Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells

Mammalian cell receptors that promote entry of intracellular bacteria into nonphagocytic cells have not been identified. We show here that multiple members of the integrin superfamily of cell adhesion receptors bind the Y. pseudotuberculosis invasin protein prior to bacterial penetration into mammalian cells. Affinity chromatography of crude detergent extracts demonstrated that integrins containing the subunit structures alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1 bound to immobilized invasin. Furthermore, phospholipid vesicles containing isolated integrin proteins were able to attach to invasin. Specificity for invasin binding to the identified integrin receptors was also demonstrated, as immunoprobing and phospholipid reconstitution studies showed that the alpha 2 beta 1 integrin, beta 2 chain integrins, and vitronectin receptor (alpha v beta 3) were not involved in cellular attachment to invasin.     

Epithelial development and differentiation in the mammary gland is not dependent on alpha 3 or alpha 6 integrin subunits

In the mammary gland, both laminin and integrins have been shown to be required for normal ductal morphogenesis during development in vivo, and for functional differentiation in culture models. Major integrin receptors for laminins in the mammary gland are alpha 3 beta 1, alpha 6 beta 1, and alpha 6 beta 4. However, the specific subunits that contribute to laminin-mediated mammary cell function and development have not been identified. In this study, we use a genetic approach to test the hypothesis that laminin-binding integrins are required for the function of the mammary gland in vivo. Rudiments of embryonic mammary gland were shown to develop in the absence of these integrin subunits. Postnatal development of the mammary gland was studied in integrin null tissue that had been transplanted into the mammary fat pads of syngeneic hosts. In mammary epithelium lacking alpha 6 integrin, the beta 4 subunit was not apparent and hemidesmosome formation was only rudimentary. However, despite this deficiency, normal ductal morphogenesis and branching of the mammary gland occurred and myoepithelial cells were distributed normally with respect to luminal cells. Mammary alveoli devoid of alpha 3 or alpha 6 integrin formed in pregnancy and were histologically and functionally identical to those in wild-type mammary gland. The tissue underwent full morphological differentiation, and the epithelial cells retained the ability to synthesize beta-casein. This work demonstrates that mammary tissue genetically lacking major laminin-binding integrin receptors is still able to develop and function.     

None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion

Antibody inhibition and alpha6beta1 ligand binding experiments indicate that the egg integrin alpha6beta1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the alpha6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of alpha6beta1. Various beta1 integrins and alphav integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using alpha3 integrin null eggs, we found that the alpha3beta1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, beta1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all beta1 integrins. We found that the beta1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse beta3 integrin function-blocking monoclonal antibody (mAb) nor alphav integrin function-blocking mAb inhibited sperm binding to or fusion with beta1 integrin null eggs. Thus, function of beta3 or alphav integrins does not seem to be involved in compensating for the absence of beta1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them.     

The collagen receptor integrins have distinct ligand recognition and signaling functions.

Distinct collagen subtypes are recognized by specific cell surface receptors. Two of the best known collagen receptors are members of the integrin family and are named alpha1beta1 and alpha2beta1. Integrin alpha1beta1 is abundant on smooth muscle cells, whereas the alpha2beta1 integrin is the major collagen receptor on epithelial cells and platelets. Many cell types, such as fibroblasts, osteoblasts, chondrocytes, endothelial cells, and lymphocytes may concomitantly express both of the receptors. We have studied the cell biology of these integrins at two levels. First, we have analyzed their ligand binding mechanism and specificity. Second, we have studied their signaling function inside three-dimensional collagen gels. This mini-review summarizes our most recent results. In conclusion, our data indicate that alpha1beta1 and alpha2beta1 integrins have differences in their ligand binding specificity. Furthermore, the two receptors are connected to distinct signaling pathways and their ligation may lead to opposite cellular responses.     

Interdomain tilt angle determines integrin-dependent function of the ninth and tenth FIII domains of human fibronectin

Integrins are an important family of signaling receptors that mediate diverse cellular processes. The binding of the abundant extracellular matrix ligand fibronectin to integrins alpha(5)beta(1) and alpha(v)beta(3) is known to depend upon the Arg-Gly-Asp (RGD) motif on the tenth fibronectin FIII domain. The adjacent ninth FIII domain provides a synergistic effect on RGD-mediated integrin alpha(5)beta(1) binding and downstream function. The precise molecular basis of this synergy remains elusive. Here we have dissected further the function of FIII9 in integrin binding by analyzing the biological activity of the FIII9-10 interdomain interface variants and by determining their structural and dynamic properties in solution. We demonstrate that the contribution of FIII9 to both alpha(5)beta(1) and alpha(v)beta(3) binding and downstream function critically depends upon the interdomain tilt between the FIII9 and FIII10 domains. Our data suggest that modulation of integrin binding by FIII9 may arise in part from its steric properties that determine accessibility of the RGD motif. These findings have wider implications for mechanisms of integrin-ligand binding in the physiological context.     

Chondrocyte integrin expression and function

The extracellular matrix (ECM) is an "information rich" environment and interactions between the chondrocyte and ECM regulate many biological processes important to cartilage homeostasis and repair including cell attachment, growth, differentiation, and survival. The integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these processes. Chondrocytes have been found to express several members of the integrin family which can serve as receptors for fibronectin (alpha 5 beta 1), types II and VI collagen (alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1), laminin (alpha 6 beta 1), and vitronectin and osteopontin (alpha V beta 3). Integrin expression can be regulated by growth factors including IGF-I and TGF-beta. By providing a link between the ECM and the cytoskeleton, integrins may be important transducers of mechanical stimuli. Integrin binding stimulates intracellular signaling which can affect gene expression and regulate chondrocyte function. Further studies are needed to more clearly define the role of integrins in cartilage.     

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.     

Involvement of transmembrane domain interactions in signal transduction by alpha/beta integrins

The alpha and beta subunits of alpha/beta heterodimeric integrins function together to bind ligands in the extracellular region and transduce signals across cellular membranes. A possible function for the transmembrane regions in integrin signaling has been proposed from structural and computational data. We have analyzed the capacity of the integrin alpha(2), alpha(IIb), alpha(4), beta(1), beta(3), and beta(7) transmembrane domains to form homodimers and/or heterodimers. Our data suggest that the integrin transmembrane helices can help to stabilize heterodimeric integrins but that the interactions do not specifically associate particular pairs of alpha and beta subunits; rather, the alpha/beta subunit interaction constrains the extramembranous domains, facilitating signal transduction by a promiscuous transmembrane helix-helix association.     

Integrins alpha2beta1 and alpha4beta1 can mediate SA11 rotavirus attachment and entry into cells

Most mammalian rotaviruses contain tripeptide amino acid sequences in outer capsid proteins VP4 and VP7 which have been shown to act as ligands for integrins alpha2beta1 and alpha4beta1. Peptides containing these sequences and monoclonal antibodies directed to these integrins block rotavirus infection of cells. Here we report that SA11 rotavirus binding to and infection of K562 cells expressing alpha2beta1 or alpha4beta1 integrins via transfection is increased over virus binding to and infection of cells transfected with alpha3 integrin or parent cells. The increased binding and growth were specifically blocked by a monoclonal antibody to the transfected integrin subunit but not by irrelevant antibodies. In our experiments, integrin activation with phorbol ester did not affect virus binding to cells. However, phorbol ester treatment of K562 parent and transfected cells induced endogenous gene expression of alpha2beta1 integrin, which was detectable by flow cytometry 16 h after treatment and quantitatively correlated with the increased level of SA11 virus growth observed after this time. Virus binding to K562 cells treated with phorbol ester 24 h previously and expressing alpha2beta1 was elevated over binding to control cells and was specifically blocked by the anti-alpha2 monoclonal antibody AK7. Virus growth in alpha4-transfected K562 cells which had also been induced to express alpha2beta1 integrin with phorbol ester occurred at a level approaching that in the permissive MA104 cell line. We therefore have demonstrated that two integrins, alpha2beta1 and alpha4beta1, are capable of acting as cellular receptors for SA11 rotavirus.     

Foot-and-mouth disease virus receptors: comparison of bovine alpha(V) integrin utilization by type A and O viruses

Three members of the alpha(V) integrin family of cellular receptors, alpha(V)beta(1), alpha(V)beta(3), and alpha(V)beta(6), have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. The virus interacts with these receptors via a highly conserved arginine-glycine-aspartic acid (RGD) amino acid sequence motif located within the betaG-betaH (G-H) loop of VP1. Other alpha(V) integrins, as well as several other integrins, recognize and bind to RGD motifs on their natural ligands and also may be candidate receptors for FMDV. To analyze the roles of the alpha(V) integrins from a susceptible species as viral receptors, we molecularly cloned the bovine beta(1), beta(5), and beta(6) integrin subunits. Using these subunits, along with previously cloned bovine alpha(V) and beta(3) subunits, in a transient expression assay system, we compared the efficiencies of infection mediated by alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), and alpha(V)beta(6) among three strains of FMDV serotype A and two strains of serotype O. While all the viruses could infect cells expressing these integrins, they exhibited different efficiencies of integrin utilization. All the type A viruses used alpha(V)beta(3) and alpha(V)beta(6) with relatively high efficiency, while only one virus utilized alpha(V)beta(1) with moderate efficiency. In contrast, both type O viruses utilized alpha(V)beta(6) and alpha(V)beta(1) with higher efficiency than alpha(V)beta(3). Only low levels of viral replication were detected in alpha(V)beta(5)-expressing cells infected with either serotype. Experiments in which the ligand-binding domains among the beta subunits were exchanged indicated that this region of the integrin subunit appears to contribute to the differences in integrin utilizations among strains. In contrast, the G-H loops of the different viruses do not appear to be involved in this phenomenon. Thus, the ability of the virus to utilize multiple integrins in vitro may be a reflection of the use of multiple receptors during the course of infection within the susceptible host.     

The ability of integrin alpha(v)beta(3) To function as a receptor for foot-and-mouth disease virus is not dependent on the presence of complete subunit cytoplasmic domains

The integrin alpha(v)beta(3) has been shown to function as one of the integrin receptors on cultured cells for foot-and-mouth disease virus (FMDV), and high-efficiency utilization of the bovine homolog of this integrin is dependent on the cysteine-rich repeat region of the bovine beta(3) subunit. In this study we have examined the role of the cytoplasmic domains of the alpha(v) and beta(3) subunits in FMDV infection. We have found that truncations or extensions of these domains of either subunit, including deletions removing almost all of the cytoplasmic domains, had little or no effect on the ability of the integrin to function as a receptor for FMDV. The lysosomotropic agent monensin inhibited viral replication in cells transfected with either intact or cytoplasmic domain-truncated alpha(v)beta(3). In addition, viral replication in transfected cells was inhibited by an alpha(v)beta(3) function-blocking antibody but not by function-blocking antibodies to three other RGD-directed integrins, suggesting that these integrins are not involved in the infectious process. These results indicate that alterations to the cytoplasmic domains of either subunit, which lead to the inability of the integrin receptor to function normally, do not abolish the ability of the integrin to bind and internalize this viral ligand.     

Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.

Branching morphogenesis of the ureteric bud (UB) [induced by the metanephric mesenchyme (MM)] is necessary for normal kidney development. The role of integrins in this complex developmental process is not well understood. However, the recent advent of in vitro model systems to study branching of UB cells and isolated UB tissue makes possible a more detailed analysis of the integrins involved. We detected integrin subunits alpha3, alpha6, beta1, and beta4 in both the UB and cells derived from the early UB. Blocking the function of each of these integrin subunits individually markedly inhibited branching morphogenesis in cell culture models. However, inhibiting individual integrin function with blocking antibodies in whole kidney and isolated UB culture only partially inhibited UB branching morphogenesis, suggesting that, in these more complex in vitro systems, multiple integrins are involved in the branching program. In whole organ and isolated bud culture, marked retardation of UB branching was observed only when both alpha3 and alpha6 integrin subunits were inhibited. The alpha6 integrin subunit can be expressed as both alpha6beta1 and alpha6beta4, and both of these beta subunits are important for UB branching morphogenesis in both cell and organ culture. Furthermore, laminin-5, a common ligand for integrins alpha3beta1 and alpha6beta4, was detected in the developing UB and shown to be required for normal UB branching morphogenesis in whole embryonic kidney organ culture as well as isolated UB culture. Together, these data from UB cell culture, organ culture, and isolated UB culture models indicate that both integrin alpha3 and alpha6 subunits play a direct role in UB branching morphogenesis, as opposed to being modulators of the inductive effects of mesenchyme on UB development. Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in UB branching during nephrogenesis. These data may help to partially explain the renal phenotype seen in integrin alpha3 and alpha3/alpha6 subunit-deficient animals.     

Integrin-mediated cell adhesion to type I collagen fibrils

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.     

Mouse primordial germ cells lacking beta1 integrins enter the germline but fail to migrate normally to the gonads

Primordial germ cells are the founder cells of the gametes. They are set aside at the initial stages of gastrulation in mammals, become embedded in the hind-gut endoderm, then actively migrate to the sites of gonad formation. The molecular basis of this migration is poorly understood. Here we sought to determine if members of the integrin family of cell surface receptors are required for primordial germ cell migration, as integrins have been implicated in the migration of several other motile cell types. We have established a line of mice which express green fluorescent protein in germline cells that has enabled us to efficiently purify primordial germ cells at different stages by flow cytometry. We have catalogued the spectrum of integrin subunit expression by primordial germ cells during and after migration, using flow cytometry, immunocytochemistry and RT-PCR. Through analysis of integrin beta1(-/-)-->wild-type chimeras, we show that embryonic cells lacking beta1 integrins can enter the germline. However, integrin beta1(-/-) primordial germ cells do not colonize the gonad efficiently. Embryos with targeted deletion of integrin subunit alpha3, alpha6, or alphaV show no major defects in primordial germ cell migration. These results demonstrate a role for beta1-containing integrins in the development of the germline, although an equivalent role for * integrin subunit(s) has yet to be established.     

Molecules mediating cell-ECM and cell-cell communication in human heart valves

The specific phenotype of different tissues depends on the interactions of cells with neighboring cells and the surrounding extracellular matrix, which is mediated by cell adhesion receptors including integrins, immunoglobulin family members, syndecans, and selectins. The aim of this study was to investigate the adhesion profile of native human valve interstitial cells (ICs) in situ and in vitro by analyzing these adhesion receptors. Flow cytometry and immunocytochemistry was used to quantify the expression of the specific receptors on ICs cultured from all human cardiac valves, and immunohistochemistry were used to profile their distribution pattern in valve tissue sections. The valve leaflets and cultured ICs from all valves expressed alpha1, alpha2, alpha3, alpha4, and alpha5 integrins to varying degrees and percentages with very little expression of alpha6 and alphaV. Valve leaflet ICs from all valves, expressed predominantly beta1 integrin but no beta3 or beta4 integrin. Syndecan-1 and Syndecan-4 were not detected. Intercellular adhesion molecule-1 was weakly detected, whereas vascular adhesion molecule-1 was barely detectable and E-selectin was not detected. This study has delineated the identity of some of the integrins synthesized and expressed by human valve ICs and the specificity of adhesion molecules with which the valve ICs interact with the extracellular matrix and mediate intercellular interactions. This pattern of expression of cell surface adhesion molecules may be considered as a basis for a fingerprint on which to base future cell alternatives and would provide useful information for valve tissue engineering.     

The role of integrins in the maintenance of endothelial monolayer integrity

This paper shows that, in confluent human umbilical vein endothelial cell (EC) monolayers, the integrin heterodimers alpha 2 beta 1 and alpha 5 beta 1, but not other members of the beta 1 subfamily, are located at cell-cell contact borders and not at cellular free edges. Also the alpha v chain, but not its most common partner beta 3, that is widely expressed in EC cell-matrix junctions, is found at cell-cell borders. In EC monolayers, the putative ligands of alpha 2 beta 1 and alpha 5 beta 1 receptors, i.e., laminin, collagen type IV, and fibronectin, are also organized in strands corresponding to cell-cell borders. The location of the above integrin receptors is not an artifact of in vitro culture since it has been noted also in explanted islets of the native umbilical vein endothelium. The integrins alpha 2 beta 1 and alpha 5 beta 1 play a role in the maintenance of endothelial monolayer continuity in vitro. Indeed, specific antibodies to alpha 2 beta 1, alpha 5 beta 1, and the synthetic peptide GRGDSP alter its continuity without any initial cell detachment. Moreover, antibodies to alpha 5 beta 1 increase the permeation of macromolecules across confluent EC monolayers. In contrast beta 3 antibodies were ineffective. It is suggested that the relocation of integrins to cell-cell borders is a feature of cells programmed to form polarized monolayers since integrins have a different distribution in nonpolar confluent dermal fibroblasts. The conclusion is that some members of the integrin superfamily collaborate with other intercellular molecules to form lateral junctions and to control both the monolayer integrity and the permeability properties of the vascular endothelial lining. This also suggest that integrins are adhesion molecules provided with a unique biochemical adaptability to different biological functions.     

β1-Integrins in the cartilage matrix

Integrins are cell-surface receptors that mediate cell attachment to extracellular matrix components. The pericellular matrix in cartilage not only is a mechanical framework, but is also important for chondrocyte differentiation and stabilization of the phenotype. The interaction between chondrocytes and pericellular matrix is mediated, in part, by integrin receptors. We have previously demonstrated the presence of beta1-integrins in the cartilage matrix of organoid culture of limb buds from 12-day-old mouse embryos by immunohistological methods. In order to corroborate these findings, we have further investigated the distribution of integrins in the cartilage matrix by immunoelectron microscopy and by immunoprecipitation methods. Cartilage tissue of limb buds of 17-day-old mouse embryos was treated with collagenase and the cell-free and cellular protein-free supernatant was removed and used for immunoprecipitation experiments. Immunoprecipitation with antibodies against beta1-, alpha1-, alpha3-, and alpha5beta1-integrins and collagen type II, followed by immunoblotting with the same antibodies, demonstrated the presence of these integrins and collagen type II in the supernatant. The integrins found in the cartilage matrix could have been either secreted or shed by the cells. The question as to whether they have a function in the cartilage matrix, such as interlinking, in the matrix organization or in the stabilization of matrix components remains to be elucidated.     

Collagen-binding I domain integrins--what do they do?

Collagens are the most abundant proteins in the mammalian body and it is well recognized that collagens fulfill an important structural role in the extracellular matrix in a number of tissues. Inactivation of the collagen alpha 1(I) gene in mice results in embryonic lethality and collagen mutations in humans cause defects leading to disease. Integrins constitute a major group of receptors for extracellular matrix components, including collagens. Currently four collagen-binding I domain-containing integrins are known, namely alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1 and alpha 11 beta 1. Unlike the undisputed role of collagens as structural elements, the biological importance of integrin mediated cell-collagen interactions is far from clear. This is in part due to the limited information available on the most recent additions of the integrin family, alpha 10 beta 1 and alpha 11 beta 1. Future studies using gene inactivation of individual and multiple integrin genes will allow testing of the hypothesis that collagen-binding integrins have redundant functions but will also shed light on their importance in pathological conditions. In this review we will describe what is currently known about the collagen-binding integrins and discuss their biological functions.     

Identification of novel interaction partners for the conserved membrane proximal region of alpha-integrin cytoplasmic domains

The alpha3Abeta1 integrin is a laminin receptor with a broad specificity for different laminin isoforms. Furthermore, it regulates the function of other integrins, like alpha2beta1, alpha5beta1 and alpha6Abeta1. In a yeast two hybrid screen of a human placenta cDNA library, we identified cDNAs coding for four different proteins that strongly interact with the conserved region of the cytoplasmic domain of the alpha3A integrin subunit. In addition to the cDNA for nucleotide exchange factor Mss4 and the putative tumour suppressor protein BIN1, two novel cDNAs were identified. Association analysis with different integrin subunits revealed them as cDNAs that encode binding proteins which react with a broad spectrum of alpha subunits. The conserved membrane proximal region of the alpha3A chain was identified as the binding site for all four proteins. They, therefore, may be involved in the regulation of general functions of integrins.     

Cell adhesion to extracellular matrix regulates the life cycle of integrins.

The expression of alpha 5 beta 1 integrin on the surface of fibroblasts requires adhesion to substratum. We have examined the basis for this adhesion-dependent surface expression by comparing the life cycle of integrins in parallel cultures of adherent and nonadherent cells. Results of biosynthetic labeling experiments in NRK fibroblasts showed that the synthesis and biosynthetic processing of the beta 1 integrin subunit proceed in the absence of cell attachment; however, when examining the behavior of preexisting cell surface integrins, we observed that the alpha beta 1 integrins are internalized and degraded when adhesion to substratum is blocked. A kinetic analysis of integrin internalization in cycloheximide-treated NRK cells showed that each of the fibroblast integrins we examined (in both the beta 1 and beta 3 families) are lost from the cell surface after detachment from substratum. Thus, the default integrin life cycle in fibroblasts involves continuous synthesis, processing, transport to the cell surface, and internalization/degradation. Interestingly, studies with NIH-3T3 cells expressing alpha 1 beta 1 integrin showed that the loss of cell-surface alpha 5 beta 1 integrin is blocked by adhesion of cells to dishes coated with type IV collagen (a ligand for alpha 1 beta 1 integrin) as well as fibronectin. Similarly, adhesion of these cells to dishes coated with type IV collagen stabilizes the surface expression of alpha 5 beta 1 as well as alpha 1 beta 1 integrin. We propose that the adhesion of fibroblasts to extracellular matrix protein alters the integrin life cycle and permits retention of these proteins at the cell surface where they can play important roles in transmitting adhesion-dependent signals.