Laminin Isoforms Containing the γ3 Chain Are Unable to Bind to Integrins due to the Absence of the Glutamic Acid Residue Conserved in the C-terminal Regions of the γ1 and γ2 Chains*

Laminins are the major cell adhesive proteins in basement membranes, and consist of three subunits termed α, β, and γ. Recently, we found that the Glu residue at the third position from the C termini of the γ1 and γ2 chains is critically involved in integrin binding by laminins. However, the γ3 chain lacks this Glu residue, suggesting that laminin isoforms containing the γ3 chain may be unable to bind to integrins. To address this possibility, we expressed the E8 fragment of laminin-213 and found that it was incapable of binding to integrins. Similarly, the E8 fragment of laminin-113 was expressed and also found to be inactive in binding to integrins, confirming the distinction between the integrin binding activities of γ3 chain-containing isoforms and those containing the γ1 or γ2 chain. To further address the importance of the Glu residue, we swapped the C-terminal four amino acids of the γ3 chain with the C-terminal nine amino acids of the γ1 chain, which contain the Glu residue. The resulting chimeric E8 fragment of laminin-213 became fully active in integrin binding, whereas replacement with the nine amino acids of the γ1 chain after substitution of Gln for the conserved Glu residue failed to restore the integrin binding activity. These results provide both loss-of-function and gain-of-function evidence that laminin isoforms containing the γ3 chain are unable to bind to integrins due to the absence of the conserved Glu residue, which should play a critical role in integrin binding by laminins.

Integrins are ␣␤ transmembrane receptors that play critical roles in cell matrix adhesion in multicellular organisms. Several members of the integrin family proteins, including ␣3␤1, ␣6␤1, ␣6␤4, and ␣7␤1, serve as laminin receptors on a variety of cell types (10). The putative binding sites for these integrins have been mapped to the globular (G) 3 domain of the laminin ␣ chains (11)(12)(13)(14)(15)(16), although trimerization with ␤ and ␥ chains is necessary for the G domain to exert its integrin binding activity (17)(18)(19). Recently, we found that the C-terminal regions of the ␥ chains are critically involved in integrin binding by laminins (20). Briefly, deletion of the C-terminal three but not two amino acids of the ␥1 chain completely abrogated the integrin binding activity of laminin-511 (␣5␤1␥1), while substitution of Gln for Glu-1607, the amino acid residue at the third position from the C terminus of the ␥1 chain, also abolished the integrin binding activity; thereby underscoring a critical role of Glu-1607 in integrin binding by this laminin. Furthermore, a Glu residue is conserved between the ␥1 and ␥2 chains at the third position from the C terminus. Deletion of the C-terminal three amino acids from the ␥2 chain or substitution of Gln for this Glu in the ␥2 chain completely abrogated the integrin binding activity of laminin-332 (␣3␤3␥2), suggesting that the same mechanism operates in the modulation of the integrin binding activities of laminins containing either the ␥1 or ␥2 chain.
A novel ␥ chain isoform, ␥3, is the eleventh laminin subunit to be identified (21)(22)(23). Studies on the tissue distribution of the ␥3 chain have shown that it is broadly expressed in the skin, kidney, retina, and testis (21, 22, 24 -26). It has been reported that the ␥3 chain associates with the ␣2 and ␤1 chains to form laminin-213 (␣2␤1␥3) in the placenta (21) and with the ␣3 and ␤3 chains to form laminin-333 (␣3␤3␥3) in adult rat testes (27). The predicted primary and secondary structures of the ␥3 chain suggest that it is more closely related to ␥1 than to ␥2 (21). However, the C-terminal region of the human laminin ␥3 chain consists of only four amino acid residues after the Cys residue conserved among the three ␥ chains, and lacks the Glu residue conserved in the C-terminal regions of the ␥1 and ␥2 chains (see Fig. 1). The unique features of the ␥3 chain, i.e. the short C-terminal tail and the absence of the Glu residue, are conserved among the ␥3 homologues from humans, mice, rats, and zebrafish. Furthermore, there is no evidence for alternative splicing that confers the critical Glu residue on the C-terminal region of the ␥3 chain. The absence of the conserved Glu residue in the ␥3 chain raises the possibility that laminin isoforms containing the ␥3 chain may be unable to bind to integrins, although the ␥3 chain-containing laminins have never been directly examined for their cell adhesive and integrin binding activities.
In the present study, we expressed and purified recombinant laminin isoforms containing the ␥3 chain and several mutant ␥3 chains, and examined their integrin binding and cell adhesive activities. Our results clearly showed that ␥3 chain-containing laminins are devoid of integrin binding and cell adhesive activities, and that the Glu residue at the third position from the C terminus is a prerequisite for laminin recognition by integrins.
Immunoprecipitation and Western Blotting-293-F cells were simultaneously transfected with different combinations of expression vectors encoding various ␣, ␤, and ␥ chains using 293fectin (Invitrogen). At 72 h after transfection, the cells were collected and washed with phosphate-buffered saline. Cell pel- A, schematic diagrams of recombinant laminin, its E8 fragment and the C-terminal amino acid sequences of its ␤1 and ␥1 chains. Cys residues are circled in black, and disulfide bonds are depicted by broken lines. The C-terminal Glu residue of the ␥1 chain is shaded. B, C-terminal amino acid sequences of the human laminin ␥1, ␥2, and ␥3 chains. Cys residues are underlined. The black boxes represent the Glu residues conserved between the ␥1 and ␥2 chains. lets were lysed with SDS sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 6% ␤-mercaptoethanol) and sonicated. The conditioned media were clarified by centrifugation and immunoprecipitated with anti-FLAG M2 agarose (Sigma) for 6 h at 4°C. Equal amounts of cell lysates and immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 4 or 5% gels under reducing or nonreducing conditions, followed by immunoblotting with mAbs against the FLAG tag or individual laminin subunits. Immunoreactive proteins were detected using Can-Get-Signal Immunoreaction Enhance Solution (Toyobo, Tokyo, Japan) and enhanced chemiluminescence (ECL Plus; GE Healthcare, Piscataway, NJ).
Expression and Purification of Recombinant Proteins-Recombinant laminins were produced using the Free-Style TM 293 Expression System (Invitrogen) as described previously (16). Recombinant ␣6␤1 and ␣7X2␤1 integrins were produced using the same expression system and purified from conditioned media using anti-FLAG columns (Sigma) as described previously (10,33). Recombinant E8 fragments of laminin-111, -113, -211, and -213, and their mutants were produced using the same expression system and purified from conditioned media as described previously (20). After dialysis against Tris-buffered saline (TBS; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl), the purities of the recombinant proteins were verified by SDS-PAGE followed by Coomassie Brilliant Blue staining or immunoblotting.
Integrin Binding Assays-Solid-phase integrin binding assays of recombinant E8 fragments and their mutants were performed using purified recombinant ␣6␤1 and ␣7X2␤1 integrins (10,33). Briefly, 96-well microtiter plates were coated with recombinant E8 fragments of laminin-111, -113, -211, and -213 and their mutants at the indicated concentrations. The amounts of the recombinant proteins adsorbed on the plates were quantified with an anti-His 6 mAb to confirm the presence of equal amounts of the adsorbed proteins. After blocking with 3% bovine serum albumin (BSA), the plates were incubated with 20 nM recombinant ␣6␤1 or ␣7X2␤1 integrin in the presence of 5 mM Mn 2ϩ at 37°C for 1 h. After washing with TBS containing 5 mM Mn 2ϩ and 0.05% Tween 20, bound proteins were quantified by sequential incubations with the biotinylated anti-ACID/BASE antibody and horseradish peroxidase-conjugated streptavidin.
Cell Adhesion Assay-Cell adhesion assays were performed as described previously (16,20) using K562 human leukemia cells transfected with a cDNA encoding an ␣7X2 integrin subunit (35), which were a generous gift from Dr. Arnoud Sonnenberg (The Netherlands Cancer Institute, Amsterdam, The Netherlands). The cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum and used without Mn 2ϩ pretreatment. After fixation with formaldehyde and staining with Diff-Quik (International Reagents Corp., Kobe, Japan), attached cells were counted in three independent wells using Scion Image software (Scion Corp., Frederick, MD).

Production of Recombinant Laminins Containing the ␥3
Chain-The ␥3 chain was originally identified in heterotrimers with the ␣2 and ␤1 chains, i.e. laminin-213 (21). To elucidate the role of the ␥3 chain in integrin binding by laminins, we first attempted to express recombinant laminin-213 by cotransfecting 293-F cells with a cDNA encoding the ␥3 chain with an N-terminal FLAG-tag and cDNAs encoding the ␣2 and ␤1 chains. Secretion of endogenous ␥3 chain-containing laminins by 293-F cells was undetectable (data not shown). We also expressed recombinant laminin-211, in which the ␥1 chain was N-terminally FLAG-tagged, in 293-F cells as a control. Immunoblotting with an anti-FLAG mAb detected both the ␥1 and ␥3 chains in cell lysates, whereas only the ␥1 chain was detected in the culture medium after immunoprecipitation of the recombinant ␥ chains with an anti-FLAG antibody (supplemental Fig. S1). Failure of recombinant laminin-213 secretion by cells was also observed after transfection of other cell lines, including JAR human choriocarcinoma cells originating from the placenta, A204 human rhabdomyosarcoma cells originating from skeletal muscle cells and NIH-3T3 cells (data not shown).
Given the scarcity of laminin-213 secreted by transfected 293-F cells, we sought to more comprehensively identify the ␣ and ␤ subunits that could assemble with the ␥3 chain to yield ␣␤␥ heterotrimers for purification of ␥3 chain-containing laminins. We transfected 293-F cells with FLAG-tagged ␥1 chain (positive control) or ␥3 chain in combination with different ␣ (␣1-␣5) and ␤ (␤1-␤3) chains, and analyzed the secreted laminin heterotrimers by immunoprecipitation with an anti-FLAG antibody followed by immunoblotting with antibodies against individual laminin subunit chains.
Immunoblotting with antibodies against ␣ chains demonstrated that laminins containing the ␥1 chain were immunoprecipitated from conditioned media with the anti-FLAG antibody irrespective of the types of ␣ and ␤ chains in the combinations, whereas laminins containing the ␥3 chain were either only faintly detected after immunoprecipitation (laminins containing the ␣1, ␣2, and ␣4 chains) or undetectable even when 8-fold more culture medium was used for immunoprecipitation (laminins containing the ␣3 and ␣5 chains) ( Fig. 2A). It was noted that the amounts of secreted laminin isoforms containing the ␤2 and ␥3 chains (i.e. laminin-123, -223, and -423) were lower than those containing the ␤1 and ␥3 chains (laminin-113, -213, and -413). A similar bias toward ␤1 chain-containing isoforms was also observed between the isoforms containing the ␤1/␥1 and ␤2/␥1 chains, suggesting that the ␤2-containing laminins were less efficient than the ␤1-containing laminins in assembly into heterotrimers and/or secretion of the resulting heterotrimers. Although the antibodies against the ␣3 and ␣5 chains failed to detect ␥3 chain-containing heterotrimers in the immunoprecipitates, this could be due to the limited sensitivities of these antibodies in immunoblotting.
Integrin Binding Activities of the E8 Fragments of Laminin-213 and -113-Because the recombinant ␥3 chain-containing laminins were not amenable for purification in sufficient quantities for functional assays, we set out to produce a recombinant E8 fragment of laminin-213 consisting of the three truncated subunits, modeled after the E8 fragment of laminin-111 (17)(18)(19). The E8 fragment of laminin-211 was also produced as a control. To obtain the heterotrimeric E8 fragments, the E8 frag-ments of both laminin-211 and -213 were enriched by sequential affinity purification using a nickel column that captured the truncated ␣2 chain with an N-terminal His 6 tag and an anti-FLAG column that captured the truncated ␥1 and ␥3 chains with an N-terminal FLAG tag. The authenticities of the resulting recombinant E8 fragments were verified by SDS-PAGE and Coomassie Brilliant Blue staining. Under reducing conditions, each recombinant protein gave three bands, one corresponding to the truncated ␣2 chain and two lower bands corresponding to the truncated ␤1 and ␥1 or ␥3 chains (Fig. 3A). The truncated ␤1 chain of the E8 fragment of laminin-213 gave a band that migrated slightly faster than the truncated ␤1 chain of laminin-211, suggesting that the ␤1 chains were differentially glycosylated or modified by other types of post-translational modifications when assembled with different ␥ chains. Under nonreducing conditions, the E8 fragments of laminin-211 and -213 gave two bands, one corresponding to the truncated ␣2 chain and the other corresponding to a heterodimer of the truncated ␤1/␥1 or ␤1/␥3 chains. The relative intensities of these two bands were essentially the same for the E8 fragments of laminin-211 and -213. These results indicated that both E8  fragments were purified as heterotrimers, although the truncated ␣2 chain was not covalently linked to the truncated ␤1/␥1 and ␤1/␥3 heterodimers.
To further investigate this possibility, we expressed and purified the E8 fragment of laminin-113 (Fig. 4A). The E8 fragment of laminin-111, which is a ligand for ␣6␤1 integrin (10), was also produced as a control. As expected, the E8 fragment of laminin-111 was active in binding to ␣6␤1 integrin, whereas the E8 fragment of laminin-113 was almost devoid of binding activity (Fig. 4B). These results are consistent with the conclusion that laminin isoforms containing the ␥3 chain are incapable of binding to integrins.

Replacement of the C-terminal Region of the ␥3 Chain with the C-terminal Region of the ␥1 Chain Restores Integrin Binding
Activity to Laminin-213-Because the C-terminal tail of the ␥3 chain is truncated and lacks the Glu residue that is conserved between the ␥1 and ␥2 chains and critical for integrin binding activity, we hypothesized that the ␥3 chain-containing laminins would regain integrin binding activities when furnished with a Glu residue at the appropriate position in the ␥3 chain. To address this possibility, we produced two mutant forms of the E8 fragment of laminin-213: one mutant containing the ␥3 chain in which the C-terminal four amino acids were replaced with the C-terminal nine amino acids of the ␥1 chain (designated ␥3 ϩ 9AA), and another mutant containing a similar ␥3 chain to ␥3 ϩ 9AA, except that the Glu residue at the third position from the C terminus was replaced with Gln (designated ␥3 ϩ 9AA(EQ), Fig. 5A). The purities and authenticities of these mutant E8 fragments were verified by SDS-PAGE and Coomassie Brilliant Blue staining of the gels (Fig. 5B). The control and mutant E8 fragments of laminin-213 gave two bands  under nonreducing conditions, one corresponding to the truncated ␣2 chain and another corresponding to a heterodimer of the truncated ␤1 and ␥3 chains. Given that both the ␤1 and ␥3 chains of the E8 fragment contain only one Cys residue conserved near their C termini, these results confirmed that heterodimerization of the ␤1 and ␥3 chains through a disulfide bridge was not compromised by the mutation introduced into the C-terminal region of the ␥3 chain.
As expected, solid-phase binding assays revealed that the mutant E8 fragment containing the ␥3 ϩ 9AA chain was fully active in binding to ␣7X2␤1 integrin (Fig. 5C). The other mutant E8 fragment containing the ␥3 ϩ 9AA(EQ) chain did not show any residual integrin binding activity, similar to the case for the control E8 fragment of laminin-213. These results clearly demonstrated that the failure of ␥3 chain-containing laminins to bind integrins was due to the absence of the C-terminal Glu residue conserved between the ␥1 and ␥2 chains, thereby endorsing the importance of this Glu residue for integrin binding by laminins.
Cell-adhesive Activities of Laminin-213 and Its Mutants-The importance of the Glu residue for integrin binding was further verified by cell adhesion assays using K562 cells stably transfected with ␣7X2 integrin (35). The control E8 fragment of laminin-211 and the E8 fragment of the laminin-213 mutant containing the ␥3 ϩ 9AA chain were fully active in mediating cell adhesion of K562 cells expressing ␣7X2␤1 integrin, whereas the E8 fragments of laminin-213 and its mutant containing the ␥3 ϩ 9AA(EQ) chain were almost devoid of cell adhesive activity (Fig. 6). Control K562 cells that were not transfected with ␣7X2 integrin did not adhere to any of the recombinant E8 fragments, confirming the specificity of the ␣7X2␤1 integrin-dependent cell adhesion. These results further supported our conclusion that ␥3 chain-containing laminins are unable to bind to integrins due to the absence of the conserved Glu residue at the third position from the C terminus.

DISCUSSION
No previous studies have addressed the functions of ␥3 chain-containing laminins at the protein level, other than their immunohistochemical localizations in tissues. Here, we have provided evidence that the ␥3 chain-containing laminins, typically laminin-213, are functionally distinct from other laminin isoforms containing the ␥1 or ␥2 chain, and lack the ability to bind to integrins. Our data have revealed that the failure of the ␥3 chain-containing laminins to bind integrins is due to the absence of the Glu residue conserved in the other ␥ chains at the third position from their C termini, since integrin binding activity was restored to laminin-213 upon substitution of the C-terminal four amino acids of the ␥3 chain with the C-terminal nine amino acids of the ␥1 chain. Only the intact nineamino acid sequence, and not that with substitution of Gln for the Glu residue, could fully restore the integrin binding activity to laminin-213, underscoring the importance of the conserved Glu residue in laminin recognition by integrins. To the best of our knowledge, this is the first demonstration of a functional distinction between the ␥3 chain-containing laminins and other laminin isoforms.
Despite the importance of the Glu residue at the C-terminal regions of the ␥ chains for integrin binding by laminins (Ref. 20 and this study), it remains unclear whether this Glu residue is directly involved in laminin recognition by integrins through coordination with the metal ion within the MIDAS (metal ion-dependent adhesion site) motif of laminin-binding integrins, as has been demonstrated for the Glu residue in the GFOGER integrin-binding motif in collagens (37,38) and the Asp residue in many cell-adhesive proteins containing the RGD motif (39 -42). Given that not only the Glu residue within the ␥1 or ␥2 chain but also the laminin G-like (LG) 1-3 modules of the ␣ chain are indispensable for integrin binding by laminins (11)(12)(13)(14)(15)(16)(17)(18)(19)43), the LG1-3 modules, but not the ␥ chains, may well provide the critical acidic residues that coordinate the metal ion in the MIDAS motif of integrins, while the Glu residue in the ␥ chains could be required for stabilization of the functionally active conformation of the LG1-3 modules, possibly through direct interaction with the LG1-3 modules. However, our preliminary results indicated that substitution of any of the acidic amino acid residues conserved within the LG1-3 modules of the different laminin ␣ chains with Ala did not compromise the integrin binding activity of the E8 fragment of laminin-511 as severely as Gln substitution for the conserved Glu residue of the ␥1 or ␥2 chain, unless the Ala substitutions severely impaired the conformational integrity of the LG1-3 modules, as evidenced by significantly reduced reactivities toward mAb 4C7 recognizing the LG1-3 domains of the ␣5 chain (Refs. 16,33) 4 ; thereby arguing against the possibility that the LG1-3 modules harbor a critical acidic residue that coordinates the metal ion in the MIDAS motif of integrins. Recently, Navdaev et al. (44) reported that C-terminal truncation of the ␥2 chain induced opening of the compact supradomain assembly of the LG1-3 modules of the E8 fragment of laminin-332, as revealed by electron microscopy, and proposed a role for the C-terminal tail of the ␥2 chain in stabilizing the integrin binding-competent conformation of the LG1-3 modules. However, it remains to be explored whether a single mutation of the Glu residue within the C-terminal region of the ␥2 chain can destabilize the supradomain assembly of the LG1-3 modules of laminin-332, because some other amino acid residues within the C-terminal nine amino acid residues are also conserved between the ␥1 and ␥2 chains.
Unlike other laminin isoforms, the ␥3 chain-containing laminins lack integrin binding activity, a hallmark of laminins in basement membranes. This unique feature of the ␥3 chaincontaining laminins raises a question as to their physiological functions in vivo. It is widely accepted that laminins serve as potent ligands for integrins in basement membranes and thereby provide anchorage-dependent signals that prevent apoptosis, regulate cell proliferation and differentiation, and maintain tissue integrity. One of the likely consequences of the absence of integrin binding activity in ␥3 chain-containing laminins could be loosened anchorage of cells onto the basement membrane and resultant weakening of integrin-mediated signaling events. Thus, the ␥3 chain-containing laminins may function as dominant-negative laminins that negatively regulate cell-basement membrane interactions during embryonic development and in tissue organization. Given that the LG4 -5 modules of the laminin ␣ chains are capable of binding to cell surface receptors other than integrins, i.e. ␣-dystroglycan, heparan sulfate proteoglycans, and sulfated glycolipids (9, 11-13, 15, 16, 45, 46), it is conceivable that the ␥3 chain-containing laminins compromise integrin-mediated signals but maintain their functions exerted through non-integrin laminin receptors. It is interesting to note that a variant form of the ␥2 transcript (variant-2), which arises by alternative splicing of the 3Ј-most exon, encodes a ␥2 chain that is shorter than the normal ␥2 chain and lacks the critical Glu residue at the third position from the C terminus (47,48). The laminin isoform containing the variant-2 ␥2 transcript is therefore assumed to be incapable of binding to integrins due to the absence of the Glu residue required for integrin binding by laminin-332 (20,44). Airenne et al. (49) showed that the variant-2 ␥2 transcript was expressed in the mesenchyme of the developing kidney, whereas the normal ␥2 transcripts were exclusively detected in the epithelium of the Wolffian duct and ureteric buds, suggesting distinctive functions for the two ␥2 chain variants in early kidney development. Although no transcript variants have been reported for the ␥1 and ␥3 chains, the expression of two kinds of laminin isoforms with and without integrin binding activity may be a novel mechanism for modulating the interactions of cells with basement membranes possessing both ␥1 and ␥3 chain-containing laminins.
It has been reported that Lamc1 and Lamc2 knock-out mice both exhibit severe phenotypes due to failure of endoderm differentiation (50) and skin blistering (51), respectively, whereas Lamc3 knock-out mice appear to be fertile and do not show any apparent developmental defects (52). Given the similarity in the protein structures between the ␥1 and ␥3 chains, deficiency of the ␥3 chain may be largely compensated for by the ␥1 chain. Because Lamc3 knock-out mice did not show any apparent phenotypes, the ␥3 chain-containing laminins may be dispensable for the maintenance and function of basement membranes throughout embryonic development. Recently, Brunken and co-workers (52) reported that the combined absence of ␤2 and ␥3 chain-containing laminins was associated with substantial alterations in the development of inner retinal neurons, particularly those positive for tyrosine hydroxylase activity, disruption of the basement membrane that lines the vitreal surface of the retina, and marked disarray of the vitreal end feet of Müller cells. These alterations are similar to those observed in the ␤2-null retina (52). Because the ␥3 chain is most prominently expressed in the nervous system, including the mouse retina (24,52,53), it may participate in the development of inner retinal neurons as an auxiliary basement membrane component that modulates the functions of ␤2 chain-containing laminins.
The laminin ␥3 chain was originally identified as a novel ␥ chain that combines with the ␣2 and ␤1 chains in the placenta (21). Because the ␥3 chain was absent from the testes in ␣2 chain-deficient mice (26), the expression of ␥3 chaincontaining laminins may be at least partially dependent on the expression of the ␣2 chain. Consistent with this possibility, the ␥3 chain was secreted by 293-F cells as a heterotrimer when cotransfected with the ␣2 and ␤1 chains. Our data also showed that the ␥3 chain could be expressed and secreted by 293-F cells as laminin-113/123, -223, and -413/423, while combinations with the other ␣ and ␤ chains did not yield detectable amounts of ␥3 chain-containing laminin heterotrimers. These results do not necessarily exclude the occurrence of ␥3 chain-containing isoforms that failed to be secreted by 293-F cells in tissues, since such isoforms have been detected in hippocampal synapses (laminin-323), testes (laminin-333), and photoreceptor synapses (laminin-523) (24,27,53). The failure to detect secretion of ␥3 chain-containing isoforms by 293-F cells could simply arise because the secretion levels of these isoforms were below the threshold for detecting laminins by immunoblotting.
Despite the low efficiency of secretion from 293-F cells, ␥3 chain-containing laminins have been detected at the basement membranes of various mouse tissues (21, 22, 24 -26), indicating that mouse isoforms containing the ␥3 chain are competent in secretion from cells and deposition at basement membranes. Although the reason for this apparent discrepancy remains to be elucidated, the human and mouse ␥3 chain isoforms may differ in their efficiencies of secretion and/or assembly with other subunit chains. In this respect, it should be noted that there are only two potential N-glycosy-lation sites in the coiled-coil domain of the human ␥3 chain, compared with four sites in the same domain of the mouse ␥3 chain. Because the coiled-coil domain is responsible for the heterotrimeric assembly of laminin subunit chains, the reduced N-glycosylation at the coiled-coil domain of the human ␥3 chain may lead to decreased stabilities of ␥3 chain-containing heterotrimers and hence significant reductions in their secretion efficiencies.
In conclusion, we have provided evidence that laminin isoforms containing the ␥3 chain are unable to bind to integrins due to the absence of the Glu residue at the third position from the C terminus, which underscores the critical role of this Glu residue conserved in the C-terminal regions of the ␥1 and ␥2 chains in integrin binding by laminins. Although the physiological functions of the ␥3 chain-containing laminins remain to be elucidated, the spatiotemporally regulated expression of such apparently dormant laminins in basement membranes may be an auxiliary mechanism that regulates the adhesive interactions of cells with underlying basement membranes during embryonic development. Further studies on the signals transduced from these laminins as well as their effects on cell proliferation, migration and apoptosis should bring about new insights into the regulatory mechanisms operating in cell-basement membrane interactions during physiological as well as pathological processes.