Abstract. We have studied the function and distribution of the o~1/31, o~5/31 and 0~6~1 heterodimers on type-1 astrocytes with antibodies specific for integrin subunits (or1, ors, or6, and/31). The c~1/31 heterodimer mediates adhesion to laminin and collagen, the otsfll to fibronectin in an RGD-dependent manner, The 0~5/~1 integrin is found in focal contacts in long-term cultures of well-spread astrocytes colocalizing with vinculin and the termini of actin stress fibers. 0tl/31 heterodimers can occasionally be found as small aggregates within focal contacts but they do not accumulate there. Instead, 0~1/31 integrins are found in punctate deposits called point contacts which are distributed over the upper and the lower cell surfaces whether laminin, collagen, fibronectin or polylysine is used as a substratum. Unlike focal contacts, point contacts contain clathrin but rarely codistribute with actin or vinculin.Two observations indicate that these point contacts are functional. First, mAb 3A3, directed against the rat o~ subunit, inhibits the attachment of astrocytes to laminin and collagen. Second, during the spreading of astrocytes, a band of point contacts forms around the cell perimeter at a time when no focal contacts are visible. While o~/31 integrins are found only in point contacts in astrocytes, the otd31 integrin, another laminin receptor, is localized within focal contacts. Moreover, o,/31 heterodimers accumulate in focal contacts in fibroblasts. Thus, the ot subunit contributes, independent of its ligand, to functional integrin heterodimer accumulation in focal contacts or in point contacts. This accumulation varies among different cell types with apparently identical heterodimers as well as with the motile state (spreading vs. flattened) of the same cells.

A monoclonal antibody (3A3) raised against a rat neural cell line (PC12) was shown previously to bind to the surfaces of these cells, inhibiting substratum adhesion. Immunochemical and other data indicated that the heterodimer recognized by 3A3 was a member of the integrin family of adhesive receptors and had a β 1 subunit. The relationship of the α subunit to other integrins was unknown. Here we show that 3A3 recognizes in rat tissues a heterodimer (~185 kDa, ~110 kDa; unreduced) that is electrophoretically and immunochemically indistinguishable from the antigen in PC12 cells. Immunoaffinity purification of the heterodimer from neonatal rats and protein microsequencing indicate that the α subunit is identical at 11 or 13 N-terminal residues with VLA-1, an integrin on human hematopoietic cells. Monoclonal antibody 3A3 inhibits the attachment of rat astrocytes to laminin or collagen but not to fibronectin or polylysine. These data suggest strongly that the integrin recognized by 3A3 is the rat homologue of VLA-1, i.e., α 1 β 1 , and that α 1 β 1 is a dual laminin/collagen receptor.Previous reports described attachment and neurite outgrowth of a neural cell line (PC12) on laminin, collagen, and other adhesive proteins Tomaselli et al., 1987Tomaselli et al., , 1988. These functional studies suggested that PC12 cells possess a dual laminin/collagen receptor . Subsequently, monoclonal antibody (mab) 1 3A3 generated against PC12 cells was found to inhibit adhesion to and neurite outgrowth on laminin and collagen . mab 3A3 immunoprecipitates two proteins of ~ 110 and ~ 185 kDa. First, functional and, later, immunochemical data suggested these proteins were members of the integrin family (Hynes, 1987;Buck & Horwitz, 1987;Ruoslahti & Pierschbacher, 1987) of heterodimeric adhesive receptors. Tomaselli et al. (1987Tomaselli et al. ( , 1988 . While the β subunit of this receptor has been identified as β 1 , the identity of the α subunit has not been determined. Since the α subunits in the β 1 integrin family largely determine the ligand binding specificity of each heterodimer (Hynes, 1987;Ruoslahti & Pierschbacher, 1987), it was important to ascertain whether the α subunit had a homologue among the several integrins identified in other species to which its functional properties could be related.Here we show that the heterodimer in PC12 cells recognized by mab 3A3 is expressed in rat tissues where it is electrophoretically and immunologically indistinguishable from that in PC12 cells. Having this rich source of receptor has permitted purication and microsequencing of the larger subunit. The amino acid sequence shows unambiguously that this is an integrin α subunit identical at 11 of the first 13 N-terminal amino acid residues with the α 1 subunit of human VLA-1, an integrin in human hematopoietic cells (Hemler, 1988). These data, together with those of others (Kramer & Marks, 1989;Ignatius & Reichardt, 1988; discussed below), argue strongly that the α 1 β 1 integrin is a dual laminin/collagen integrin is hematopoi...

Wound healing is critically affected by age, ischemia, and growth factors such as TGFbeta1. The combined effect of these factors on fibroblast migration, an essential component of wound healing, is poorly understood. To address this deficiency, we examined expression of TGFbeta receptor type I and II (TGFbetaRI and RII) under normoxia or hypoxia (1% O(2)) in cultured human dermal fibroblasts (HDFs) from young (ages 24-33) and aged (ages 61-73) adults. TGFbetaRI and RII expression was similar in both groups under normoxia. Hypoxia did not alter receptor levels in young HDFs but significantly decreased TGFbetaRI in aged cells (12 and 43%, respectively). Additionally, young cells displayed a 50% increase in activation of p42/p44 mitogen-activated kinase by TGFbeta1 (2-200 pg/ml) under hypoxia while aged cell levels of active p42/p44 decreased up to 24%. To determine functional outcomes of these findings, we measured the migratory capacity of the cells on type I collagen using a gold salt migration assay. Hypoxia increased the migratory index (MI) of young HDFs over normoxia by 30% but had no effect on aged cells. Under normoxia, TGFbeta1 (1-1000 pg/ml) increased young HDF migration in a concentration-dependent manner up to 109% over controls but minimally increased aged HDF migration (37%). Under hypoxia, TGFbeta1 significantly increased young cell MI at all concentrations but was without effect on the aged HDF response. These data demonstrate that aged fibroblasts have an impaired migratory capacity with complete loss of responsiveness to hypoxia and deficits in the migratory and signal transduction responsiveness to TGFbeta1 that may partly explain diminished healing capabilities often observed in aged patients.