thapsigargin, which causes leakage of Ca 2+ from the ER by inhibiting the ER sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase (SERCA) pump (Thastrup et al., 1990). It has been shown previously that treating cells with such a low concentration of thapsigargin depletes free Ca 2+ , preventing the folding of the Ca 2+ -binding LDL receptor, but does not cause a general defect in protein folding (Pena et al., 2010).
HT1080 cells were either untreated or depleted of free Ca 2+ from the ER by treating with thapsigargin during the 30 minute starvation, 30 minute pulse and for various periods of chase (Fig.  4). As previously described, in the absence of Ca 2+ depletion, TS2/16 immunoisolated correctly-folded ER-localised 1-integrins as well as -integrin heterodimers that had been transported to the Golgi (Fig. 4A). However, following Ca 2+ depletion, only a small fraction of 1-integrins were isolated by this antibody (Fig.  4B). However, 1-integrins were still recognised by 9EG7 following Ca 2+ depletion (Fig. 4D). In contrast to the situation in the absence of Ca 2+ depletion, the ER form was immunoisolated throughout the time course by 9EG7, indicating that no ER to Golgi transport had occurred. In support of this conclusion, JB1A immunoisolated only the ER form of 1-integrins throughout the time course, with no Golgi form observed or co-isolation of integrins (Fig. 4F). These results demonstrate a dramatic effect of Ca 2+ depletion on 1-integrin folding, assembly and trafficking. Folding of the monomer was compromised, because very little reactivity was seen with TS2/16. The 9EG7 epitope was formed, but no transport was seen from the ER and no assembly with integrin chains. These results indicate that Ca 2+ is required early in the folding pathway of the integrin receptor, is loaded onto the molecule in the ER and is required to ensure assembly with the integrin subunit.
To further explore the assembly of the 1-integrin with a specific -integrin subunit, immunoisolation was carried out with the 5integrin-specific monoclonal antibody mAb11 (Clark et al., 2005) (Fig. 5A). Assembly of 51-integrin was seen in HT1080 cells as evidenced by the co-isolation of 1-integrins with mAb11 (Fig.  5A, lanes 3,4). In Ca 2+ -depleted cells, only the 5-integrin band was observed (Fig. 5A, lanes 5-8), showing lack of 51-integrin assembly in absence of Ca 2+ . These results demonstrate that correct folding and association of 5-integrin with 1-integrin subunits requires Ca 2+ in the ER and that depletion of Ca 2+ inhibits trafficking of 51-integrin molecules to the Golgi.
It has been shown previously that the folding of influenza virus haemagglutinin is not affected by the conditions of Ca 2+ depletion used in our experiments (Pena et al., 2010). To determine the effect of Ca 2+ depletion on the folding machinery in the ER, the folding, assembly and Golgi transport of endogenous MHC Class I molecules was studied. During the biosynthesis of MHC Class I molecules, the heavy chain is first translocated across the ER membrane before assembly with 2-microglobulin and loading with peptide in the ER (Morrice and Powis, 1998). Both the initial assembly with 2microglobulin, peptide loading and the subsequent transport to the Golgi is dependent upon the action of Ca 2+ -dependent ER chaperones, such as calnexin (Tector and Salter, 1995) and calreticulin (Sadasivan et al., 1996). Following pulse labelling of HT1080 cells, 2microglobulin was immunoisolated to determine whether it was associated with the assembled heavy chain. In the absence of Ca 2+ depletion, MHC Class I heavy chain was co-isolated at both 0 and 120 minutes of chase (Fig. 5B). After Ca 2+ depletion, heavy chain was still isolated, demonstrating that thapsigargin treatment did not grossly affect the ER folding machinery. In addition, a conformationspecific antibody (W6/32) that only recognises correctly assembled and peptide-loaded MHC Class I molecules (Barnstable et al., 1978), was able to immunoisolate heavy chains even after Ca 2+ depletion (Fig. 5C). To follow transport of newly synthesised MHC Class I molecules to the Golgi, the immunoisolated heavy chains were treated with endoglycosidase H (Endo-H). In the absence of Ca 2+ depletion, heavy chains were Endo-H sensitive after 0 minutes of chase (Fig. 5C, lane 2). Heavy chains become Endo-H resistant after 120 minutes of chase (Fig. 5C, lane 4), indicating modification of their oligosaccharide side chains in the Golgi. Similarly, in thapsigargin-treated cells, MHC class I molecules were Endo-H sensitive after 0 minutes of chase, but became resistant to digestion after 120 minutes, indicating their successful transport to the Golgi. As the Ca 2+ depletion had little effect on the folding of MHC Class I, we conclude that the dramatic effect on 1-integrin folding and assembly was due to the requirement for Ca 2+ during folding of this protein in the ER, rather than a general defect in the protein folding machinery.

Discussion
In this study, we used conformation-sensitive monoclonal antibodies to track the folding and assembly of 1-integrin, and investigate the conformational state of newly synthesised integrin molecules. Our major findings are: (1) 1-integrins adopt a bent, inactive conformation after assembly with an -integrin subunit in the ER, before transport to the Golgi; (2) correct folding and assembly are dependent on the binding of Ca 2+ ions, which have to be present from the start of the process; and (c) the integrin molecule remains in an inactive form throughout the secretory pathway. Although the role of cations during the activation of integrins at the cell surface is well established, our results show that the binding of cations is also important to prevent any activation during intracellular trafficking.  1-4) or presence (lanes 5-8) of thapsigargin. Cell lysates were collected at varying time points as indicated, and 5-integrin subunits were immunoisolated with mAb11 and then subjected to SDS-PAGE and autoradiography. The identity of radiolabelled proteins is as described previously. Arrow indicates non-specific protein bands that were pulled down by Protein-G-Sepharose alone. (B,C)HT1080 cells were starved and pulse-labelled for 30 minutes, and chased for up to 2 hours in the absence or presence of thapsigargin as indicated. (B)Cell lysates were collected and immunoisolated with an antibody against 2-microglobulin and then subjected to SDS-PAGE and autoradiography. Black arrowhead indicates MHC I co-precipitated with 2-microglobulin. (C)Cell lysates were prepared and treated with Endo-H as indicated and then immunoisolated with anti-MHC I antibody W6/32. Black arrowhead indicates undigested MHC I and the line below indicates MHC I digested with EndoH enzyme.
Monoclonal antibodies have become essential tools to study the structure and function of integrins because they recognise distinct conformational states of the receptors (Mould, 1996). Many of these antibodies have been used to study changes in the ligandbinding affinity of integrins on the cell surface (Humphries, 2000); however, in this study, we used their specificities to report defined conformational states during 1-integrin biosynthesis. In particular, we noted that two conformation-specific antibodies, 8E3 and 9EG7, which have been shown to recognise the unbent form of 1integrins, also react with monomeric 1-integrin subunits. 9EG7 was of particular interest because it seems to recognise an epitope that requires the formation of a disulphide and once this disulphide is formed, the epitope is not lost even after denaturation. The epitope has been mapped previously to within a cysteine-rich stretch (residues 495-602) at the back of the 1-integrin knee region (Bazzoni et al., 1995). Because this epitope also appears following Ca 2+ depletion in the ER, we can conclude that this region of the integrin can still fold under these conditions. The epitope for TS2/16 has been mapped to the 1-integrin A-domain (Hemler et al., 1984;Tsuchida et al., 1997), therefore the dramatic effect on the assembly of 1-integrin and -integrin in the absence of free Ca 2+ would indicate that a lack of folding of the A-domain results in a lack of assembly and subsequent transport from the ER. Given that we did not see a dramatic effect on the folding of other ER proteins we can conclude that lack of assembly was the direct result of a lack of Ca 2+ binding.
Our results demonstrate that the majority of integrin molecules expressed by HT1080 cells are in an inactivate state, as judged by a lack of 9EG7 epitope recognition. This regulation of activation is very important for their biological function, as is most evident from considering integrins present on circulating blood cells. The major platelet integrin IIb3 is present at high density on circulating platelets, where it is inactive. If it were not, platelets would bind their ligand fibrinogen from the plasma and aggregate. Integrins carry signals from the outside to the inside of the cell and vice versa. In inside-out signalling, integrin activity is regulated by binding of regulatory proteins to the short cytoplasmic domains of integrins . So far, two major protein families, talins and kindlins, have been reported to regulate the activity of integrins from within the cells by binding the tails of the -integrin subunit (Moser et al., 2009). Because these intracellular proteins can bind to integrins and activate them inside the cells, how cells avoid unwanted intracellular signalling is still an unresolved question. Our results suggest tight control of cell surface molecules by cation binding. A similar study has looked at the expression of 9EG7 on various 1-integrin heterodimers at the cell surface, and demonstrated that removal of Ca 2+ with EDTA or EGTA induced expression of the 9EG7 epitope (Bazzoni et al., 1998). This is consistent with other inhibitory effects of Ca 2+ on 1-integrin heterodimers at the cell surface (Sonnenberg et al., 1988;Staatz et al., 1989;Mould et al., 1995). Our results extend these observations and provide evidence that Ca 2+ helps to stabilise an inactive, bent conformation of 1-integrin heterodimers inside the cell. Ca 2+ is a predominant ion present in mM concentrations in the ER (Montero et al., 1995) and many ER-resident proteins involved in protein folding bind Ca 2+ through high-affinity binding sites (Macer and Koch, 1988). This previously unreported intracellular function of Ca 2+ might allow 1-integrin heterodimers to maintain an inactive state in the presence of high free Ca 2+ concentrations in the secretory pathway.
1678 Journal of Cell Science 124 (10) Our pulse-chase experiments using TS2/16 and JB1A antibodies show that the 1-integrin associates with the -integrin before transport from the ER to the Golgi. Such a requirement for assembly before transport is in agreement with previous studies on leukocyte integrin biosynthesis; these showed that the transport of -integrin and -integrin subunits from the ER to Golgi is dependent upon the formation of -integrin heterodimers (Ho and Springer, 1983;Kishimoto et al., 1987). In addition, it has been reported that the -integrin propeller domain of -integrins and the A-domain of integrins associate with one another and both are mutually dependent on the formation of -integrin for folding . The Ca 2+ -binding motifs in integrin -subunits are predicted to be close to one another on the lower surface of the -integrin propeller domain, which, although it is not involved in interaction with the -integrin subunit, appears to stabilise the tertiary structure of this fold (Tuckwell et al., 1992). It is also very interesting that Ca 2+ ions are shown to stabilise integrin association: removal of Ca 2+ resulted in dissociation of integrin and -integrin in detergent-solubilised IIb3 integrin (Jennings and Phillips, 1982) and made and -integrin subunits susceptible to dissociation by high pH in L2 integrin (Dustin et al., 1992). Our results extend these studies by showing that after the depletion of Ca 2+ from the ER, the 1-integrin subunit cannot attain its native fold and does not associate with the -integrin subunit. Furthermore, the transportation of both the and integrins from the ER to the Golgi was blocked in absence of Ca 2+ in the ER, emphasising the importance of these ions for the proper folding and trafficking of 1-integrins. Taken together, our data shed light on integrin folding during biosynthesis and provide strong evidence for a key mechanism by which inappropriate intracellular signalling is prevented.

Metabolic labelling, pulse chase and immunoisolation
Approximately 3ϫ10 6 HT1080 cells were starved of essential amino acids for 30 minutes before pulse labelling for 30 minutes using 11 Ci/ml of [ 35 S]methionine protein labelling mix (PerkinElmer). Cells were washed and incubated in complete DMEM3 medium for various chase times. Cells were lysed in buffer [50 mM Tris-HCl buffer, pH 7.4 containing 1% (v/v) Triton X-100, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA) and 0.5 mM phenylmethylsulphonyl fluoride (PMSF)]. Clarified lysates were incubated for 1 hour at 4°C with Protein-G-Sepharose beads (Sigma) to remove proteins that may bind non-specifically, before being incubated with antibodies with protein-G-Sepharose beads overnight at 4°C. Beads were washed three times with RIPA buffer (50 mM Tris-HCl buffer, pH 8.0 containing 1% (w/v) deoxycholic acid, 0.1% (w/v) SDS, 1% (v/v) Triton X-100, 500 mM NaCl and 0.5 mM PMSF) before being resuspended in SDS-PAGE sample buffer containing 50 mM DTT and boiling for 5 minutes. Samples were separated by SDS-PAGE under reducing conditions on a 7% Tris-acetate gel, which was dried and exposed to Kodak BioMax MR film (GRI, Braintree, UK).

In vitro transcription and translation
Human 1-integrin was transcribed from a pSPUTK vector linearised with EcoRV. SP6 polymerase was used for transcription reactions. RNA transcripts were translated in a rabbit reticulocyte lysate (Flexilysate, Promega) in the presence of SP-cells and [ 35 S]methionine (PerkinElmer) as described previously (Jessop et al., 2007). Initiation of protein synthesis was allowed to proceed for 5 minutes at 30°C before inhibition with 1 mM ATCA (Sigma), followed by incubation at 30°C for 2 hours to allow elongation and post-translational modification. Translation products were immunoisolated with anti-1-integrin monoclonal antibodies before electrophoresis. Samples were separated by SDS-PAGE under reducing conditions on a 7% Trisacetate gel, which was dried and exposed to Kodak BioMax MR film (GRI).

Biotinylation of cell surface proteins
Approximately 10 7 HT1080 cells were detached and resuspended in 1 ml PBS (without Ca 2+ and Mg 2+ ). NHS-sulfo-Biotin (Sigma) was added to cells at 0.25 mg/ml final concentration and incubated at room temperature for 30 minutes. Cells were washed twice with PBS and then with Tris-buffered saline. Cells were divided into four aliquots and then lysis was performed in lysis buffer (see above) containing different cation compositions [without EDTA and with 1 mM CaCl 2 , MnCl 2 or MnCl 2 with 50 K fragment of fibronectin (10 g/ml)] for 15 minutes at 4°C. Lysates were centrifuged at 14,400 g for 5 minutes. Integrin subunits were immunoisolated with anti-5-integrin or anti-1-integrin monoclonal antibodies before electrophoresis. Samples were separated by SDS-PAGE and blotting was performed using extravidin-conjugated HRP.

Flow cytometry
Cells were detached, washed and resuspended to a final concentration of 10 6 -10 7 cells/ml in HEPES-buffered saline (HBS) containing 4.5 g/l glucose and 1% FBS. Cell aliquots (50 l) were incubated with primary antibody 9EG7 or rat IgG (control) at a final concentration of 10 g/ml in the same buffer supplemented with 1 mM MnCl 2 or CaCl 2 with or without 10 g/ml 50 K fragment of fibronectin for 1 hour at 4°C. Cells were washed in the respective buffers three times and isolated by centrifugation at 1100 g for 30 minutes. FITC-conjugated anti-rat secondary antibody (50 l) diluted to 5 g/ml in PBS + 1% FBS, was added followed by incubation for a further 30 minutes at 4°C. Cells were washed and made up to a final volume of 300 l in HBS before immediate analysis on a Dako Cyan flow cytometer using an excitation wavelength of 488 nm and a 530/40 nm emission filter.

Depletion of Ca 2+ from the ER
To deplete Ca 2+ from the ER, 100 nM of the Ca 2+ -ATPase inhibitor thapsigargin was added to starvation, pulse, and chase media. The cells were starved for 30 minutes and pulse-labelled for 30 minutes with 11 Ci/ml of [ 35 S]methionine protein labelling mix (PerkinElmer). After various chase times, the cells were treated with 20 mM Nethylmaleimide (NEM) to block free sulphydryl groups and prevent any disulfide bond formation and lysis was performed in lysis buffer (see above) with 20 mM NEM. After centrifugation the cell lysates were used for immunoisolation as described above.
The work presented in this paper was funded by The Wellcome Trust grant #082041. We would like to acknowledge Mike Jackson (University of Manchester) who carried out the FACS analysis. Deposited in PMC for release after 6 months.