_:b40355 "Since hemidesmosomes appeared normal at the ultrastructural level in \u03B13-null skin, we also examined the expression and distribution of \u03B16\u03B24, a component of hemidesmosomes (Stepp et al., 1990; Jones et al., >>1991<<). The \u03B16 subunit also associates with the \u03B21 subunit in some cells (Hynes, 1992)." . _:b207962613 . _:b207962609 . _:b40356 "The \u03B16 subunit also associates with the \u03B21 subunit in some cells (Hynes, >>1992<<). However, most or all \u03B16 in the epidermis appears to be associated with \u03B24 (Watt and Hertle, 1994), and little or no \u03B16 was detected in skin from \u03B24-null mice (van der Neut et al., 1996; Dowling et al., 1996)." . . _:b40409 . _:b207962534 . _:b207962577 . _:b40380 _:b40381 . _:b40380 _:b40382 . _:b40380 _:b40383 . . _:b207962540 . . . _:b207962603 . _:b207962543 . _:b207962614 . _:b207962700 . _:b207962557 . _:b40387 "These roles for \u03B13\u03B21 and \u03B16\u03B24 have been demonstrated previously in function-blocking studies of human foreskin keratinocytes (Xia et al., 1996), and have been suggested for a murine mammary tumor cell line (Sonnenberg et al., >>1993<<). Differences in function between these two integrins reflect their distinct roles in vivo; \u03B16\u03B24 is required for stable adhesion of the epidermis to the basement membrane, and \u03B13\u03B21 appears to have a post-adhesion role in basement membrane" . _:b40368 "As expected for basal keratinocytes lacking \u03B16\u03B24 (van der Neut et al., 1996; Georges-Labouesse et al., 1996; Dowling et al., >>1996<<), these cells were detached from the basement membrane, since laminin-5 distributed only to the dermal side of the separation (compare arrowheads in Fig." . . . _:b40353 . _:b40331 . _:b40416 . _:b40380 _:b40404 . _:b40341 "To prepare laminin-5\u2013rich ECM, human epidermal keratinocytes (HEKs) were either prepared from neonatal foreskin as described (Rheinwald and Green, >>1975<<) or purchased from Clonetics (San Diego, CA) and grown, respectively, in either FAD medium (1:" . _:b40380 _:b40405 . _:b40380 _:b40406 . _:b40380 _:b40407 . _:b40380 _:b40400 . _:b40380 _:b40401 . . _:b207962574 . _:b40380 _:b40402 . _:b40380 _:b40403 . _:b40345 "1, A and B, arrows), as expected for this integrin (Peltonen et al., 1989; Carter et al., 1990a ,b; Hertle et al., 1991)." . _:b40380 _:b40412 . _:b40380 _:b40413 . _:b207962656 . . _:b40380 _:b40414 . _:b40380 _:b40415 . _:b40372 . _:b40380 _:b40408 . _:b40380 _:b40409 . . _:b40397 "a broad range of matrix proteins, including fibronectin (Wayner and Carter, 1987; Elices et al., 1991), laminin-1 (Wayner and Carter, 1987; Gehlsen et al., 1988; Elices et al., 1991), collagens (Wayner and Carter, 1987; Elices et al., >>1991<<), and entactin/nidogen (Dedhar et al., 1992; Wu et al., 1995), and shows post-adhesion localization to focal contacts in cells cultured on most of these proteins (DiPersio et al., 1995)." . _:b40380 _:b40410 . _:b40380 _:b40411 . _:b40380 _:b40388 . . _:b40380 _:b40389 . _:b40380 _:b40390 . _:b40380 _:b40391 . . _:b40380 _:b40384 . _:b40380 _:b40385 . _:b40394 "Consistent with this notion, \u03B13\u03B21 can interact weakly with a broad range of matrix proteins, including fibronectin (Wayner and Carter, 1987; Elices et al., 1991), laminin-1 (Wayner and Carter, 1987; Gehlsen et al., >>1988<<; Elices et al., 1991), collagens (Wayner and Carter, 1987; Elices et al., 1991), and entactin/nidogen (Dedhar et al., 1992; Wu et al., 1995), and shows post-adhesion localization to focal contacts in cells cultured on most of these" . _:b40380 _:b40386 . _:b207962658 . _:b40380 _:b40387 . _:b40380 _:b40396 . . _:b40380 _:b40397 . . _:b40380 _:b40398 . _:b40380 _:b40399 . _:b40380 _:b40392 . _:b40380 _:b40393 . _:b40380 _:b40394 . . . _:b40380 _:b40395 . _:b40348 . . . . _:b207962622 . _:b207962559 . . _:b207962634 . _:b40380 _:b40416 . _:b40380 _:b40417 . . _:b40380 _:b40418 . _:b40380 _:b40419 . _:b40389 "In contrast, \u03B16\u03B24 is necessary for assembly of hemidesmosomes but not for formation of a continuous lamina densa (Georges-Labouesse et al., 1996; van der Neut et al., 1996; Dowling et al., >>1996<<)." . . _:b207962586 . . . . . . _:b207962516 . _:b40332 "Rabbit antisera against the cytoplasmic domains of the human \u03B21 subunit and the chicken \u03B13A subunit were prepared as described (Marcantonio and Hynes, 1988; Hynes et al., 1989; DiPersio et al., >>1995<<). Rabbit antiserum against the cytoplasmic domain of the \u03B16A subunit, and the monoclonal antibody 346-11A against the \u03B24 subunit, were kindly provided by V." . . . _:b207962585 . . _:b207962528 . _:b40335 . _:b40358 "Double-label immunofluorescence of wild-type skin, using anti\u2013laminin-5 serum and the anti-\u03B16 monoclonal antibody GoH3 (Sonnenberg et al., >>1987<<), showed that \u03B16 subunit was concentrated at the basal surfaces of keratinocytes (Fig." . _:b40375 "Each of these fragments showed faster migration in the mouse keratinocytes than has been reported in human cells (Hemler et al., >>1989<<), possibly due to species-specific differences in glycosylation of \u03B24." . _:b207962549 . _:b207962578 . _:b207962584 . _:b207962600 . _:b207962565 . _:b40379 "sufficient for attachment of mouse keratinocytes to laminin-5, \u03B13\u03B21 is essential for postattachment cell spreading on this ligand, consistent with previous, integrin-blocking studies using human foreskin keratinocytes (Xia et al., >>1996<<)." . _:b207962536 . _:b207962567 . _:b207962655 . . _:b40398 . . _:b40376 "We subcultured primary mouse keratinocytes on laminin5-rich ECM prepared from human keratinocyte cultures, as described in the Materials and Methods section; this ECM supports \u03B13\u03B21-mediated cell attachment (Carter et al., >>1991<<; Weitzman et al., 1993). Both \u03B13-positive cells (in this case from a mouse heterozygous for the \u03B13-null mutation) and \u03B13-null cells attached to laminin-5\u2013rich ECM within 15 min." . . _:b40417 . _:b40405 "\u03B13\u03B21-mediated adhesion can induce the tyrosine phosphorylation of focal adhesion kinase, or FAK (Kornberg et al., >>1991<<; Jewell et al., 1995), as well as of other unidentified proteins in mouse keratinocytes (DiPersio, C.M., and R.O. Hynes, unpublished data)." . _:b207962520 . _:b40410 "ECM structure/composition and the activities of matrix degrading enzymes has been demonstrated in other systems (Sympson et al., 1994; Tremble et al., 1994) and can be regulated through integrins (Huhtala et al., 1995; Brooks et al., >>1996<<). Ligand binding by \u03B13\u03B21 may be required to signal the presence of basement membrane and provide positive or negative feedback to the cell that regulates basement membrane production." . . _:b207962629 . _:b40399 . . _:b40330 "Rabbit antisera against the cytoplasmic domains of the human \u03B21 subunit and the chicken \u03B13A subunit were prepared as described (Marcantonio and Hynes, >>1988<<; Hynes et al., 1989; DiPersio et al., 1995). Rabbit antiserum against the cytoplasmic domain of the \u03B16A subunit, and the monoclonal antibody 346-11A against the \u03B24 subunit, were kindly provided by V." . _:b207962628 . . . . _:b207962631 . _:b207962615 . _:b207962703 . _:b207962630 . _:b207962683 . . . . _:b207962625 . _:b207962675 . _:b207962679 . . _:b40377 "primary mouse keratinocytes on laminin5-rich ECM prepared from human keratinocyte cultures, as described in the Materials and Methods section; this ECM supports \u03B13\u03B21-mediated cell attachment (Carter et al., 1991; Weitzman et al., >>1993<<). Both \u03B13-positive cells (in this case from a mouse heterozygous for the \u03B13-null mutation) and \u03B13-null cells attached to laminin-5\u2013rich ECM within 15 min." . _:b40366 . _:b207962624 . _:b40408 . _:b207962627 . . _:b207962626 . . _:b40336 . . _:b207962637 . _:b40395 "with this notion, \u03B13\u03B21 can interact weakly with a broad range of matrix proteins, including fibronectin (Wayner and Carter, 1987; Elices et al., 1991), laminin-1 (Wayner and Carter, 1987; Gehlsen et al., 1988; Elices et al., >>1991<<), collagens (Wayner and Carter, 1987; Elices et al., 1991), and entactin/nidogen (Dedhar et al., 1992; Wu et al., 1995), and shows post-adhesion localization to focal contacts in cells cultured on most of these proteins (DiPersio et al.," . . _:b207962636 . _:b207962523 . _:b207962694 . _:b207962618 . . _:b207962639 . _:b207962638 . _:b207962561 . _:b207962686 . _:b207962633 . _:b207962639 . _:b207962632 . _:b207962705 . _:b40356 . _:b207962635 . _:b207962634 . _:b207962508 . _:b40353 "To determine the distribution of laminin-5 within blisters of \u03B13-null skin, we stained frozen skin sections from neonatal mice with a polyclonal antiserum against laminin-5 (Marinkovich et al., >>1992<<). Surprisingly, laminin-5 was detected at both the epidermal and dermal sides of blisters, and occasionally in what appeared to be remnants of matrix extending through the blisters (Fig." . . _:b207962697 . _:b207962645 . _:b207962680 . _:b207962642 . _:b40383 "The latter two mutations appear to cause detachment of basal epidermal keratinocytes from the basement membrane, since laminin-5 localized only to the dermal sides of blisters in both cases (Georges-Labouesse et al., >>1996<<; van der Neut et al., 1996; Dowling et al., 1996)." . _:b207962644 . _:b40385 "The chemical composition and physical organization of the epidermal basement membrane are complex (for reviews see Borradori and Sonnenberg, >>1996<<; Uitto et al., 1996). At the ultrastructural level, the dermal-epidermal junction is resolved into the lamina lucida, which abuts the plasma membranes of basal keratinocytes in the epidermis, and the underlying lamina densa." . _:b207962674 . _:b207962647 . _:b207962654 . _:b207962612 . _:b207962646 . . _:b207962641 . _:b40408 "Indeed, a balance between ECM structure/composition and the activities of matrix degrading enzymes has been demonstrated in other systems (Sympson et al., 1994; Tremble et al., >>1994<<) and can be regulated through integrins (Huhtala et al., 1995; Brooks et al., 1996)." . _:b207962640 . _:b40418 "At least two cases of JEB with pyloric atresia are associated with loss of \u03B16\u03B24 integrin through mutations in the \u03B24 integrin subunit (Vidal et al., >>1995<<; Niessen et al., 1996)." . _:b207962643 . _:b207962642 . _:b207962602 . . _:b207962653 . _:b207962669 . _:b40334 "Rabbit antiserum specific for the EIIIB segment of fibronectin was raised against an EIIIB-GST fusion protein and immunopurified, as described (Peters et al., >>1995<<)." . _:b40352 "Indeed, in \u03B16-null or \u03B24-null skin, laminin-5 remains associated with the dermal sides of blisters (Georges-Labouesse et al., 1996; van der Neut et al., 1996; Dowling et al., >>1996<<). Therefore, the blistering phenotype in the \u03B13-null epidermis appeared consistent with a role for \u03B13\u03B21 as a receptor for laminin-5 or other basement membrane ligands." . _:b207962570 . _:b207962652 . _:b40418 . _:b207962655 . _:b40401 . _:b207962654 . _:b40393 "Consistent with this notion, \u03B13\u03B21 can interact weakly with a broad range of matrix proteins, including fibronectin (Wayner and Carter, 1987; Elices et al., 1991), laminin-1 (Wayner and Carter, >>1987<<; Gehlsen et al., 1988; Elices et al., 1991), collagens (Wayner and Carter, 1987; Elices et al., 1991), and entactin/nidogen (Dedhar et al., 1992; Wu et al., 1995), and shows post-adhesion localization to focal contacts in cells cultured" . _:b207962644 . . _:b40403 . _:b207962513 . _:b207962649 . _:b40380 "discussion" . _:b40384 . _:b40400 . _:b40415 "Similarly, patients with bullous pemphigoid, an acquired blistering disease, develop antibodies against either BPAG-1e (bullous pemphigoid antigen-1e) or BPAG-2, both components of hemidesmosomes (Diaz et al., >>1977<<; Mueller et al., 1989; Sawamura et al., 1991)." . _:b207962648 . _:b40337 "Epidermal keratinocytes were prepared from neonatal mice essentially as described previously (Dlugosz et al., >>1995<<). Briefly, newborn mice were sacrificed by CO2 narcosis, washed in 10% iodine solution (Mallinckrodt, Paris, KY) in PBS for 10 min, rinsed with PBS, washed in 70% ethanol for 10 min, and then rinsed in PBS." . . _:b207962651 . . . . _:b207962682 . _:b207962650 . _:b207962690 . _:b207962661 . . _:b207962666 . _:b207962660 . . _:b40389 . . _:b207962663 . _:b207962672 . . _:b40391 . _:b207962661 . _:b207962662 . . _:b207962657 . _:b207962656 . _:b40370 "\u03B16\u03B24 was detected using a polyclonal antiserum against the \u03B16 integrin subunit (lane 3), which coimmunoprecipitated a series of bands expected for the \u03B16 subunit (\u223C140 kD) and various derivatives of the \u03B24 subunit (Hemler et al., >>1989<<; Kajiji et al., 1989; Adams and Watt, 1991)." . _:b207962659 . . _:b207962658 . . . _:b207962669 . . . . _:b40393 . . . _:b207962668 . . _:b207962552 . . _:b207962671 . _:b207962687 . _:b207962670 . _:b40396 . _:b207962619 . _:b207962665 . . _:b207962695 . _:b207962664 . _:b40406 . . . . _:b207962667 . . _:b207962652 . . . _:b207962666 . . . . _:b207962607 . _:b207962677 . _:b207962604 . _:b40365 "seen at birth; fibronectin was detected throughout the dermis up to the dermal-epidermal junction, and in some sections it was considerably more concentrated at this junction (data not shown), as reported previously (Peters and Hynes, >>1996<<)." . _:b207962676 . _:b207962508 . . _:b207962509 . . _:b40377 . _:b207962510 . _:b207962511 . _:b207962679 . . _:b207962678 . . _:b40359 "4 B), as expected for \u03B16\u03B24 (Carter et al., 1990b ). Although \u03B16 showed the same distribution in \u03B13-null epidermis, it did not codistribute with laminin-5 when the latter was detected below the plane of the basement membrane in regions of disorganized matrix (compare Fig." . _:b207962532 . _:b207962673 . _:b207962520 . _:b207962521 . _:b40362 . _:b207962522 . . _:b40385 . _:b207962672 . _:b207962523 . _:b207962524 . _:b207962525 . _:b207962526 . _:b207962527 . _:b40398 "fibronectin (Wayner and Carter, 1987; Elices et al., 1991), laminin-1 (Wayner and Carter, 1987; Gehlsen et al., 1988; Elices et al., 1991), collagens (Wayner and Carter, 1987; Elices et al., 1991), and entactin/nidogen (Dedhar et al., >>1992<<; Wu et al., 1995), and shows post-adhesion localization to focal contacts in cells cultured on most of these proteins (DiPersio et al., 1995)." . _:b207962675 . _:b207962512 . _:b207962513 . _:b40376 . . _:b207962514 . _:b207962515 . _:b207962674 . _:b207962516 . _:b207962517 . _:b40410 . _:b207962518 . _:b40413 . . _:b207962685 . _:b207962519 . . _:b207962536 . _:b207962537 . _:b207962538 . _:b40358 . . _:b207962537 . _:b207962539 . _:b207962684 . . _:b207962540 . _:b207962541 . . _:b207962542 . _:b207962543 . _:b207962687 . _:b207962528 . _:b207962529 . _:b207962593 . _:b207962530 . _:b40386 "These roles for \u03B13\u03B21 and \u03B16\u03B24 have been demonstrated previously in function-blocking studies of human foreskin keratinocytes (Xia et al., >>1996<<), and have been suggested for a murine mammary tumor cell line (Sonnenberg et al., 1993)." . _:b207962667 . _:b207962531 . _:b207962686 . . _:b207962532 . _:b207962533 . _:b207962534 . _:b207962535 . _:b207962681 . _:b207962552 . . _:b40371 "using a polyclonal antiserum against the \u03B16 integrin subunit (lane 3), which coimmunoprecipitated a series of bands expected for the \u03B16 subunit (\u223C140 kD) and various derivatives of the \u03B24 subunit (Hemler et al., 1989; Kajiji et al., >>1989<<; Adams and Watt, 1991)." . _:b207962553 . _:b207962554 . . _:b207962680 . _:b207962657 . _:b207962555 . . _:b207962556 . _:b207962530 . _:b207962557 . _:b207962558 . _:b207962559 . _:b207962683 . _:b207962544 . _:b207962562 . _:b207962545 . _:b40369 "To address directly the importance of \u03B13\u03B21 as a laminin-5 receptor in the epidermis, we isolated primary keratinocytes from wild-type and \u03B13-null, neonatal mice (Dlugosz et al., >>1995<<; for details see Materials and Methods)." . _:b207962546 . _:b207962547 . _:b207962682 . . . _:b207962548 . . _:b207962549 . _:b207962550 . _:b207962551 . _:b207962693 . _:b207962568 . _:b207962569 . _:b207962570 . _:b40332 . . _:b207962692 . _:b207962571 . _:b207962572 . _:b207962573 . _:b207962574 . . _:b207962575 . _:b207962695 . _:b207962560 . . _:b207962561 . _:b207962562 . _:b207962546 . _:b40392 "Consistent with this notion, \u03B13\u03B21 can interact weakly with a broad range of matrix proteins, including fibronectin (Wayner and Carter, 1987; Elices et al., >>1991<<), laminin-1 (Wayner and Carter, 1987; Gehlsen et al., 1988; Elices et al., 1991), collagens (Wayner and Carter, 1987; Elices et al., 1991), and entactin/nidogen (Dedhar et al., 1992; Wu et al., 1995), and shows post-adhesion localization" . _:b40346 "1, A and B, arrows), as expected for this integrin (Peltonen et al., 1989; Carter et al., 1990a ,b; Hertle et al., >>1991<<). Preimmune serum did not stain the epidermis (Fig. 1, C and D). In \u03B13-null mice, stratification of the epidermis appeared normal (Figs. 1 E and 2 B). However, staining of the basal keratinocytes of \u03B13-null epidermis with anti-\u03B13 (Fig. 1" . _:b207962694 . _:b207962563 . _:b207962564 . . _:b207962565 . _:b207962566 . _:b207962567 . _:b207962689 . _:b207962584 . _:b207962585 . . _:b207962586 . _:b40378 . _:b207962587 . _:b207962688 . _:b207962588 . _:b207962589 . _:b207962590 . _:b207962591 . _:b40332 . _:b207962691 . _:b207962576 . _:b207962577 . _:b207962578 . _:b207962579 . _:b40333 . _:b207962690 . _:b207962580 . _:b207962581 . _:b207962582 . _:b207962598 . _:b207962583 . _:b40334 . _:b207962701 . . . _:b207962599 . _:b207962590 . _:b207962555 . _:b207962600 . _:b207962601 . _:b207962602 . _:b207962603 . _:b40335 . _:b207962700 . _:b207962604 . _:b207962605 . _:b207962606 . _:b40341 . _:b207962607 . _:b207962703 . _:b207962592 . _:b207962593 . _:b207962594 . _:b40349 "mutations of either the \u03B16 (Georges-Labouesse et al., 1996) or the \u03B24 subunit (van der Neut et al., 1996; Dowling et al., 1996) and with human blistering diseases caused by defects in laminin-5 (Domloge-Hultsch, 1992; Baudoin et al., >>1994<<; Kirtschig et al., 1995), the ligand for both \u03B16\u03B24 and \u03B13\u03B21." . _:b207962595 . _:b40335 "Embryonic or neonatal offspring were collected from matings of mice heterozygous for a null mutation of the \u03B13 integrin gene (Kreidberg et al., >>1996<<), and their tails were removed and digested overnight at 56\u00B0C in 0.2% SDS, 100 mM Tris, pH 6.8, 200 mM NaCl, 5 mM EDTA, and 0.1 mg/ ml proteinase K." . _:b207962702 . _:b207962596 . _:b40329 . _:b207962597 . _:b207962649 . _:b207962598 . _:b207962599 . _:b40330 . _:b207962697 . . _:b207962616 . _:b207962617 . _:b207962618 . _:b207962619 . _:b40331 . _:b207962696 . _:b207962620 . _:b207962621 . _:b207962622 . _:b40404 . _:b40336 "to be stained with anti-EIIIB were incubated at 37\u00B0C for 2 h with N-glycanase (PNGase F, New England Biolabs, Beverly, MA) at 50,000 U/ml in reaction buffer provided by the manufacturer, as described previously (Peters and Hynes, >>1996<<). Sections were fixed in 3.7% formaldehyde for 10 min and then incubated in blocking buffer (0.1% BSA, 0.2% Triton X-100, 0.1% glycine in PBS/ Ca2+Mg2+) for at least 30 min." . _:b207962623 . _:b40340 . _:b207962699 . _:b207962608 . _:b207962609 . _:b40342 "results" . _:b207962610 . _:b207962611 . _:b40341 . _:b207962698 . _:b207962673 . _:b207962612 . _:b207962524 . _:b207962613 . _:b207962614 . _:b207962615 . _:b40342 . . . _:b40343 . . _:b40342 . _:b40336 . . _:b40329 . _:b40337 . . _:b40347 "Epidermal blistering in the \u03B13-null mice is reminiscent of more severe blistering phenotypes associated with loss of \u03B16\u03B24 integrin through null mutations of either the \u03B16 (Georges-Labouesse et al., >>1996<<) or the \u03B24 subunit (van der Neut et al., 1996; Dowling et al., 1996) and with human blistering diseases caused by defects in laminin-5 (Domloge-Hultsch, 1992; Baudoin et al., 1994; Kirtschig et al., 1995), the ligand for both \u03B16\u03B24 and" . . _:b40338 . _:b207962705 . _:b207962526 . _:b207962605 . _:b207962704 . _:b40339 . . . _:b40348 . _:b207962707 . . _:b207962706 . _:b40349 . _:b40338 "Mouse keratinocyte cultures were grown for 7 d, and monolayers were surface-labeled with 0.5 mCi/10-cm plate of Na[125I] (New England Nuclear, Boston, MA) using the lactoperoxidase-glucose oxidase method (Hynes, >>1973<<). Cells were washed four times with 50 mM NaI in PBS/ Ca2+Mg2+ and lysed for 15 min on ice in 1 ml of a detergent buffer containing 200 mM octyl-\u03B2-d-glucopyranoside (Calbiochem), 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 2" . _:b40380 . . . _:b40343 . _:b40350 . _:b207962627 . _:b207962636 . _:b40351 . _:b207962707 . _:b40344 . _:b40373 "immunoprecipitates from human cells, the fulllength \u03B24 subunit migrates at \u223C210 kD (a fragment), and smaller fragments of 165 kD (b fragment), 125 kD (c fragment), and 85 kD are thought to result from proteolysis of \u03B24 (Hemler et al., >>1989<<). Indeed, this pattern of proteolysis has been useful as a diagnostic tool in studies of \u03B24 integrins (Hemler et al., 1989)." . . . . _:b40345 . _:b40346 . . _:b40346 . . _:b207962626 . . _:b40347 . _:b207962676 . _:b40356 . _:b40386 . _:b40363 "5, A and B) and occasionally showed concentrated staining in the basement membrane zone (not shown), as reported previously during embryonic development (Peters and Hynes, >>1996<<). An antiserum specific for the EIIIB segment of fibronectin produced similar staining patterns (Fig. 5, D and E). In \u03B13-null skin, total fibronectin (Fig. 5 C) and EIIIB+ fibronectin (Fig. 5 F) were each detected at both the epidermal" . _:b40357 . _:b40358 . _:b40361 "Type VII collagen and entactin, both components of the epidermal basement membrane (Martin, 1987; Timpl, 1989; Burgeson, >>1993<<), were each detected in the basement membrane of wild-type epidermis (Fig. 4, C and D, respectively); entactin was also detected in the basement membranes of capillaries throughout the dermis (Fig." . _:b40334 . _:b207962509 . _:b40359 . . _:b207962508 . _:b40352 . . _:b207962511 . . _:b40353 . _:b207962510 . _:b207962629 "2"^^ . _:b207962596 . _:b40354 . _:b40329 "materials and methods" . _:b207962628 "2"^^ . _:b207962515 . _:b40355 . _:b40330 . _:b207962631 "2"^^ . _:b40364 "Since levels of EIIIB+ fibronectin are normally very low in serum (Peters et al., >>1995<<), these staining patterns suggest that fibronectin present in the blisters is derived from extracellular matrix rather than from infiltrating serum." . _:b40364 . _:b207962630 "2"^^ . _:b40365 . _:b207962693 . _:b207962625 "2"^^ . _:b40366 . _:b207962517 . _:b207962624 "2"^^ . _:b207962691 . _:b40367 . _:b207962685 . _:b207962516 . _:b207962627 "2"^^ . _:b207962643 . _:b40360 . . _:b207962589 . _:b207962519 . _:b40366 "The relative distributions of \u03B16\u03B24 and laminin-5 were determined by double-label immunofluorescence using a monoclonal antibody against the \u03B24 subunit (346-11A; Kennel et al., >>1989<<) and anti\u2013laminin-5 serum (Fig." . _:b207962626 "2"^^ . _:b207962580 . _:b40361 . _:b207962678 . _:b207962518 . _:b207962637 "2"^^ . _:b40362 . . _:b207962635 . _:b207962513 . _:b207962636 "2"^^ . . _:b40363 . _:b40362 "Similar staining (not shown) was seen with an antiserum expected to recognize laminin isoforms containing the \u03B11, \u03B21, or \u03B31 chain (see Burgeson et al., >>1994<<), including the basement membrane protein laminin-6 (\u03B13, \u03B21, and \u03B31), but not laminin-5 (\u03B13, \u03B23, and \u03B32)." . . . _:b207962511 . _:b207962639 "2"^^ . _:b207962512 . _:b40372 . _:b40397 . _:b40396 "interact weakly with a broad range of matrix proteins, including fibronectin (Wayner and Carter, 1987; Elices et al., 1991), laminin-1 (Wayner and Carter, 1987; Gehlsen et al., 1988; Elices et al., 1991), collagens (Wayner and Carter, >>1987<<; Elices et al., 1991), and entactin/nidogen (Dedhar et al., 1992; Wu et al., 1995), and shows post-adhesion localization to focal contacts in cells cultured on most of these proteins (DiPersio et al., 1995)." . _:b207962515 . _:b207962638 "2"^^ . _:b40373 . . _:b207962514 . _:b40407 "Indeed, a balance between ECM structure/composition and the activities of matrix degrading enzymes has been demonstrated in other systems (Sympson et al., >>1994<<; Tremble et al., 1994) and can be regulated through integrins (Huhtala et al., 1995; Brooks et al., 1996)." . _:b207962633 "2"^^ . _:b207962582 . _:b207962509 . _:b40374 . . _:b207962525 . _:b207962632 "2"^^ . . _:b40375 . . _:b207962648 . _:b207962524 . _:b207962635 "2"^^ . _:b40368 . . . . _:b40343 "Frozen sections were prepared from the limbs of \u03B13-null or wild-type mice and stained by immunofluorescence with an antiserum against the cytoplasmic domain of the \u03B13 subunit (DiPersio et al., >>1995<<). In wild-type mice, \u03B13 expression was restricted to the basal layer of keratinocytes in the epidermis (Fig." . _:b207962634 "2"^^ . . _:b207962527 . _:b40369 . _:b40392 . . _:b207962645 "2"^^ . _:b207962526 . _:b40370 . _:b40395 . _:b207962521 . _:b40402 . _:b40361 . _:b207962644 "2"^^ . . _:b40371 . _:b40406 "\u03B13\u03B21-mediated adhesion can induce the tyrosine phosphorylation of focal adhesion kinase, or FAK (Kornberg et al., 1991; Jewell et al., >>1995<<), as well as of other unidentified proteins in mouse keratinocytes (DiPersio, C.M., and R.O. Hynes, unpublished data)." . . _:b207962647 "2"^^ . _:b207962520 . _:b207962511 "10"^^ . _:b40380 . _:b207962647 . _:b207962523 . _:b207962646 "2"^^ . _:b40381 . . _:b207962510 "11"^^ . . _:b207962632 . _:b207962522 . _:b207962641 "2"^^ . _:b207962633 . _:b207962634 . _:b40382 . _:b207962635 . _:b207962636 . _:b207962671 . . _:b207962640 "2"^^ . _:b207962509 "13"^^ . _:b207962637 . _:b207962533 . _:b207962638 . _:b40383 . . . _:b207962665 . _:b207962639 . _:b207962624 . . _:b40403 "Thus, \u03B16\u03B24 may be a primary receptor for keratinocyte attachment to the basement membrane, while \u03B13\u03B21 functions as a relatively weak, postadhesion receptor (DiPersio et al., >>1995<<) required for basement membrane assembly." . _:b207962643 "2"^^ . _:b207962625 . _:b207962532 . _:b207962626 . _:b40376 . _:b207962508 "14"^^ . _:b207962627 . _:b207962628 . _:b207962535 . _:b207962642 "2"^^ . _:b207962629 . _:b207962630 . _:b40419 "At least two cases of JEB with pyloric atresia are associated with loss of \u03B16\u03B24 integrin through mutations in the \u03B24 integrin subunit (Vidal et al., 1995; Niessen et al., >>1996<<). The blistering phenotype associated with mice lacking integrin \u03B13\u03B21 suggests that this basement membrane receptor may also be a potential target of human blistering diseases. \u03B13-null mice provide a useful system in which to study the" . _:b40377 . _:b207962631 . _:b207962648 . _:b207962534 . _:b207962653 "2"^^ . _:b207962649 . . _:b40378 . _:b207962650 . _:b207962651 . _:b207962652 . _:b40339 . _:b207962529 . _:b207962652 "2"^^ . _:b207962588 . _:b207962653 . _:b207962654 . _:b40379 . _:b207962655 . _:b207962670 . _:b207962640 . _:b207962601 . . _:b207962655 "2"^^ . _:b207962641 . _:b207962528 . _:b207962642 . _:b40388 . _:b207962643 . _:b207962644 . _:b207962531 . _:b207962654 "2"^^ . _:b207962620 . _:b207962517 "5"^^ . _:b207962645 . _:b207962646 . _:b40389 . _:b207962647 . _:b207962664 . _:b207962530 . _:b207962692 . _:b207962649 "2"^^ . _:b207962516 "5"^^ . _:b207962665 . . _:b40390 . _:b207962651 . _:b207962666 . . _:b207962667 . _:b40375 . . _:b207962668 . _:b207962541 . _:b40371 . _:b207962648 "2"^^ . _:b207962519 "5"^^ . _:b207962669 . . _:b40391 . _:b207962670 . _:b207962671 . _:b40374 . _:b40367 "As expected for basal keratinocytes lacking \u03B16\u03B24 (van der Neut et al., 1996; Georges-Labouesse et al., >>1996<<; Dowling et al., 1996), these cells were detached from the basement membrane, since laminin-5 distributed only to the dermal side of the separation (compare arrowheads in Fig." . _:b207962656 . _:b207962518 "5"^^ . _:b207962651 "2"^^ . _:b207962657 . _:b207962540 . . _:b40384 . _:b207962658 . _:b207962659 . _:b40373 . . _:b40364 . _:b207962660 . _:b207962661 . _:b40349 . _:b207962650 "2"^^ . _:b207962543 . _:b207962662 . _:b40385 . _:b207962663 . _:b207962531 . _:b207962680 . _:b207962681 . _:b207962663 . _:b207962661 "2"^^ . _:b207962542 . _:b207962682 . _:b40386 . _:b207962683 . _:b207962684 . _:b207962512 "8"^^ . _:b207962685 . _:b207962660 "2"^^ . _:b207962537 . _:b207962515 "5"^^ . _:b207962686 . _:b40387 . _:b207962687 . _:b40390 "Analysis of kidneys from \u03B13-null mice also revealed disorganization of the glomerular basement membrane (Kreidberg et al., >>1996<<), suggesting that \u03B13\u03B21 may be generally important for organization of extracellular matrices." . _:b40370 . _:b207962684 . _:b207962672 . _:b207962556 . _:b207962513 "8"^^ . _:b207962673 . _:b207962663 "2"^^ . _:b207962536 . _:b207962514 "5"^^ . _:b207962674 . _:b40396 . _:b207962544 . _:b207962675 . _:b207962676 . _:b207962646 . _:b207962525 "4"^^ . _:b207962677 . _:b207962662 "2"^^ . _:b207962539 . _:b207962678 . _:b40397 . _:b207962662 . _:b207962679 . _:b207962696 . _:b207962697 . _:b207962657 "2"^^ . _:b207962597 . _:b207962538 . _:b207962524 "5"^^ . _:b207962698 . _:b40398 . . _:b207962699 . _:b207962700 . _:b207962527 "4"^^ . . _:b207962656 "2"^^ . _:b207962549 . _:b207962701 . _:b207962702 . _:b40399 . _:b207962703 . _:b207962688 . _:b207962526 "4"^^ . _:b207962689 . _:b207962659 "2"^^ . _:b207962621 . _:b207962548 . _:b207962690 . _:b40392 . _:b207962691 . _:b207962692 . _:b207962693 . _:b207962658 "2"^^ . _:b207962551 . _:b207962521 "5"^^ . _:b207962694 . _:b40393 . . _:b207962695 . . _:b207962520 "5"^^ . _:b207962669 "2"^^ . _:b207962550 . _:b40394 . _:b207962523 "5"^^ . _:b207962668 "2"^^ . _:b207962545 . _:b40395 . . . _:b207962704 . _:b207962705 . _:b207962671 "2"^^ . _:b207962544 . _:b207962522 "5"^^ . _:b207962706 . _:b40404 . _:b207962699 . . _:b207962707 . . _:b207962533 "4"^^ . _:b207962640 . . _:b207962547 . _:b207962670 "2"^^ . . _:b40405 . _:b207962532 "4"^^ . _:b40409 "a balance between ECM structure/composition and the activities of matrix degrading enzymes has been demonstrated in other systems (Sympson et al., 1994; Tremble et al., 1994) and can be regulated through integrins (Huhtala et al., >>1995<<; Brooks et al., 1996). Ligand binding by \u03B13\u03B21 may be required to signal the presence of basement membrane and provide positive or negative feedback to the cell that regulates basement membrane production." . _:b207962529 . _:b207962546 . _:b207962665 "2"^^ . _:b40406 . . _:b207962535 "4"^^ . . _:b207962664 "2"^^ . _:b207962557 . _:b40407 . _:b40407 . _:b40391 "Consistent with this notion, \u03B13\u03B21 can interact weakly with a broad range of matrix proteins, including fibronectin (Wayner and Carter, >>1987<<; Elices et al., 1991), laminin-1 (Wayner and Carter, 1987; Gehlsen et al., 1988; Elices et al., 1991), collagens (Wayner and Carter, 1987; Elices et al., 1991), and entactin/nidogen (Dedhar et al., 1992; Wu et al., 1995), and shows" . _:b207962534 "4"^^ . _:b207962556 . _:b207962667 "2"^^ . _:b40350 . _:b40400 . _:b207962529 "4"^^ . _:b207962559 . _:b207962666 "2"^^ . _:b40401 . _:b207962542 . _:b207962528 "4"^^ . _:b207962551 . . . _:b207962558 . _:b207962677 "2"^^ . . _:b40402 . . _:b207962701 . _:b207962531 "4"^^ . _:b40333 . _:b207962553 . _:b207962676 "2"^^ . _:b40369 . _:b40403 . _:b207962530 "4"^^ . _:b40351 . _:b207962552 . _:b207962679 "2"^^ . _:b40412 . _:b207962541 "3"^^ . _:b207962631 . _:b207962555 . _:b207962678 "2"^^ . . _:b40413 . _:b40333 "Rabbit antiserum against rat plasma fibronectin was prepared as described (Mautner and Hynes, >>1977<<). Rabbit antiserum specific for the EIIIB segment of fibronectin was raised against an EIIIB-GST fusion protein and immunopurified, as described (Peters et al., 1995)." . _:b207962540 "3"^^ . _:b40402 "In the epidermal basement membrane, entactin binds to the type IV collagen network (Aumailley et al., >>1989<<). Therefore, certain bridging interactions between matrix proteins that require \u03B13\u03B21 may be absent from \u03B13-null skin, thereby compromising basement membrane integrity and weakening the dermal-epidermal junction. Thus, \u03B16\u03B24 may be a" . . _:b207962673 "2"^^ . _:b207962554 . . _:b40414 . _:b207962543 "3"^^ . _:b207962677 . _:b207962565 . _:b40344 . _:b207962672 "2"^^ . _:b40415 . _:b207962542 "3"^^ . _:b40347 . _:b40357 "However, most or all \u03B16 in the epidermis appears to be associated with \u03B24 (Watt and Hertle, 1994), and little or no \u03B16 was detected in skin from \u03B24-null mice (van der Neut et al., 1996; Dowling et al., >>1996<<). In addition, we detected no \u03B16\u03B21 in primary mouse keratinocytes (discussed below), and the distributions of \u03B16 and \u03B24 were identical in wild-type and \u03B13-null skin. Double-label immunofluorescence of wild-type skin, using anti\u2013laminin-5" . _:b207962564 . _:b207962675 "2"^^ . _:b40408 . _:b40415 . _:b207962537 "4"^^ . . _:b207962567 . _:b207962674 "2"^^ . _:b40350 "\u03B16 (Georges-Labouesse et al., 1996) or the \u03B24 subunit (van der Neut et al., 1996; Dowling et al., 1996) and with human blistering diseases caused by defects in laminin-5 (Domloge-Hultsch, 1992; Baudoin et al., 1994; Kirtschig et al., >>1995<<), the ligand for both \u03B16\u03B24 and \u03B13\u03B21." . _:b40409 . _:b207962583 . _:b207962536 "4"^^ . _:b207962623 . _:b207962533 . "PMC0" . _:b207962685 "2"^^ . _:b207962566 . _:b40410 . . _:b207962539 "4"^^ . . _:b207962561 . _:b207962684 "2"^^ . . _:b40411 . _:b207962581 . . _:b207962550 . . _:b207962538 "4"^^ . _:b40413 "For example, some forms of JEB have been attributed to defects in the \u03B13, \u03B23, or \u03B32 chain of laminin-5 (Aberdam et al., 1994; Pulkkinen et al., 1994a ,b; Uitto et al., 1994; Kivirikko et al., >>1995<<), and dystrophic forms of JEB are caused by mutations in the gene for type VII collagen (Uitto et al., 1994)." . . _:b207962560 . _:b207962687 "2"^^ . _:b207962592 . _:b40416 "Similarly, patients with bullous pemphigoid, an acquired blistering disease, develop antibodies against either BPAG-1e (bullous pemphigoid antigen-1e) or BPAG-2, both components of hemidesmosomes (Diaz et al., 1977; Mueller et al., >>1989<<; Sawamura et al., 1991). At least two cases of JEB with pyloric atresia are associated with loss of \u03B16\u03B24 integrin through mutations in the \u03B24 integrin subunit (Vidal et al., 1995; Niessen et al., 1996)." . _:b207962549 "3"^^ . . _:b40412 . _:b207962563 . _:b40355 . _:b207962686 "2"^^ . _:b207962548 "3"^^ . . _:b207962681 "2"^^ . _:b207962562 . _:b207962551 "3"^^ . _:b207962579 . . . _:b207962573 . _:b207962680 "2"^^ . _:b207962550 "3"^^ . . _:b207962572 . _:b40411 . _:b207962683 "2"^^ . _:b207962606 . _:b40416 . _:b207962545 "3"^^ . . _:b207962575 . _:b207962682 "2"^^ . _:b40417 . _:b207962544 "3"^^ . _:b207962574 . _:b207962693 "2"^^ . _:b40418 . _:b207962547 "3"^^ . _:b40331 "Rabbit antisera against the cytoplasmic domains of the human \u03B21 subunit and the chicken \u03B13A subunit were prepared as described (Marcantonio and Hynes, 1988; Hynes et al., >>1989<<; DiPersio et al., 1995). Rabbit antiserum against the cytoplasmic domain of the \u03B16A subunit, and the monoclonal antibody 346-11A against the \u03B24 subunit, were kindly provided by V." . _:b207962569 . _:b207962692 "2"^^ . . _:b40419 . _:b207962659 . _:b207962546 "3"^^ . _:b207962641 . _:b40367 . _:b207962568 . _:b207962695 "2"^^ . . _:b207962557 "3"^^ . _:b207962571 . _:b207962694 "2"^^ . _:b207962702 . _:b207962556 "3"^^ . . . _:b207962689 "2"^^ . _:b207962570 . _:b207962559 "3"^^ . _:b207962581 . _:b207962688 "2"^^ . . _:b40360 "Type VII collagen and entactin, both components of the epidermal basement membrane (Martin, 1987; Timpl, >>1989<<; Burgeson, 1993), were each detected in the basement membrane of wild-type epidermis (Fig." . _:b207962558 "3"^^ . _:b207962545 . _:b207962689 . _:b207962580 . _:b207962691 "2"^^ . . _:b207962553 "3"^^ . . . _:b207962583 . _:b40417 "with bullous pemphigoid, an acquired blistering disease, develop antibodies against either BPAG-1e (bullous pemphigoid antigen-1e) or BPAG-2, both components of hemidesmosomes (Diaz et al., 1977; Mueller et al., 1989; Sawamura et al., >>1991<<). At least two cases of JEB with pyloric atresia are associated with loss of \u03B16\u03B24 integrin through mutations in the \u03B24 integrin subunit (Vidal et al., 1995; Niessen et al., 1996)." . _:b207962690 "2"^^ . _:b207962610 . _:b40378 "The antibody GoH3, which blocks cell adhesion mediated by \u03B16 integrins (Sonnenberg et al., >>1987<<), only slightly inhibited attachment, and did not affect spreading, of wild-type keratinocytes on laminin-5 (Fig." . _:b207962552 "3"^^ . _:b207962566 . _:b207962650 . _:b207962582 . _:b207962701 "2"^^ . _:b207962555 "3"^^ . . _:b207962510 . . _:b207962577 . _:b207962700 "2"^^ . _:b207962554 "3"^^ . _:b207962575 . _:b207962576 . _:b207962703 "2"^^ . . _:b207962565 "3"^^ . _:b207962579 . _:b207962702 "2"^^ . . _:b207962564 "3"^^ . . _:b207962697 "2"^^ . _:b207962578 . _:b207962567 "2"^^ . _:b207962589 . _:b207962696 "2"^^ . _:b207962563 . _:b207962566 "2"^^ . . . _:b207962588 . _:b207962699 "2"^^ . _:b207962561 "3"^^ . _:b207962591 . _:b207962698 "2"^^ . . _:b207962517 . _:b40340 . _:b207962560 "3"^^ . _:b40381 . _:b207962590 . _:b207962563 "3"^^ . _:b207962585 . _:b40339 "Protein concentrations of supernatants were determined using a Bio-Rad kit, and equal amounts of protein were immunoprecipitated with antiintegrin antibodies as described (Marcantonio and Hynes, >>1988<<). Briefly, BSA was added to lysates (180 \u03BCg total protein) to a final concentration of \u223C3 mg/ml, followed by 5\u201310 \u03BCl of antiserum. After incubation at 4\u00B0C for 1 h, 50 \u03BCl of protein A\u2013Sepharose (1:1 slurry preabsorbed with 10 mg/ml BSA in" . _:b40337 . _:b207962562 "3"^^ . _:b40401 "Indeed, Wu et al. (>>1995<<) recently showed that \u03B13\u03B21 in transfected cells can mediate assembly of both fibronectin and entactin into the ECM." . _:b40383 . _:b207962584 . _:b207962573 "2"^^ . _:b207962587 . _:b207962572 "2"^^ . _:b207962576 . . _:b40340 "Nonreducing SDS-PAGE was performed by the method of Laemmli (>>1970<<) using 5% acrylamide and a 3% stacking gel." . _:b207962586 . _:b207962705 "2"^^ . . _:b40414 "Patients with cicatricial pemphigoid that develop anti\u2013laminin-5 antibodies also form skin blisters (Kirtschig et al., >>1995<<). Similarly, patients with bullous pemphigoid, an acquired blistering disease, develop antibodies against either BPAG-1e (bullous pemphigoid antigen-1e) or BPAG-2, both components of hemidesmosomes (Diaz et al., 1977; Mueller et al.," . _:b40405 . _:b207962575 "2"^^ . _:b40399 "and Carter, 1987; Elices et al., 1991), laminin-1 (Wayner and Carter, 1987; Gehlsen et al., 1988; Elices et al., 1991), collagens (Wayner and Carter, 1987; Elices et al., 1991), and entactin/nidogen (Dedhar et al., 1992; Wu et al., >>1995<<), and shows post-adhesion localization to focal contacts in cells cultured on most of these proteins (DiPersio et al., 1995)." . . . _:b207962704 "2"^^ . _:b207962597 . _:b207962574 "2"^^ . _:b207962568 . _:b207962596 . _:b207962707 "2"^^ . _:b207962569 "2"^^ . _:b40348 "mice is reminiscent of more severe blistering phenotypes associated with loss of \u03B16\u03B24 integrin through null mutations of either the \u03B16 (Georges-Labouesse et al., 1996) or the \u03B24 subunit (van der Neut et al., 1996; Dowling et al., >>1996<<) and with human blistering diseases caused by defects in laminin-5 (Domloge-Hultsch, 1992; Baudoin et al., 1994; Kirtschig et al., 1995), the ligand for both \u03B16\u03B24 and \u03B13\u03B21." . _:b207962638 . _:b207962564 . _:b207962625 . . _:b207962706 "2"^^ . _:b207962599 . . _:b207962568 "2"^^ . . . _:b207962598 . . _:b207962571 "2"^^ . _:b207962514 . _:b40354 . . _:b40387 . _:b207962593 . _:b207962522 . _:b207962570 "2"^^ . _:b207962592 . _:b207962581 "2"^^ . . _:b207962595 . _:b207962580 "2"^^ . _:b207962594 . . _:b207962583 "2"^^ . . _:b207962668 . . . _:b207962605 . _:b207962582 "2"^^ . . _:b207962604 . _:b207962577 "2"^^ . _:b207962607 . . _:b207962576 "2"^^ . . _:b207962616 . _:b207962558 . _:b207962606 . . _:b207962579 "2"^^ . _:b40394 . _:b207962601 . _:b207962578 "2"^^ . _:b207962600 . _:b40412 "For example, some forms of JEB have been attributed to defects in the \u03B13, \u03B23, or \u03B32 chain of laminin-5 (Aberdam et al., 1994; Pulkkinen et al., 1994a ,b; Uitto et al., 1994; Kivirikko et al., 1995), and dystrophic forms of JEB are caused by mutations in the gene for type VII collagen (Uitto et al., 1994)." . _:b207962629 . _:b207962589 "2"^^ . . _:b207962603 . _:b207962588 "2"^^ . _:b40379 . _:b207962595 . . _:b207962602 . _:b207962591 "2"^^ . _:b207962613 . _:b207962590 "2"^^ . _:b40400 "(Wayner and Carter, 1987; Elices et al., 1991), and entactin/nidogen (Dedhar et al., 1992; Wu et al., 1995), and shows post-adhesion localization to focal contacts in cells cultured on most of these proteins (DiPersio et al., >>1995<<)." . _:b207962547 . _:b207962612 . _:b207962585 "2"^^ . . . _:b40388 . _:b207962660 . . _:b207962615 . _:b207962584 "2"^^ . _:b207962614 . _:b40351 "Indeed, in \u03B16-null or \u03B24-null skin, laminin-5 remains associated with the dermal sides of blisters (Georges-Labouesse et al., >>1996<<; van der Neut et al., 1996; Dowling et al., 1996)." . _:b207962554 . _:b207962587 "2"^^ . . . _:b207962609 . _:b207962617 . _:b207962586 "2"^^ . _:b207962624 . . _:b207962594 . _:b207962608 . _:b207962553 . _:b207962597 "2"^^ . . . _:b207962611 . _:b207962539 . _:b207962596 "2"^^ . _:b40388 "In contrast, \u03B16\u03B24 is necessary for assembly of hemidesmosomes but not for formation of a continuous lamina densa (Georges-Labouesse et al., >>1996<<; van der Neut et al., 1996; Dowling et al., 1996)." . _:b207962610 . _:b207962527 . _:b207962599 "2"^^ . . _:b40374 "Indeed, this pattern of proteolysis has been useful as a diagnostic tool in studies of \u03B24 integrins (Hemler et al., >>1989<<). Bands corresponding to the a, b, and c fragments of \u03B24 in mouse keratinocytes are indicated in Fig. 8; an unidentified band of \u223C70 kD may correspond to the 85-kD fragment seen in human cells. Each of these fragments showed faster" . _:b207962621 . _:b207962541 . _:b207962598 "2"^^ . _:b207962548 . . _:b207962620 . _:b207962593 "2"^^ . _:b40414 . . _:b207962623 . _:b207962592 "2"^^ . _:b207962622 . _:b207962569 . _:b207962595 "2"^^ . . _:b207962617 . _:b207962594 "2"^^ . _:b207962518 . _:b207962616 . _:b207962605 "2"^^ . _:b207962525 . . _:b40342 _:b40376 . _:b40342 _:b40377 . _:b207962619 . _:b40342 _:b40378 . _:b207962628 . _:b207962604 "2"^^ . . _:b40342 _:b40379 . _:b40342 _:b40372 . _:b40342 _:b40373 . _:b40359 . _:b207962618 . _:b40342 _:b40374 . _:b207962607 "2"^^ . _:b207962512 . _:b40342 _:b40375 . . _:b40342 _:b40368 . _:b40342 _:b40369 . _:b207962519 . _:b207962606 "2"^^ . _:b40342 _:b40370 . _:b40342 _:b40371 . _:b207962560 . _:b207962521 . _:b40344 "1, A and B, arrows), as expected for this integrin (Peltonen et al., >>1989<<; Carter et al., 1990a ,b; Hertle et al., 1991)." . . _:b40342 _:b40364 . _:b40342 _:b40365 . _:b40372 "against the \u03B16 integrin subunit (lane 3), which coimmunoprecipitated a series of bands expected for the \u03B16 subunit (\u223C140 kD) and various derivatives of the \u03B24 subunit (Hemler et al., 1989; Kajiji et al., 1989; Adams and Watt, >>1991<<). In nonreduced immunoprecipitates from human cells, the fulllength \u03B24 subunit migrates at \u223C210 kD (a fragment), and smaller fragments of 165 kD (b fragment), 125 kD (c fragment), and 85 kD are thought to result from proteolysis of \u03B24" . . _:b40342 _:b40366 . _:b207962601 "2"^^ . _:b40342 _:b40367 . _:b40384 "to cause detachment of basal epidermal keratinocytes from the basement membrane, since laminin-5 localized only to the dermal sides of blisters in both cases (Georges-Labouesse et al., 1996; van der Neut et al., 1996; Dowling et al., >>1996<<). In contrast, blistering in \u03B13-null epidermis appeared to result from defects within the basement membrane itself, since laminin-5 distributed to both the epidermal and the dermal sides of the split." . _:b40342 _:b40360 . _:b40342 _:b40361 . _:b40342 _:b40362 . _:b207962600 "2"^^ . . _:b40338 . _:b207962587 . _:b40342 _:b40363 . _:b40342 _:b40356 . _:b207962688 . _:b40342 _:b40357 . _:b40342 _:b40358 . _:b207962603 "2"^^ . _:b40342 _:b40359 . _:b207962572 . _:b40342 _:b40352 . _:b40342 _:b40353 . _:b207962573 . _:b40342 _:b40354 . _:b207962602 "2"^^ . _:b40342 _:b40355 . _:b40342 _:b40348 . _:b40342 _:b40349 . . _:b40342 _:b40350 . _:b207962633 . _:b207962613 "2"^^ . . _:b40342 _:b40351 . _:b40342 _:b40344 . _:b40342 _:b40345 . _:b40342 _:b40346 . _:b207962612 "2"^^ . . _:b207962611 . _:b40342 _:b40347 . . _:b207962615 "2"^^ . _:b40342 _:b40343 . . _:b207962637 . _:b207962614 "2"^^ . _:b207962706 . _:b207962609 "2"^^ . _:b40345 . _:b40419 . _:b207962608 "2"^^ . _:b40390 . . _:b40352 . _:b207962681 . _:b207962611 "2"^^ . . _:b207962610 "2"^^ . . _:b207962664 . _:b207962621 "2"^^ . _:b207962591 . _:b207962620 "2"^^ . . _:b40329 _:b40330 . _:b207962623 "2"^^ . _:b40329 _:b40331 . _:b207962696 . _:b207962653 . . _:b40357 . _:b40329 _:b40332 . _:b40329 _:b40333 . _:b40404 "such distinct functions for fibronectin receptors in an embryonic stem cell line, where \u03B1v\u03B23 was dominant in focal contact formation, while \u03B15\u03B21 was primarily responsible for fibronectin matrix assembly (Wennerberg et al., >>1996<<)." . _:b40329 _:b40334 . _:b207962622 "2"^^ . _:b40329 _:b40335 . . . _:b40329 _:b40336 . _:b40329 _:b40337 . . _:b207962617 "2"^^ . _:b40329 _:b40338 . _:b40329 _:b40339 . _:b40329 _:b40340 . _:b40329 _:b40341 . _:b207962616 "2"^^ . _:b40360 . _:b207962571 . _:b207962619 "2"^^ . _:b207962538 . _:b207962618 "2"^^ . . _:b40382 . _:b207962632 . _:b207962704 . . _:b207962535 . . . _:b40368 . _:b40363 . . . _:b40381 "A deficiency in \u03B16\u03B24 through null mutation of either the \u03B16 subunit (Georges-Labouesse et al., >>1996<<) or the \u03B24 subunit (van der Neut et al., 1996; Dowling et al., 1996) also causes epidermal blistering, although the blistering phenotype in these mice is much more severe than in the \u03B13-null mice." . . _:b207962630 . . _:b207962645 . _:b207962608 . _:b40411 "For example, some forms of JEB have been attributed to defects in the \u03B13, \u03B23, or \u03B32 chain of laminin-5 (Aberdam et al., >>1994<<; Pulkkinen et al., 1994a ,b; Uitto et al., 1994; Kivirikko et al., 1995), and dystrophic forms of JEB are caused by mutations in the gene for type VII collagen (Uitto et al., 1994)." . _:b40365 . _:b40382 "A deficiency in \u03B16\u03B24 through null mutation of either the \u03B16 subunit (Georges-Labouesse et al., 1996) or the \u03B24 subunit (van der Neut et al., 1996; Dowling et al., >>1996<<) also causes epidermal blistering, although the blistering phenotype in these mice is much more severe than in the \u03B13-null mice." . _:b207962698 . _:b40354 "Since hemidesmosomes appeared normal at the ultrastructural level in \u03B13-null skin, we also examined the expression and distribution of \u03B16\u03B24, a component of hemidesmosomes (Stepp et al., >>1990<<; Jones et al., 1991). The \u03B16 subunit also associates with the \u03B21 subunit in some cells (Hynes, 1992)." .