Functional Comparison of the α3A and α3B Cytoplasmic Domain Variants of the Chicken α3 Integrin Subunit

Experimental Cell Research 268, 45– 60 (2001) doi:10.1006/excr.2001.5273, available online at http://www.idealibrary.com on

Functional Comparison of the ␣3A and ␣3B Cytoplasmic Domain Variants of the Chicken ␣3 Integrin Subunit C. Michael DiPersio, 1 Jane E. Trevithick, and Richard O. Hynes Howard Hughes Medical Institute, Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

appears to be prevented by ER-retention signals in the ␣ and ␤ subunits that are repressed in the intact ␣␤ heterodimer [2– 4]. Both subunits have large extracellular domains (700 to 1000 amino acids) that together bind extracellular ligands and in most cases, a relatively short cytoplasmic domain (13 to 75 amino acids). Integrin domain structures have been conserved among vertebrates and invertebrates; however, vertebrates have evolved a much greater diversity of integrins [5]. Indeed, mammals express at least 8 ␤ subunits and 18 ␣ subunits that dimerize in limited combinations to form more than 20 functionally distinct integrins. Further diversity is generated by alternative splicing of the transcripts for certain ␣ and ␤ subunits, resulting in integrin variants that differ in their extracellular and/or cytoplasmic domains (for a review, see [6]). Some integrins are recruited to focal contacts and initiate signaling pathways that regulate cell growth and differentiation, cell migration and invasion, cell survival, and ECM assembly and remodeling [7]. Integrin cytoplasmic domains couple directly and indirectly with cytoskeletal and signaling proteins, enabling integrins to transmit signals in both directions across the plasma membrane [8, 9]. Studies with mutant integrins or chimeric receptors have shown that ␤ subunit cytoplasmic domains are necessary and sufficient for receptor localization to focal contacts or other adhesion sites [10 –15] and initiation of many integrin signaling functions [16, 17]. Therefore, it is not surprising that alternative splicing of ␤ cytoplasmic domains affects diverse cell functions, including integrin binding to ECM, integrin– cytoskeleton binding, signal transduction, and export of integrins to the cell surface [6]. The cytoplasmic domains of ␣ subunits confer distinct functions to integrins [18 –21], some of which may be regulated through binding of cytoplasmic proteins to the ␣ subunit [22–25]. However, a major role of ␣ cytoplasmic domains is to suppress functions of the ␤1 cytoplasmic domain and keep the integrin in an “inactive” state when it is not bound to ligand [2, 10, 12, 13]. The evolutionarily related ␣3, ␣6, and ␣7 subunits share approximately 40% identity, and the transcript for each can be alternatively spliced to generate “A”

Integrin ␣3␤1 can be alternatively spliced to generate ␣3A and ␣3B cytoplasmic domain variants that are conserved among vertebrates. To identify distinct functions of these variants, we transfected cells with intact ␣3 integrins or chimeric receptors. ␣3A␤1 and ␣3B␤1 each localized to focal contacts in keratinocytes on an extracellular matrix rich in laminin-5, to which both are known to bind with high affinity. However, ␣3B accumulated intracellularly in keratinocytes on collagen, suggesting that laminin binding may stabilize ␣3B␤1 surface expression. Neither ␣3 cytoplasmic domain affected recruitment of chimeric ␣5 integrins to fibronectin-induced focal contacts, and either substituted for the ␣5 cytoplasmic domain in ␣5␤1-mediated cell migration. However, the ␣5/␣3B chimera localized to cell– cell borders in MDCK or CHO cells to a lesser extent than did the ␣5/␣3A chimera. To determine whether the ␣3 cytoplasmic domains conferred distinct localization to a nonintegrin protein, we transfected cells with interleukin-2 receptor (IL-2R) chimeras containing the ␣3 cytoplasmic domains. The IL-2R/␣3A chimera was expressed efficiently on the cell surface, while the IL-2R/␣3B chimera accumulated intracellularly. Our findings suggest that the ␣3B cytoplasmic domain harbors a retention signal that is regulated in an intact integrin and can alter cell surface expression and distribution of ␣3␤1. © 2001 Academic Press

Key Words: integrin ␣3␤1; ␣ cytoplasmic variants; integrin transport.

INTRODUCTION

Integrin receptors for the extracellular matrix (ECM) are heterodimeric, transmembrane proteins consisting of an ␣ and a ␤ subunit [1]. Subunit dimerization occurs in the endoplasmic reticulum (ER), and export of nondimerized subunits to the cell surface 1

To whom correspondence and reprint requests should be addressed at Center for Cell Biology and Cancer Research, Albany Medical College, Mail Code 165, Room MS-326, 47 New Scotland Avenue, Albany, NY 12208-3479. Fax: (518) 262-5669. E-mail: [email protected]. 45

0014-4827/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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and “B” cytoplasmic domain variants [26 –28]. The A and B cytoplasmic domains share no similarity with one another beyond the membrane-proximal “KXGFFKR” motif (X is a nonconserved residue) that is conserved in all ␣ subunits. Studies comparing the functions of ␣6A␤1 and ␣6B␤1 revealed similar ligandbinding specificities but distinct postadhesion effects on cell spreading, cell migration, and signal transduction [29 –33]. Like the ␣6 integrin variants, ␣3A␤1 and ␣3B␤1 showed the same ligand-binding specificity, and both variants were activated by PMA or an anti-␤1 stimulatory antibody [34]. In contrast with the ␣6 variants, distinct functions have not been described for the ␣3A and ␣3B subunit variants. Integrin ␣3␤1 is expressed in a broad range of normal tissues, and ␣3␤1-deficient mice display developmental defects in several organ systems, including skin, kidney, lungs, and brain (for a review, see [35]). ␣3A is by far the more common ␣3 variant, being expressed in skin, kidney, lung, brain, intestine, skeletal muscle, and other tissues [34]. In marked contrast, the ␣3B transcript was detected in only a few mammalian tissues, including brain, heart, and skeletal muscle [26, 34], and the ␣3B protein was detected in only a subset of endothelial cells in heart and brain [34]. The amino acid sequence of the ␣3B variant, as well as the splicing event that generates it, has been conserved in mammals [6]. In the current study, we demonstrate that the splicing event that generates ␣3B has also been conserved in nonmammalian vertebrates. To compare ␣3A␤1 and ␣3B␤1 functions, we transfected cells with intact integrins, chimeric integrin subunits, and single-subunit interleukin-2 receptor (IL-2R) chimeras. In the context of a single-subunit IL-2R chimera, the ␣3B cytoplasmic domain inhibited receptor expression on the cell surface. However, this function of the ␣3B cytoplasmic domain was regulated in the context of an intact integrin ␣␤ heterodimer. Our results suggest that alternative ␣3 cytoplasmic domains may regulate cell surface distribution and expression levels of nonligand-bound ␣3␤1 in some cell types. MATERIALS AND METHODS Cloning of ␣3 cDNAs and construction of recombinant ␣3A and ␣3B. A complete set of overlapping cDNAs for ␣3A were cloned from chicken embryo fibroblasts (CEFs), as described previously [36, 37]. cDNAs were cloned into pBluescript II (SK-) (Invitrogen, Carlsbad, CA) and overlapping deletions were generated using exonuclease III [38]. Both DNA strands were sequenced by the dideoxy nucleotide termination method [39]. In some cases, oligonucleotides corresponding to ␣3 sequences were used as primers. The open reading frame (ORF) for the ␣3B cytoplasmic domain is retained within the ␣3A transcript downstream of the termination codon allowing us to design primers to clone a portion of the ␣3B cDNA by RT-PCR, as outlined in Fig. 2. Total CEF mRNA was used as template for AMV reverse transcriptase (Stratagene, La Jolla, CA) to transcribe from reverse primer R2, which hybridized within the ␣3B ORF. The resulting cDNA was used as template in a first round of

PCR with forward primer F1 and reverse primer R2. The resulting F1-R2 PCR product was used as template in a second round of PCR with F1 and a nested reverse primer R1. PCR conditions were as follows: denaturation at 94°C for 1 min; extension at 55°C for 2 min; annealing at 72°C for 4 min; 35 amplification cycles. F1-R1 PCR products included a 396-base-pair (bp) fragment from the ␣3A cDNA, and a 211-bp fragment that encompassed the spliced junction of the transmembrane and ␣3B cytoplasmic domains (Fig. 2B). The latter product was digested at internal Hinf1 and BamH1 sites, subcloned into pBluescript II (SK-), and confirmed by DNA sequencing. Subsequently, downstream ␣3B cDNA sequences (cloned separately within cDNA fragments) were incorporated using the BamH1 site in the ␣3B cytoplasmic domain (Fig. 2C). Finally, full-length cDNAs for ␣3A or ␣3B were constructed by fusing the extracellular and transmembrane domains from human ␣3 to the ␣3A or ␣3B cytoplasmic domains from chicken at a conserved Bcl1 site (Fig. 2C, and arrowhead in Fig. 1). Human ␣3 cDNA was a generous gift from Dr. M. Hemler (Dana-Farber Cancer Institute, Boston, MA). For transfections, ␣3 cDNAs were inserted downstream of the CMV promoter in the expression vectors pCMVneo and pcDNA3.1/Zeo(⫹) (Invitrogen), which contain genes for neomycin or zeocin resistance, respectively. Construction of chimeric integrins and interleukin-2 (IL-2) receptors. ␣5/␣3 chimeric integrin subunits were constructed to juxtapose the ␣5 transmembrane domain and the ␣3A or ␣3B cytoplasmic domain, resulting in perfect substitution of the KXGFFKR and downstream cytoplasmic sequences of ␣5 with those of either ␣3A or ␣3B (shown schematically in Fig. 4A). To accomplish this, we exploited a naturally occurring HindIII site in human ␣5 that constitutes the codons for the last two residues (KL) of the transmembrane domain. HindIII sites were engineered into the homologous positions of ␣3A and ␣3B using synthetic primers that hybridized to ␣3A or ␣3B coding sequences beginning with the first codon of the GFFRR or DFFQR, respectively. Primer sequences, with HindIII sites in bold and codon sequences underlined, were as follows: ␣3A, 5⬘-TGGAAGCTTGGTTTCTTCCGGCGG-3⬘; ␣3B, 5⬘-TGGAAGCTTGACTTCTTCCAGCGGACGCGTTATTACCGG-3⬘. These primers were used with appropriate reverse primers to PCR amplify ␣3A or ␣3B cytoplasmic domain sequences from cDNA clones. Each PCR product was then digested at the 5⬘ HindIII site and fused to the HindIII site in human ␣5. A truncated ␣5 mutant (␣5⌬cyto) lacking cytoplasmic residues including and downstream of the GFFKR was generated by blunt-end ligation of the filled-in HindIII site to a filled-in XbaI site in pBluescript II, which introduced a stop codon after the final KL residue pair of the transmembrane domain. ␣5 mutants and chimeras were confirmed by DNA sequencing and inserted downstream of the SV40 enhancer/human metallothionine promoter in the pLENneo expression vector, which contains a neomycin resistance gene. IL-2R/integrin chimeras are shown schematically in Fig. 9A. The cDNA for the 55-kDa subunit of the human IL-2 receptor was a generous gift from Dr. S. LaFlamme (Albany medical College, Albany, NY). We ligated a HindIII site at the end of the IL-2R transmembrane domain to the above described HindIII site of the ␣5, ␣3A, or ␣3B cytoplasmic domain, to juxtapose the IL-2R transmembrane domain and each cytoplasmic domain. A truncated IL-2R mutant (IL-2R⌬cyto) lacking a cytoplasmic domain was generated by bluntend ligation of the filled-in HindIII site to a filled-in XbaI site, which introduced a stop codon after the final KL codon pair encoded by the HindIII site. IL-2R/integrin chimeras were inserted into the pCMVneo expression vector. Antibodies. Monoclonal antibodies against human ␣3 (P1B5) or ␣5 (P1D6) were from GIBCO BRL (Gaithersburg, MD). Monoclonal anti-vinculin was from Sigma (St. Louis, MO). The monoclonal antibody 4E3 against the 55-kDa subunit of the human IL-2 receptor was from Boehringer Mannheim Biochemicals (Indianapolis, IN). Rabbit polyclonal antisera were raised against synthetic peptides corresponding to the cytoplasmic domain of the ␣3A, ␣3B, ␣5, or ␤1 integrin subunit, as described previously [37, 40, 41]. Immunogenic

AVIAN ␣3␤1 INTEGRIN VARIANTS peptides for anti-␣3 sera were as follows: ␣3A, YEAKGQKAEMRIQPSETERLTDDY; ␣3B, RYYRVMPKYHAVRIRQEQRYPPAGPRRKKHWVTSWQQPDKYY. Antisera were specific for the respective integrins. Stable transfection and FACS analysis. MDCK and NIH 3T3 cells were grown in DME plus 10% fetal bovine serum (FBS) or 10% calf serum, respectively. CHO cells and the CHO variant B2 (a gift from Dr. R. Juliano, Univ. North Carolina, Chapel Hill, NC, [42]) were grown in MEM␣ plus 10% FBS. Recombinant plasmids were introduced into the latter cell lines by calcium phosphate-mediated transfection, using 25 ␮g of DNA per 0.5–1.0 ⫻ 10 6 cells. The keratinocyte cell line MK-5.4.6 was derived from ␣3-null mice and maintained as described previously [43]. MK-5.4.6 cells were transfected with calcium phosphate as described previously [43], or with Lipofectamine (GIBCO BRL) according to the manufacturer’s directions, using a mix of 10 ␮g of DNA and 30 ␮l lipofectamine reagent per 10-cm dish of cells. Stable transfectants were selected in G418 (0.4 mg/ml for NIH 3T3 cells; 0.6 mg/ml for MDCK and CHO cells) or zeocin (Invitrogen; 200 ␮g/ml for MK-546 cells; 250 ␮g/ml for MDCK cells), as appropriate. In some cases transfected cell populations or clones were selected by FACS with P1B5 (␣3 transfectants), P1D6 (␣5 chimera transfectants), or 4E3 (IL-2R chimera transfectants) and expanded. For FACS, primary antibodies were used at 1:500 dilution, followed by FITC-conjugated goat anti-mouse IgG (BioSource International, Inc., Camarillo, CA) at 1:1000 dilution. Cell surface iodination and immunoprecipitation. Cell monolayers were surface-labeled with 0.5 mCi Na- 125I (New England Nuclear, Boston, MA) using the lactoperoxidase-glucose oxidase method, and cell lysates were prepared in detergent buffer (200 mM octyl-␤-Dglucopyranoside, 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl 2, 1 mM MgCl 2, 2 mM PMSF, 12.5 ␮g/ml leupeptin, 15 ␮g/ml aprotinin), as described previously [44]. Immunoprecipitations were performed as described [44] with antisera against integrin cytoplasmic domains (see above), or with monoclonal antibodies P1B5 (anti-human ␣3), P1D6 (anti-human ␣5), or 4E3 (anti-IL-2R). Samples were analyzed by SDS–PAGE on nonreducing 5% gels for integrin immunoprecipitations or on reducing (100 mM DTT) 10% gels for IL-2R immunoprecipitations. Western blotting. Cell lysates were prepared in RIPA buffer (0.1% SDS, 1% Triton X-100, 1% NaDOC, 158 mM NaCl, 10 mM Tris, pH 7.3, 2 mM EGTA, 2 mM PMSF, 12.5 ␮g/ml leupeptin, 15 ␮g/ml aprotinin) and quantitated using the Bio-Rad protein assay. For each sample, 50 ␮g of protein was subject to reducing SDS/PAGE on a 10% gel, transferred to nitrocellulose, and incubated with anti-␣3A or anti-␣3B serum (1:500 dilution) followed by peroxidase (HRP)-conjugated goat anti-rabbit IgG as secondary antibody. Chemiluminescence was performed with the Renaissance kit (NEN, Life Sciences Products). Immunofluorescence. Glass coverslips were coated with fibronectin (20 –50 ␮g/ml; Collaborative Research, Bedford, MA), vitronectin (40 ␮g/ml; GIBCO BRL), EHS laminin-1 (50 ␮g/ml; GIBCO BRL), collagen (30 ␮g/ml; Cohesion Technologies, Palo Alto, CA), or laminin-5-rich ECM prepared from SCC-25 cells as described previously [43], then blocked with 2 mg/ml BSA. Cells were grown on coated coverslips for several hours to overnight, and then prepared for immunofluorescence as described previously [41, 44]. Briefly, cells were fixed in 4% paraformaldehyde/PBS for 10 min, permeabilized in 0.5% NP40/PBS for 10 min, blocked in 10% normal goat serum/PBS, and then incubated with primary antibodies (1:100 dilutions for rabbit polyclonal sera; 1:50 dilution for anti-vinculin; 1:500 dilutions for P1B5, P1D6, or 4E3). Fluorescein-conjugated goat anti-rabbit IgG, fluorescein-conjugated goat anti-mouse IgG, and rhodamineconjugated goat anti-mouse IgG were used as secondary antibodies (BioSource International, Inc., Camarillo, CA). Filamentous actin was stained with fluorescein-conjugated phalloidin (Sigma) at a 1:5000 dilution. Representative fields were photographed on a Zeiss Axiophot microscope.

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Cell migration assays. B2 cells were transfected with full-length ␣5 subunit, or with ␣5/␣3A or ␣5/␣3B chimeras, and then compared for fibronectin-induced migration using a modified Boyden Chamber assay. Bottom chambers contained 5.0 ␮g/ml fibronectin in serumfree MEM␣; 8 ⫻ 10 4 B2 cells were seeded onto 8-␮m-pore filters in serum-free medium and incubated at 37°C for 6 h. Filters were fixed in methanol and stained with Giemsa, and nonmigratory cells were removed from upper surfaces with a cotton swab. Cells that migrated to the underside of the filter were counted by eye. For each transfectant, the average number of migrated cells and standard deviation were calculated for at least eight fields from two to three filters.

RESULTS

Alternative Splicing Events That Generate the ␣3A and ␣3B Subunit Variants Are Conserved from Birds to Mammals A full-length cDNA for the ␣3 integrin subunit was cloned from CEFs and sequenced (GenBank Accession No. AF330146). Figure 1A shows the predicted amino acid sequence of chicken ␣3 aligned with ␣3 from human [45], mouse [46], and Xenopus [47]. The ␣3 subunit is 88% identical between human and mouse [46] and has been conserved highly among vertebrates in general, showing percentage identities of 65% between human and chicken, 56% between chicken and Xenopus, and 57% between human and Xenopus. The ␣3A cytoplasmic domain has been particularly well conserved, showing 86% identity between human and chicken (Fig. 1A). Other highly conserved features include three putative metal-binding domains, the transmembrane domain, and 17 extracellular cysteines (Fig. 1A). Chicken and human ␣3 show 61% identity over a 135-amino acid region that has been shown to interact with the TM4SF protein CD151 [48] (Fig. 1A). The ␣3A and ␣3B cytoplasmic domains are conserved highly among mammals, suggesting that distinct functions for these variants have also been conserved in evolution [26, 34]. The spliced transcript for ␣3B has been detected in certain tissues from mouse [26] and human [34], but it has not been known whether this splicing event occurs in nonmammalian vertebrates. It was important to address this issue, since some integrin splice variants are not conserved even among mammals [49]. As reported previously for mammalian ␣3 [26], we found that the 3⬘-untranslated region of the chicken ␣3A transcript contained a potential ORF for a peptide that is 73% identical to the human ␣3B cytoplasmic domain (shown schematically in Fig. 2A; GenBank Accession No. AF358261). To determine whether the splicing event that generates the ␣3B transcript has been conserved in avian evolution, we used CEF mRNA as template for RT-PCR as outlined in Fig. 2A (refer to Materials and Methods for details). Primers F1 and R1, which flanked the putative site of alternative splicing, amplified a 396-bp product expected for the ␣3A cDNA as well as a 211-bp product (Fig. 2B). Sequencing of the 211 bp product

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FIG. 2. Summary of the strategies used to clone chicken ␣3B and reconstruct ␣3A and ␣3B subunits. (A) Schematic representation of the chicken ␣3A cDNA. The transmembrane domain (black box), the ␣3A cytoplasmic domain (box labeled “A”), and the termination codon (asterisk) are indicated. The exon encoding the ␣3B cytoplasmic domain (box labeled “B”) is retained in the 3⬘ untranslated sequences of the ␣3A transcript. The enlargement shows regions of hybridization of a forward primer (F1) and two reverse primers (R1 and R2), and the number of nucleotides between them. Relevant restriction sites are indicated. (B) RT-PCR was used to clone a portion of the spliced ␣3B cDNA. CEF mRNA was reverse-transcribed from primer R2, and the resulting cDNA was used as template to PCR-amplify products from the primers F1 and R1. The ethidium bromide-stained gel shows F1-R1 PCR products (CEF), which include the 396-bp fragment expected for ␣3A and a 211-bp fragment encompassing the spliced junction of the transmembrane and ␣3B cytoplasmic domains. Both fragments were absent from a control reaction that lacked template (⫺). M, molecular weight markers. (C) Full-length cDNAs for ␣3A or ␣3B were reconstructed by fusing the ␣3 extracellular and transmembrane domains from human to the ␣3A or ␣3B cytoplasmic domain from chicken at a conserved Bcl1 site in the transmembrane domain (see arrowhead in Fig. 1 for exact fusion). Termination codons are indicated by asterisks.

FIG. 1. (A) Comparison of ␣3 integrin subunits from human (HUMAN, GenBank M59911, [45]), mouse (MOUSE, GenBank NM013565, [46]), chicken (CHICK ␣3A, GenBank AF330146; ␣3B, Genbank AF358261), and Xenopus (XENO, GenBank L43057, [47]), beginning with the N-terminus of the mature peptide; numbers correspond to amino acid positions within human ␣3. Regions of identity with human ␣3 are shaded. Gaps in alignment are indicated by dashes. In one chicken ␣3 cDNA clone, a single nucleotide difference at position 840 resulted in substitution of a threonine (T) for a conserved proline (shown as a boxed P); however, it was not determined whether this discrepancy represents a polymorphism or a cloning artifact. Putative metal-binding domains are indicated with brackets. Seventeen cysteines in the extracellular domain are conserved in all four species (black dots); the cysteine at human position 71 is absent from chicken (open dot). The putative dibasic cleavage site is indicated by plus signs (⫹). The dashed line indicates human ␣3 sequences that bind CD151 [48]. The transmembrane domain is boxed; the arrowhead marks the position where human and chicken ␣3 were fused in recombinant subunits (see Fig. 2C). The ␣3A and ␣3B cytoplasmic domains are shown (␣3A cyto and ␣3B cyto, respectively) and are aligned one above the other with respect to the “KXGFFKR” motif (indicated by asterisks); the mouse ␣3B sequence is partial (GenBank S66294, [26]). Junctions of cytoplasmic domains with the transmembrane domain are indicated with a bracket. The amino acid sequence of the human ␣3B cytoplasmic domain was deduced from 3⬘ untranslated sequences of the human ␣3A cDNA [45]. The closest match to a potential alternative cytoplasmic domain in Xenopus is shown in italics, and was deduced from 3⬘ untranslated sequences of the Xenopus ␣3A cDNA [47]. (B) Alignment of the “KXGFFKR” motif in mammalian and Drosophila integrins, with highly conserved residues shaded. The asterisk indicates the aspartic acid (D) in ␣3B that replaces the glycine (G) that is conserved in all other known vertebrate ␣ subunits [8,63-68] and four of five known Drosophila ␣ subunits [5] (www.celera.com).

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confirmed that it was a partial cDNA encoding the ␣3 transmembrane domain juxtaposed to the ␣3B cytoplasmic domain (Fig. 2B). Thus, both the splicing event that generates ␣3B and the amino acid sequence of the ␣3B cytoplasmic domain are conserved in birds. Interestingly, the 3⬘-untranslated region of the published Xenopus ␣3A cDNA does not contain an obvious ORF for an ␣3B cytoplasmic domain [47]. A potential ORF for a short cytoplasmic domain, including a GFFHR sequence, does occur in the 3⬘-untranslated region of the Xenopus ␣3 transcript; however, the predicted peptide is only 23 residues long and shows very limited homology with avian or mammalian ␣3B (Fig. 1A, sequence shown in italics). Thus, the ␣3B variant may have been lost from (or diverged significantly in) Xenopus, or it may have arisen in other vertebrates after their divergence from amphibians. Alternatively, splicing of ␣3 may have evolved in Xenopus in such a way that the ORF for the ␣3B cytoplasmic domain is not retained in the ␣3A transcript. The ␣3A and ␣3B cytoplasmic domains share no homology with one another beyond the membrane proximal KXGFFKR motif that is present in all integrin ␣ subunits (Fig. 1B). It is remarkable, however, that ␣3B contains a D residue at the third position of this motif, while every other known vertebrate ␣ subunit and four of the five known Drosophila ␣ subunits contain a G residue at this position (Fig. 1B). This substitution of D for G is conserved in ␣3B from birds and mammals (Fig. 1A), suggesting that this unusual version of the KXGFFKR sequence may confer on ␣3B a function that is unique among ␣ subunits (see Discussion).

␣3A␤1 and ␣3B␤1 Localize to Focal Contacts in Keratinocytes on Laminin-5 ␣3A␤1 and ␣3B␤1 each mediate cell adhesion to laminin-5 [34]. To compare the intracellular localization of the ␣3 variants, we stably transfected ␣3A or ␣3B into the ␣3␤1-deficient MK-546 cell line, derived previously from ␣3-null keratinocytes [43]. In order to exploit antibodies specific for the human ␣3 subunit, we replaced the extracellular domain of chicken ␣3 with the homologous sequences from human ␣3 (Fig. 2C); the fusion site within the transmembrane domain is indicated by an arrowhead in Fig. 1A. Transfected MK-546 cells were cultured on laminin-5-rich ECM at subconfluent densities to maximize spreading. Immunofluorescence with the monoclonal antibody P1B5, specific for the human ␣3 subunit, revealed that ␣3A␤1 and ␣3B␤1 were each recruited to focal contacts at the periphery of the cell (Figs. 3A and 3B, respectively); in some cases, focal contacts were in a fine brush-stroke pattern near the cell edge. Double-label immunofluorescence with P1B5 (to stain ␣3) and FITC-conjugated phalloidin (to stain actin) confirmed that actin stress

FIG. 3. ␣3␤1-deficient MK-546 cells were transfected with ␣3A (A, C, E, G) or ␣3B (B, D, F, H), and stained by immunofluorescence. (A, B) Cells cultured on laminin-5-rich ECM for 2.5 h were stained with P1B5 to detect transfected ␣3 variants. Focal contacts are marked by arrowheads. (C–F) Cells cultured on laminin-5-rich ECM were double-stained with P1B5 and FITC-conjugated phalloidin, as indicated. Image pairs are C and E, D and F; arrowheads mark colocalization between focal contacts and stress fiber termini. (G, H) Cells cultured on collagen for 2 days were stained with P1B5. Size bar, 50 ␮m.

fibers terminated at these ␣3-positive focal contacts (arrowheads in Figs. 3C and 3E, 3D and 3F). These focal contacts were often difficult to visualize (i.e., Fig. 3A) or not visible in many transfected cells, probably because they were obscured by high levels of diffusely distributed ␣3 [41]. When transfected MK-546 cells were grown for 2 days on collagen, ␣3-positive focal contacts were detected rarely. Instead, ␣3A showed a diffuse distribution with some concentration near cell edges (Fig. 3G). In contrast, a higher proportion of transfected ␣3B was detected in a perinuclear pattern that resembled ER staining (Fig. 3H), possibly suggesting that when ␣3B␤1 was not stabilized on the surface by ligand binding it accumulated intracellularly. Consistent with this notion, transfected MK-546 cells that were

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FIG. 4. Transfection of NIH 3T3 cells with ␣5/␣3 integrin chimeras. (A) Schematic representation of intact ␣5 integrin subunit, and of ␣5/␣3A and ␣5/␣3B chimeras. The ␣5 extracellular domain is shown as a dark gray box; the gap indicates the relatively large size of this domain. The ␣5 transmembrane domain (TM) is shown as a light gray box. The amino acid sequences are shown for each of the ␣5, ␣3A, and ␣3B cytoplasmic domains beginning with the residue at which the sequences begin to diverge between constructs. (B) Untransfected NIH 3T3 cells (control) or cells transfected with the indicated constructs were cultured on fibronectin and stained by double-label immunofluorescence with anti-vinculin (top row) and antiserum against the cytoplasmic domains (bottom row) of ␣5 (for control and ␣5-transfectants), ␣3A (for ␣5/␣3A-transfectants), or ␣3B (for ␣5/␣3B-transfectants). Arrowheads point to examples of colocalization with vinculin at focal contacts. Size bar, 50 ␮m.

detached showed surface expression of ␣3B that was approximately twofold lower than that of ␣3A by FACS (V. Iyer and C. M. DiPersio, unpublished data). The ␣3A and ␣3B Cytoplasmic Domains Substitute Functionally for the ␣5 Cytoplasmic Domain in an Intact Integrin To compare ␣3 cytoplasmic domain functions in the context of an integrin that does not bind laminin, we constructed ␣5/␣3 chimeric subunits by replacing the cytoplasmic domain of human ␣5 with that of either ␣3A or ␣3B (Fig. 4A). Intracellular localization of the ␣5/␣3A and ␣5/␣3B chimeras were compared to that of intact ␣5 in transfected NIH 3T3 cells cultured on fibronectin. Immunofluorescence with the anti-human ␣5 antibody P1D6, which does not recognize murine ␣5, stained focal contacts in transfected cells but not untransfected cells (data not shown). As expected, double-label immunofluorescence with anti-vinculin and

antiserum that recognizes the ␣5 cytoplasmic domain across species showed that ␣5␤1 was present in vinculin-positive focal contacts in either untransfected cells (control) or ␣5-transfected cells (␣5). Similarly, doublestaining for vinculin and the ␣3A or ␣3B cytoplasmic domain showed that ␣5/␣3A and ␣5/␣3B, respectively, each localized to focal contacts (Fig. 4B). When we double-stained with anti-␣5 cytoplasmic domain (to label endogenous murine ␣5) and P1D6 (to label transfected ␣5/␣3 chimeras), we observed that endogenous ␣5 was displaced from focal contacts in cells expressing high levels of chimera (data not shown), showing that the ␣5/␣3 chimeras competed with endogenous ␣5 for recruitment to focal contacts. To determine directly whether the ␣5 chimeras could mediate adhesion to fibronectin, we transfected them into a variant CHO cell line, B2, which lacks endogenous ␣5 and adheres poorly to fibronectin [42]. We also transfected B2 cells with a mutant ␣5 that lacked

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cytoplasmic domain residues including and downstream of the GFFKR (␣5⌬cyto). Transfected pools that had been selected by one round of FACS with P1D6 were 125I-surface-labeled and assayed by immunoprecipitation for surface expression of ␣5 derivatives (Fig. 5A). Antiserum against the ␤1 cytoplasmic domain immunoprecipitated endogenous ␤1 and associated ␣ subunits from wild-type CHO cells (lane 1). As expected, B2 cells showed dramatically reduced levels of ␤1 integrin and lacked an ␣ subunit the size of ␣5 (lane 2). In transfected B2 cells, human ␣5, ␣5/␣3A, and ␣5/␣3B were each co-immunoprecipitated with the ␤1 subunit (lanes 3–5), indicating that each dimerized with endogenous ␤1 on the cell surface. This result was confirmed by immunoprecipitation with anti-human ␣5 (lanes 7–10) or antiserum specific for the cytoplasmic domain of the transfected ␣ subunit (lanes 12–17). Surface expression of the ␣5⌬cyto mutant was considerably lower than that of intact ␣5 (compare lanes 3 and 6, 8 and 11), in agreement with the previous finding that the cytoplasmic domain of ␣5 is required for efficient surface expression of ␣5␤1 [50]. In contrast, surface levels of ␣5/␣3A and ␣5/␣3B were comparable to that of intact ␣5 (compare lanes 3–5, 8 –10), which was confirmed by FACS analysis with P1D6 (Fig. 5B). Thus, either ␣3 cytoplasmic domain can substitute for that of ␣5 to confer stable surface expression of the ␣5␤1 heterodimer. In a previous study by Juliano and co-workers [50], B2 cells did not migrate in response to fibronectin, while transfection with full-length ␣5 restored B2 migration to levels seen for wild-type CHO cells. In contrast, transfection with a truncated ␣5 lacking the GFFKR motif and downstream residues restored migration to only 15–35% of wild-type levels, demonstrating a requirement for the ␣5 cytoplasmic domain in efficient cell migration. We used a similar assay to show that fibronectin-induced migration was restored in B2 cells transfected with ␣5, ␣5/␣3A, or ␣5/␣3B (Fig. 5C), demonstrating that either the ␣3A or the ␣3B cytoplasmic domain can substitute for that of ␣5 in cell migration. The ␣3A and ␣3B Cytoplasmic Domains Confer Overlapping, but Distinguishable, Intracellular Localization to Integrins That Are Not Bound to ECM In normal epidermis ␣3␤1 is localized to the basal cell surface that contacts the basement membrane, as well as to the lateral and apical cell surfaces that contact other cells [51]. Similarly, in cultured epithelial cells ␣3␤1 is concentrated heavily along cell– cell borders [52, 53]. The concentration of ␣3␤1 at cell– cell borders is usually greater when the integrin is not engaged with ECM ligands. For example, during wound healing much of the ␣3␤1 in an epithelial cell

FIG. 5. Transfection of ␣5␤1-deficient B2 cells with intact ␣5, ␣5/␣3A or ␣5/␣3B chimera, or truncated ␣5 (␣5⌬cyto). (A) Cells were surface-labeled and assayed by immunoprecipitation for integrin expression. Samples are from untransfected CHO cells, untransfected B2 cells, or B2 cells transfected with ␣5, ␣5/␣3A, ␣5/␣3B, or ␣5⌬cyto, as indicated. Immunoprecipitations were performed with antiserum against the cytoplasmic domain of ␤1 (␤1-cyto), ␣5 (␣5cyto), ␣3A (␣3A-cyto), or ␣3B (␣3B-cyto), or with monoclonal antibody against human ␣5 (P1D6). Positions of endogenous ␤1 and ␣5 derivatives are indicated. (B) FACS analysis of transfected B2 cells with P1D6 confirmed high surface expression levels of ␣5, ␣5/␣3A, or ␣5/␣3B; untransfected cells were included as a negative control. (C) Migration of untransfected (control) or transfected B2 cells (as indicated) in response to fibronectin was compared after 6 h using a modified Boyden Chamber assay. The average number of migrated cells plus standard deviation was calculated.

shifts from cell– cell borders to the basal cell surface as the integrin engages newly deposited laminin-5 [54]. Localization of ␣3␤1 to cell– cell borders may be a result of its displacement from the basal cell surface by another receptor with higher affinity for limiting amounts of laminin. For example, ␣3␤1 shifts from a basolateral distribution in normal epidermis to a basal distribution in epidermis of ␣6␤4-deficient mice, presumably because more laminin-5 is available for ␣3␤1 binding in the absence of ␣6␤4 [55]. Alternatively, or in addition, ␣3␤1 may interact with resident proteins at cell– cell borders. As shown in Fig. 3, ␣3A␤1 and ␣3B␤1 each localized to focal contacts in keratinocytes grown on laminin-5. To determine whether the ␣3 variants show differences in localization when ␣3␤1 is not engaged with ECM

AVIAN ␣3␤1 INTEGRIN VARIANTS

FIG. 6. Localization of ␣3 variants in transfected MDCK cells. (A) Cells were assayed for surface expression of transfected integrins as described for Fig. 5. Samples are from untransfected cells (control), or from cells transfected with ␣3A or ␣3B (as indicated). For each sample, 300 ␮g of lysate was immunoprecipitated with antiserum against the cytoplasmic domain of ␤1 (anti-␤1), ␣3A (anti-␣3A), or ␣3B (anti-␣3B), with monoclonal antibody against human ␣3 (P1B5), or with preimmune serum (preimm). Positions of endogenous ␤1 and of endogenous and transfected ␣ subunits are indicated. (B) MDCK cells transfected with vector alone, ␣3A, or ␣3B (as indicated) were cultured on fibronectin for 1 day and stained by immunofluorescence with P1B5. Part of the panel for ␣3B-transfectants is enlarged to show the granular staining pattern (arrow). Size bar, 50 ␮m.

ligand, we transfected full-length ␣3A or ␣3B into MDCK cells and cultured the cells on fibronectin. Immunoprecipitation of 125I-labeled integrins showed that each ␣3 variant was expressed on the cell surface (Fig. 6A). Anti-␣3A serum immunoprecipitated both endogenous canine ␣3A␤1 (lane 2) and transfected human ␣3A (lane 5; note ␣ band doublet). As expected, the monoclonal antibody P1B5 immunoprecipitated the slightly larger human ␣3A subunit from transfected cells (lane 4), but did not cross-react with canine ␣3A (lane 1). P1B5 and anti-␣3B serum each immunoprecipitated ␣3B␤1 from ␣3B-transfected cells (lanes 8, 11) but not from control cells (lanes 1, 7). Reduced levels of endogenous ␣3A␤1 in transfected cells indicated that transfected ␣3 was expressed at the expense

53

of the endogenous integrin (i.e., compare lanes 2 and 9). Intracellular localization of transfected ␣3 variants was compared by immunofluorescence. P1B5 stained control transfectants at background levels (Fig. 6B, vector). In MDCK cells cultured on fibronectin, which is not a ligand for ␣3␤1, staining of transfected ␣3A was concentrated heavily along cell– cell borders (Fig. 6B, ␣3A) in a pattern similar to that for endogenous ␣3A (not shown) and other integrins expressed in MDCK cells [56]. Transfected ␣3B was also concentrated along cell– cell borders, although to a noticeably lesser extent (Fig. 6B, ␣3B). In addition, P1B5 produced a punctate, dorsal staining pattern in ␣3B-transfected cells that was much less prominent in, or absent from, ␣3Atransfected cells and may represent staining of microvilli (Fig. 6B, arrow in enlargement). Similar experiments were performed using MDCK cells transfected with the ␣5/␣3 chimeras. Staining with P1D6 showed that ␣5, ␣5/␣3A, and ␣5/␣3B each colocalized to cell– cell borders with endogenous ␤1 subunit (Fig. 7). Presumably, high levels of transfected ␣5 derivatives saturated fibronectin binding at the basal cell surface, allowing excess integrin to accumulate at lateral surfaces. ␣5/␣3B was also detected in a more punctate pattern, similar to that observed for the ␣3B subunit (Fig. 6B). To determine the influence of ECM binding on ␣5/␣3 chimera localization, we cultured transfected CHO cells on fibronectin (where the ␣5␤1 chimera is engaged with ECM) or on vitronectin (where the ␣5␤1 chimera is not engaged with ECM). P1D6 produced low

FIG. 7. Localization of ␣5/␣3 integrin chimeras in transfected MDCK cells. Cells transfected with intact ␣5 or the ␣5/␣3A and ␣5/␣3B chimeras (as indicated) or vector alone (control) were cultured on fibronectin for 2 days and stained by double-label immunofluorescence with P1D6 against human ␣5 (top row) and antiserum against the ␤1 cytoplasmic domain (bottom row). Size bar, 50 ␮m.

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FIG. 8. Localization of ␣5/␣3 integrin chimeras in transfected CHO cells. Untransfected cells (control), or cloned transfectants expressing intact ␣5, ␣5/␣3A, or ␣5/␣3B (as indicated) were cultured on fibronectin (FN) or vitronectin (VN) and stained by immunofluorescence with P1D6 against human ␣5. Size bar, 50 ␮m.

levels of nonspecific, perinuclear staining in untransfected CHO cells (Fig. 8, control). CHO cells cultured on fibronectin showed a well-spread morphology, and P1D6 staining showed that ␣5, ␣5/␣3A, and ␣5/␣3B were not concentrated at cell– cell borders (Fig. 8, FN). CHO cells cultured on vitronectin were less spread with increased cell– cell contacts, and P1D6 staining showed that ␣5/␣3A localized efficiently to cell– cell borders, while ␣5/␣3B and intact ␣5 showed cell– cell localization to a lesser extent (Fig. 8, VN). Taken together, our transfection results with intact ␣3 integrins and ␣5/␣3 integrin chimeras (Fig. 6 – 8) suggest that the ␣3A and ␣3B cytoplasmic domains can differentially influence the intracellular localization of an integrin that is not engaged with ECM ligand. The ␣3B Cytoplasmic Domain Causes Intracellular Accumulation of a Chimeric IL-2 Receptor The ␣3A and ␣3B cytoplasmic domains may interact directly with distinct intracellular proteins in different compartments. Alternatively, the ␣3A and ␣3B cytoplasmic domains may differentially regulate the ability of the ␤1 cytoplasmic domain to direct integrin localization. To test the sufficiency of the ␣3 cytoplasmic domains for receptor localization in the absence of the ␤1 subunit, we constructed chimeric proteins containing the extracellular and transmembrane domains of the interleukin-2 receptor fused to either the ␣3A or the ␣3B cytoplasmic domain (IL-2R/␣3A and IL-2R/ ␣3B, respectively; Fig. 9A). As controls, we also constructed IL-2R with the ␣5 integrin cytoplasmic domain or lacking a cytoplasmic domain (IL-2R/␣5 and IL-2R⌬cyto, respectively; Fig. 9A). These chimeras were transfected into cells that make focal contacts (NIH 3T3 cells) or cells that make cell– cell adhesions (MDCK cells), and then assayed for intracellular localization. In NIH 3T3 cells on fibronectin (Fig. 9B), dou-

ble-label immunofluorescence showed that none of the transfected IL-2R derivatives colocalized with ␤1 integrins in focal contacts. Instead, IL-2R/␣5 and IL-2R/ ␣3A (Fig. 9B), as well as IL-2R⌬cyto (not shown), were each detected diffusely. In contrast, IL-2R/␣3B was concentrated heavily in the perinuclear region, indicating subcellular accumulation in the ER or other compartment. Double-label immunofluorescence of transfected MDCK cells (Fig. 9C) showed that IL-2R/␣5 and IL-2R/ ␣3A each co-localized with endogenous ␣3␤1 at cell– cell borders. Staining of IL-2R⌬cyto at cell– cell borders was less intense, despite high levels of expression on the cell surface (see Fig. 10), indicating that the ␣5 and ␣3A cytoplasmic domains enhanced cell– cell localization of IL-2R. In contrast, IL-2R/␣3B was completely absent from cell– cell borders and was concentrated in the perinuclear region (Fig. 9C), as seen in NIH 3T3 cells (Fig. 9B). Similar distributions were seen when these chimeras were transfected into A431 epidermoid carcinoma cells (data not shown). To assess directly the surface expression of IL-2R chimeras in the stably transfected MDCK cells, we performed FACS analysis with the anti-IL-2R antibody 4E3 (Fig. 10A). Although there were many 4E3-negative cells in each population, a large fraction of cells transfected with IL-2R/␣5, IL-2R⌬cyto, or IL-2R/␣3A were 4E3-positive (Fig. 10A). In contrast, the epitope for 4E3 was barely detectable on the surface of MDCK cells transfected with IL-2R/␣3B, even though a large fraction of this cell population stained positively with 4E3 by immunofluorescence (see Fig. 9C). Similar FACS results were obtained with the transfected NIH 3T3 cells (data not shown). Lower surface expression of IL-2R/␣3B was confirmed by immunoprecipitation of IL-2R chimeras from surface-labeled MDCK cells (Fig. 10B). Reduced surface expression of IL-2R/␣3B was not

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55

FIG. 9. Transfection of NIH 3T3 or MDCK cells with IL-2R/integrin chimeras. (A) Schematic representation of IL-2R/␣5, IL-2R/␣3A, and IL-2R/␣3B chimeras. The IL-2R⌬cyto construct lacks a cytoplasmic domain. The IL-2R extracellular domain is shown as a dark gray box; the gap indicates the relatively large size of this domain. The IL-2R transmembrane domain (TM) is shown as a light gray box. The amino acid sequences are shown for each of the ␣5, ␣3A, and ␣3B cytoplasmic domains beginning with the residue at which the sequences begin to diverge between constructs. (B) NIH 3T3 cells were mock-transfected (mock) or transfected with IL-2R/integrin chimeras (as indicated), and then cultured on fibronectin and stained by double-label immunofluorescence with the monoclonal antibody 4E3 against the IL-2R extracellular domain (top row) and antiserum against the ␤1 cytoplasmic domain (bottom row). Bright staining with 4E3 identifies transfected cells in each field. (C) MDCK cells were transfected with IL-2R derivatives as indicated, and then cultured on fibronectin and stained by double-label immunofluorescence with 4E3 (top row) and antiserum against the ␣3A cytoplasmic domain (bottom row). Arrowheads point to positions of focal contacts in (B) or cell-cell contacts in (C). Size bar, 50 ␮m.

due to reduced chimera synthesis, since western blots of MDCK cell lysates revealed comparable amounts of IL-2R/␣3A and IL-2R/␣3B in the respective transfec-

tants (Fig. 10C). Together, these results show that the ␣3B cytoplasmic domain is sufficient to cause intracellular accumulation of a heterologous surface protein.

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DISCUSSION

We have shown that the alternative splicing event that generates the ␣3B integrin variant and the sequence of the ␣3B cytoplasmic domain have been conserved from birds to mammals, indicating that ␣3B␤1specific functions have also been conserved in evolution. ␣3A␤1 and ␣3B␤1 have the same lamininbinding specificity [34], suggesting that functional differences between these variants lie in more subtle aspects of integrin function. Indeed, studies of chimeric integrins showed that swapping ␣ cytoplasmic domains altered postadhesion cellular responses to ligand without changing ligand-binding specificity of the integrin [18, 19]. Thus, alternative splicing of ␣3 may allow a cell to modulate postattachment functions such as cell migration or signaling, as has been shown for ␣6A and ␣6B [29 –33]. However, distinct postadhesion functions for the ␣3A and ␣3B variants have not yet been described. Alternative splicing may also regulate preadhesion aspects of ␣3␤1 function, such as receptor surface levels or localization. In transfected MDCK cells, ␣3Bstaining was less intense at cell– cell borders than was ␣3A-staining and occurred in an additional granular pattern that resembled microvillar staining. Similar differences in localization were seen between ␣5/␣3A and ␣5/␣3B chimeric integrins when they were not engaged with ECM ligand. In addition, we have observed that ␣3B␤1 surface expression was about twofold lower than that of ␣3A␤1 in transfected MK-546 keratinocytes (data not shown). Reduced surface expression of ␣3B␤1 may be cell-type-specific, since transfected ␣3B␤1 was expressed efficiently on the surface of MDCK cells (Fig. 6) or K562 cells (data not shown; and [34]). Together, our observations suggest that ␣3A and ␣3B cytoplasmic domains can differentially regulate integrin surface expression and distribution. The physiological importance of these observations is not known. However, a potential role for ␣3B in regulating integrin surface levels is very similar to a recently described role for the ␤1 cytoplasmic splice variants ␤1C-1 and ␤1C-2, which caused ER retention and intracellular degradation of ␤1 integrins [57]. Single-subunit IL-2R chimeras are powerful tools for FIG. 10. Cell surface expression of IL-2R chimeras in transfected MDCK cells. (A) Cells were transfected with the indicated constructs and sorted into enriched pools by one round of FACS with antibody 4E3. Surface expression of the 4E3 epitope was then compared between transfected cells (solid lines) and untransfected cells (dashed lines). (B) Surface expression of transfected IL-2R chimeras was assayed as described for Fig. 5. Samples from untransfected MDCK cells, or from cells transfected with the indicated constructs, were immunoprecipitated with 4E3 (upper gel, anti-IL2R) or with antiserum against the cytoplasmic domain of ␤1 as a control (lower gel, anti-␤1 integrin). Positions of IL-2R derivatives are indicated by the black arrowhead. A higher molecular weight product was also detected (open arrowhead) and probably represents a modified form

of the IL-2R subunit, since variations in its position and intensity correlated with differences between the known IL-2R derivatives. Endogenous ␤1 and ␣ subunits are indicated. (C) Total cell lysates from untransfected MDCK cells or from cells transfected with IL-2R/ ␣3A or IL-2R/␣3B (as indicated above each lane) were immunoblotted with antiserum against the cytoplasmic domain of either ␣3A (anti-␣3A) or ␣3B (anti-␣3B). Positions of the IL-2R derivatives are indicated; the higher and lower molecular weight products probably represent glycosylated and unglycosylated protein, respectively. Anti-␣3A serum also detects the light chain of reduced, endogenous ␣3A subunit, as indicated. Molecular weight markers are shown.

AVIAN ␣3␤1 INTEGRIN VARIANTS

analyzing functions of integrin cytoplasmic domains in isolation from other integrin domains [58]. Using this approach, we showed that the ␣3A and ␣3B cytoplasmic domains differentially regulate receptor surface expression in a way that appears to be regulated in the context of the intact integrin. The ␣3B cytoplasmic domain was sufficient to inhibit completely the surface expression of an IL-2R chimera in MDCK cells or NIH 3T3 cells, while the ␣3A cytoplasmic domain allowed efficient IL-2R chimera expression on the cell surface. Together with our data for integrin chimeras, these results suggest that inhibitory functions of the ␣3B cytoplasmic domain are constitutive in the context of the IL-2R chimera but are suppressed in the context of an intact ␣␤ heterodimer. Similar regulation has been observed for the ␤1 subunit: i.e., localization of the ␤1 cytoplasmic domain to focal contacts is constitutive in the context of an IL-2R chimera [10] but is suppressed by the ␣ subunit in the context of an intact integrin until a conformational change is induced by ligand binding [2, 13]. Thus, it seems possible that export signals in the ␤1 subunit and/or in the ␣ subunit extracellular domain function in opposition to a retention signal in the ␣3B cytoplasmic domain, resulting in a “tug-of-war” between export and retention. Cellular factors that mediate these opposing functions may vary between cell types, so that in a given cell type the balanced forces of export and retention determine ␣3B␤1 surface levels. Binding to extracellular ligand could also influence surface stability of ␣3␤1. We have not identified sequences within ␣3B that are necessary to inhibit receptor export. However, it is striking that in both birds and mammals the ␣3B cytoplasmic domain contains a D (aspartic acid) residue in place of the G (glycine) residue within the otherwise conserved KXGFFKR motif at the juxtamembrane postion. This G residue is conserved completely in all other vertebrate ␣ subunits (see Fig. 1B). Previous studies have demonstrated a requirement for an intact KXGFFKR motif in ␣␤ dimerization and integrin export [4, 50], and both natural and engineered mutations have identified specific residues within this motif that are important in these processes [59, 60]. An intriguing possibility is that the replacement of the G residue with a negatively charged D residue confers less efficient export and/or less stable surface expression on ␣3B␤1. At first glance, results of another study by de Melker et al. [59] do not support this idea, since mutation of the same G residue to A (alanine) in the KXGFFKR motif of the ␣6A subunit had no effect on dimerization with ␤1 and subsequent cell surface expression. However, different mutations of the same residue could have distinct effects on integrin export. Indeed, mutation of the last R (arginine) to either D or L (lysine) in ␣6A had no effect on ␣6A ␤1 surface expression [59], while mutation of the homologous R to Q (glutamine) in ␣IIb caused greatly reduced surface ex-

57

pression of ␣IIb␤3 [60]. Therefore, it seems possible that the acidic D residue at the “G” position in ␣3B may inhibit ␣3␤1 export, even though replacement of the G residue with a similar small amino acid (alanine) did not affect ␣6A␤1 export [59]. Mechanisms have been proposed to explain how the KXGFFKR motif may regulate integrin export. In the case of integrin ␣L␤2, the KXGFFKR confers stability to the ␣␤ heterodimer and may promote export by masking a retention element in the ␤2 subunit [4]. Thus, ␣3B may be less effective in some cells at masking a retention element in ␤1 than is ␣3A. A second possibility is suggested by the finding that talin binding to newly synthesized integrins is required for their efficient export to the cell surface [61]. It was proposed that talin binding to the ␤1 cytoplasmic domain induces a conformational change that potentiates an export signal in the KXGFFKR of the ␣ subunit. Perhaps the substitution of G to D in ␣3B abrogates this export signal. Importantly, neither of the above mechanisms explains our observation that the ␣3B cytoplasmic domain was sufficient to prevent surface expression of an IL-2R chimera. Indeed, the fact that both the IL-2R/␣3A and the IL-2R⌬cyto were expressed efficiently on the cell surface suggests that the ␣3B cytoplasmic domain contains a retention signal that is absent from ␣3A. It is possible that the unique membrane proximal sequence described above plays a direct role in protein retention. ␣3B also contains a conserved lysine pair (KK) in its cytoplasmic domain that is similar to ER retention signals in some type I transmembrane proteins. However, there appears to be a strict positional requirement for these signals, since the second lysine of the KK pair must be placed three residues from the C-terminus of the protein to cause retention [62]. The KK in ␣3B is not present in the optimal position for a functional retention signal, and potential roles in retention for these or other ␣3B residues remain to be determined. The differences in localization between intact ␣3A␤1 and ␣3B␤1, and the dramatic differences in surface expression between the IL-2R/␣3A and IL-2R/␣3B chimeras, could reflect distinct interactions of the ␣3 cytoplasmic domains with cytoplasmic factors that regulate intracellular localization. Yeast two-hybrid analysis recently identified several proteins that bind to the KXGFFKR motif or more distal sequences in the ␣3A cytoplasmic domain [22, 24]. Although many of these proteins also bound to other ␣ subunits, some of them bound differentially to ␣3A and ␣3B [22]. Not all of these interactions have been analyzed in intact integrins, and their significance remains unclear. It will be interesting to determine whether any of these ␣ subunit binding proteins are involved in differential export of IL-2R/␣3A and IL-2R/␣3B chimeras and, if so, whether these interactions are regulated in the context of an intact integrin.

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We are grateful to Drs. Mary Ann Stepp, Jasper Rees, and Ramila Patel-King for contributions to ␣3 cloning and sequencing, and to Drs. Susan LaFlamme, Martin Hemler, and Rudolph Juliano for providing critical reagents. We also thank Susan LaFlamme for critical reading of the manuscript and Dr. Douglas DeSimone for helpful discussions regarding Xenopus ␣3 integrin. We thank Glen Paradise for assistance with FACS analysis and cell sorting. This research was supported by a grant from the National Institutes of Health to R.O.H. (R01CA17007). R.O.H. is an Investigator of the Howard Hughes Medical Institute.

16.

LaFlamme, S. E., Thomas, L. A., Yamada, S. S., and Yamada, K. M. (1994). Single subunit chimeric integrins as mimics and inhibitors of endogenous integrin functions in receptor localization, cell spreading and migration, and matrix assembly. J. Cell Biol. 126, 1287–1298.

17.

Akiyama, S. K., Yamada, S. S., Yamada, K. M., and LaFlamme, S. E. (1994). Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras. J. Biol. Chem. 269, 15961–15964.

18.

Chan, B. M. C., Kassner, P. D., Schiro, J. A., Byers, H. R., Kupper, T. S., and Hemler, M. E. (1992). Distinct cellular functions mediated by different VLA integrin ␣ subunit cytoplasmic domains. Cell 68, 1051–1060.

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Briesewitz, R., Kern, A., and Marcantonio, E. E. (1993). Liganddependent and -independent integrin focal contact localization: The role of the ␣ chain cytoplasmic domain. Mol. Biol. Cell 4, 593– 604.

Kassner, P. D., Alon, R., Springer, T. A., and Hemler, M. E. (1995). Specialized functional properties of the integrin ␣4 cytoplasmic domain. Mol. Biol. Cell 6, 661– 674.

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Bennett, J. S., Kolodziej, M. A., Vilaire, G., and Poncz, M. (1993). Determinants of the intracellular fate of truncated forms of the platelet glycoproteins IIb and IIIa. J. Biol. Chem. 268, 3580 –3585.

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