Epithelial integrins

Cell Differentiation and Development, 32 (1990) 361-366 © 1990 Elsevier Scientific Publishers Ireland, Ltd. 0922-3371/90/$03.50

361

CELDIF 99919

Epithelial integrins Vito Quaranta Department of Immunology IMMS, Research Institute of Scripps Clinic, La Jolla, CA, U.S.A.

We have undertaken the study of integrins specifically or predominantly expressed in epithelial cells, as they may be involved in establishing and maintaining properties peculiar to epithelia, such as polarization and morphogenetic movements. We describe here recent results regarding two such integrins. One of these contains the novel 13 chain, 136, whose structure was deduced from cDNA clones. Some initial results on distribution and possible ct chain associated to [36 are discussed. Structural data are then summarized on another epithelial integrin, ¢t6[34. The a 6 subunit and a variant form of the [34 subunit were recently cloned in our laboratory. Adhesion assay results indicate that ct6134 may mediate attachment of epithelial cells to basement membranes.

Introduction

The main interest of my laboratory concerns the role of integrins in establishing and maintaining the epithelial phenotype (Kajiji et al., 1989; Cheresh et al., 1989; Tamura et al., 1990; DeLuca et al., 1990). While there is a vast body of information rapidly accumulating on the structure and function of integrins (Hynes, 1987; Ruoslahti and Pierschbacher, 1987), their relationship to certain landmark features of epithelial cells (Simons and Fuller, 1985) is not clearly understood. For instance, to what extent are integrins involved in the establishment of polarity in epithelial cells? What is the importance of integrin receptors in distinguishing lateral (cell-cell) from basal (cell-substratum) adhesive interactions? Do integrins control, at least in part, morphogenetic movements of epithelial sheets during development? Some or all

Correspondence address: V. Quaranta, M.D., Department of Immunology IMMS, Research Institute of Scripps Clinic, 10666 North Torrey Pines Rd., La Jolla, CA 92037, U.S.A.

of these phenomena may utilize, and/or be explained by integrin-type receptors. Another critical property of epithelial cells is their capacity to proliferate and/or migrate short distances as a response to breaks in the continuity of epithelial sheets, or as part of physiologic regeneration. To properly perform these tasks, changes in adhesive properties must be coordinated with respect to external cues and to growth stages. Importantly, a breakdown in these control mechanisms may result or contribute to malignant transformation, or altered development. Our goal is to unravel the involvement of integrins in such processes, by investigating their structures, function and modes of expression. An initial question one may ask is whether there exist epithelial-specific integrins, as such specificity in expression would immediately suggest a direct involvement in functions particular to epithelia. Thus far, we and others have described two integrin fl chains that are predominantly expressed in epithelial cells, f14 and /36, while no epithelial a chains have been reported. In other terms, the a chains that associate with /34 or/36 may also be found in other cell types, in associa-

362

tion with other/3 chains. The rest of this paper will review recent results obtained in our laboratory pertaining to structural and functional aspects of these epithelial integrins. The integrin subunit /36 was cloned from epithelial cells in a collaboration with Robert Pytela's laboratory (UCSF, San Francisco), using an approach based on the polymerase chain reaction (PCR). Primers based on nucleotide sequences corresponding to conserved regions of integrin /3 chains were used to amplify first-strand cDNA templates from bronchial epithelium cells (Sheppard et al., 1990). Amplified products were cloned and sequenced. One of them encoded an amino acid sequence homologous but not identical to known integrin/3 chains. Screening of carcinoma cDNA libraries with this probe resulted in the isolation of overlapping inserts encoding a typical, novel integrin/3 chain, which we termed/36. Optimally aligned with other /3 chains, /36 displays identities between 35 and 50%. The highest scores are found with/33 and /35. An interesting feature of the /36 cytoplasmic tail is that it contains a cysteine as its last residue. Cysteines in this position are often posttranslationally modified and may acquire a regulatory role. It is possible that some modification of this kind occurs in the /36 tail. The distribution of/36 is being studied by both Northern blotting and PCR amplification. The initial results in cultured cell lines indicate that this chain is only found in epithelial cells, e.g., pancreatic carcinoma, colon carcinoma, lung carcinoma, choriocarcinoma. Several other cell types (melanoma, fibrosarcoma, leukocytes) were negative. Some negative epithelial cells were also observed (cervical carcinoma HeLa, pancreatic carcinoma PancI), suggesting that /36 may have a very narrow distribution, and/or be expressed in a

developmentally regulated fashion. Examination of tissues should clarify this point. Since all integrins recognized to date are heterodimers, /36 should also be associated to an a chain. To identify this partner, we have made antibodies to the cytoplasmic tail of ]36. These antibodies were used to immunoprecipitate radiolabeled detergent cell lysates, and were found to react with two components, presumably associated. One has an apparent molecular weight of about 115 kDa under nonreducing conditions, which becomes slightly larger under reducing conditions. Both the size and the change in mobility suggest that this component may represent /36 itself, since its size is also consistent with that of the r6 protein deduced from cDNAs. The other component is identical or similar to a v, as judged by its size, its faster migration in reducing conditions, and its reactivity with an anti-a v monoclonal antibody (LM142). If these results are confirmed,/36 will become the fourth/3 chain partner described for a v (in addition to ill, r3 and /3s) and, by analogy with these other a v integrins, may be a receptor for matrix components, perhaps RGD-dependent. Another epithelial integrin we are studying is a6/34. We and others (Kajiji et al., 1989; Hemler et al., 1989; Suzuki and Naitoh, 1990) have previously shown that a6fl 4 is expressed predominantly in epithelia (epidermis, gut-lining epithelia, mammary gland, amniotic sac). By immunofhiorescence, we have also detected a6fl 4 in Schwann cells which, although not overtly epithelial, nonetheless are polarized and lay upon (or are wrapped by) a laminin-containing membrane. Note that a 6 can also be found in non-epithelial cells (e.g. platelets) in association with 131 (Hemler et al., 1989). While a6/31 has been described as a laminin receptor, no clear ligand has been found for a6fl 4,

Fig. 1. Alignment of r4 with other integrin fl subunits and location of fibronectin Type III repeats. A. The r4 sequence and other fl chain sequences were derived from recently published sequences retrievable from the Genbank database. Conserved residues are shaded. Cysteine residues which are conserved among all fl chains are indicated by squares. Chevrons mark positions where cysteines are conserved in all of the fl chains except for f14. The putative transmembrane region is designated by brackets above and below the sequences. Boundaries of the fibronectin Type III repeats are marked with arrows. An alternatively spliced cytoplasmic insert is shown with a stippled box. (hum = human; chk = chicken; xen = Xenopus; dros = Drosophila). B. Alignment of the r4 Type III repeats and comparison with a consensus sequence of the human fibronectin Type III repeats. In the consensus sequence, upper case designates invariant residues, and lower case designates dominant residues.

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364 although we have recently shown that antisera to 0/6/34 inhibit adhesion of normal cultured keratinocytes (see below). We have now completed the nucleotide sequence of cDNAs for a 6 (Tamura et al., 1990). The deduced amino acid sequence predicts a transmembrane protein with the expected properties of an integrin a chain. The degree of identity of a 6 is highest with ot3 (37%), while the other a chains range between 18 and 26%. There are three potential cleavage sites that can account for the proteolytic processing of a 6 during maturation, and three of the four potential cation binding sites described in integrin a chains are present. Because a 6 can complex with either/34 or/31 (Hemler et al., 1989), we are investigating whether subtle structural differences (e.g., alternatively spliced exons) may account for this property. We prepared an antiserum to the cytoplasmic tail of a 6, and used it to immunoprecipitate radiolabeled detergent-lysates from cells expressing either a6/34 or a6/31. In both cases, the antiserum was capable of immunoprecipitating the appropriate heterodimer. In addition, we have re-cloned by PCR amplification about 35% of the coding region of a 6 from an a6/31-expressing cell type, and found no difference with the sequence of ot6 which was cloned from an a6/34-expressing line. Thus, the available evidence suggests that there are no structural differences between ot6 associating with f14, and a 6 associating with/31. The basis for the preferential association of a 6 with/34 rather than /31 in cells that express both of these /3 chains remains therefore to be determined. cDNAs for/34 were also cloned in our laboratory (Tamura et al., 1990). Within the initial 710 amino acids of the deduced sequence, homology to the other integrin/3 chains is obvious, including conservation of cysteines at 47 of 56 positions. This portion is followed by a short hydrophobic stretch likely representing a transmembrane region, and an unusually large cytoplasmic domain encompassing in excess of 1,000 residues. While the predicted extracellular portion of /34 aligns with the integrin/3 chains, no significant homologies with other proteins were found for the /34 cytoplasmic sequence by searching the Genbank database (release 62) with TFASTA (University of

Wisconsin Computer Group Programs). However, dot-matrix analyses (COMPARE and DOTPLOT) revealed a repeat region in the cytoplasmic tail. A sequence of 132 amino acids is imperfectly reproduced three times (Fig. 1). A frequency matrix derived from this three-fold repeat (PROFILE) was used to search the NBRF databank (PROFILESEARCH). Significant similarity was found with the type III fibronectin repeats as previously noted also by Suzuki and Naitoh (1990). An alignment (LINEUP) of consensus sequences from 15 type III repeats of human fibronectin and of the f14 repeats is shown (Fig. 1). Two other groups have independently determined the complete amino acid sequence of/34 as deduced from cDNAs (Suzuki and Naitoh, 1990; Hogervorst et al., 1990). Some minor differences are apparent among these three sequences in the region of the cytoplasmic tail. Thus, three variants of /34 tall are possible, according to whether one of two possible inserts of 70 and 53 residues, respectively, or neither of them, are present. The insertions occur in the area of the type III fibronectin repeats, and may be generated by alternative splicing of primary transcripts. Interestingiy, by PCR amplification with diagnostic primers, we found evidence that such alternative splicing may occur in a cell-type specific fashion. The unique large cytoplasmic tail of f14, together with its epithelial expression, suggest that this integrin subunit may be of special significance to adhesive mechanisms of epithelial cells. We have therefore investigated this functional aspect by the use of an antiserum raised against purified ot6/34. Antisera of this kind have been instrumental for unveiling the ligand-binding properties of several other integrins, e.g., in assays based on inhibition of cell adhesion. Initially, we could not detect any inhibition of adhesion by anti-a6/34 antiserum (5710) among a large panel of carcinoma cells we tested on various matrix substrates. However, when we turned to testing normal epithelial cells in short-term culture, we immediately uncovered obvious activity by the 5710 antiserum (DeLuca et al., 1990). Thus, the adhesion to laminin-based substrates of normal human keratinocytes from confluent cultures was inhibited by 5710. In addition, over

365 50% of c o n f l u e n t k e r a t i n o c y t e s are d e t a c h e d b y this a n t i s e r u m in d e t a c h m e n t assays. N o effect was o b s e r v e d on d e r m a l f i b r o b l a s t s in these assays. A n t i b o d i e s to fll integrins h a d n o effect o n k e r a t i n o c y t e adhesion, while i n h i b i t i n g fibroblasts. I m m u n o f l u o r e s c e n c e staining ( D e L u c a et al., 1990) showed that, in confluent keratinocytes, ot6fl4 is expressed exclusively on the b a s a l m e m b r a n e of the cells, e.g., the one in c o n t a c t with substrate. I n constrast, the fit integrins are f o u n d on the l a t e r a l surfaces of cells, e.g., in areas of intercellular contacts. Together, these results p o i n t at a direct a n d i m p o r t a n t role for ot6fl4 in establishing adhesive contacts b e t w e e n epithelial cells a n d their basement membrane. It should, however, be n o t e d that the a d h e s i o n assays h a d to b e carried out with p r o l o n g e d times (12 h, as o p p o s e d to the 0.5 to 2 h used for, e.g., fibroblasts), b e c a u s e at shorter times little attachm e n t of k e r a t i n o c y t e s was detectable. This time r e q u i r e m e n t m a y reflect the necessity for establishing some p r o p e r epithelial traits, e.g., p o l a r i z a tion, as a prerequisite for a t t a c h m e n t . W h a t e v e r the case, these l o n g i n c u b a t i o n times m a y allow for secretion a n d / o r m o d i f i c a t i o n of m a t r i x substrate b y keratinocytes, such that no firm conclusions can b e d r a w n on the B4 l i g a n d even t h o u g h assays are carried o u t on purified m a t r i x g l y c o p r o teins. In conclusion, the structural a n d f u n c t i o n a l definition of epithelial integrins will h o p e f u l l y cont r i b u t e to the unveiling of the c o m p l e x m e c h a nisms that regulate a d h e s i o n in epithelial cells.

References Cheresh, D.A., J.W. Smith, H.M. Cooper and V. Quaranta: A novel vitronectin receptor integrin (avflx) is responsible for distinctive adhesive properties of carcinoma cells. Cell 57, 59-69 (1989). DeLuca, M., R.N. Tamura, S. Kajiji, S. Bondanza, R. Cancedda, P. Rossino, P.C. Marchisio and V. Quaranta: Polarized integrin a6fl 4 mediates keratinocytes adhesion to basal lamina. Proc. Natl. Acad. Sci. USA, 87:6888-6892 (1990). Hemler, M.E., C. Crouse and A. Sonnenberg: Association of the VLA subunit a 6 with a novel protein. A possible alternative to the common VLA fll subunit on certain cell lines. J. Biol. Chem. 264, 6529-6535 (1989). Hogervorst, F., I. Kuikman, A.E.G.Kr. yon dem Borne and A. Sonnenberg: Cloning and sequence analysis of f14 cDNA: An integrin subunit that contains a unique 118 kDa cytoplasmic domain. EMBO J. 9, 765-770 (1990). Hynes, R.O.: Integrins: A family of cell surface receptors. Cell 48, 549-554 (1987). Kajiji, S., R.N. Tamura and V. Quaranta: A novel integrin (aEfl4) from human epithelial cells suggests a fourth family of integrin adhesion receptors. EMBO J. 3, 673-680 (1989). Ruoslahti, E. and M.D. Pierschbacher: New perspectives in cell adhesion: RGD and integrins. Science 238, 491-497 (1987). Sheppard, D., C. Rozzo, L. Starr, V. Quaranta, D.J. Erie and R. Pytela: Complete amino acid sequence of a novel integrin fl subunit (fl6) identified in epithelial cells using the polymerase chain reaction. J. Biol. Chem. 265, 11502-11507 (1990). Simons, K. and S.D. Fuller: Cell surface polarity in epithelia. Annu. Rev. Cell Biol. 1, 243-288 (1985). Suzuki, S. and Y. Naitoh: Amino acid sequence of a novel integrin f14 subunit and primary expression of the mRNA in epithelial cells. EMBO J. 9, 757-763 (1990). Tamura, R.N., C. Rozzo, L. Starr, J. Chambers, L.F. Reichardt, H.M. Cooper and V. Quaranta: Epithelial integrin a6fl4: Complete primary structure of a 6 and variant forms of f14. J. Cell Biol., 111:1593-1604 (1990).