Mutational Analysis of Cell Cycle Inhibition by Integrin beta 1C*

Jere E. Meredith Jr.Dagger , William B. Kiosses, Yoshikazu Takada, and Martin Alexander Schwartz§

From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Integrin beta 1C is an alternatively spliced cytoplasmic variant of the beta 1 subunit that potently inhibits cell cycle progression. In this study, we analyzed the requirements for growth suppression by beta 1C. A chimera containing the extracellular/transmembrane domain of the Tac subunit of the human interleukin 2 receptor (gp55) fused to the cytoplasmic domain of beta 1C (residues 732-805) strongly inhibited growth in mouse 10T1/2 cells even at low expression levels, whereas chimeras containing the beta 1A, beta 1B, beta 1D, beta 3, and beta 5 cytoplasmic domains had weak and variable effects. The beta 1C cytoplasmic domain is composed of a membrane proximal region (732-757) common to all beta 1 variants and a COOH-terminal 48-amino acid domain (758-805) unique to beta 1C. The beta 1C-specific domain (758-805) was sufficient to block cell growth even when expressed as a soluble cytoplasmic green fluorescent protein fusion protein. These results indicate that growth inhibition by beta 1C does not require the intact receptor and can function in the absence of membrane targeting. Analysis of deletions within the beta 1C-specific domain showed that the 18-amino acid sequence 775-792 is both necessary and sufficient for maximal growth inhibition, although the 13 COOH-terminal residues (793-805) also had weak activity. Finally, beta 1C is known to be induced in endothelial cells in response to tumor necrosis factor and is down-regulated in prostate epithelial cells after transformation. The green fluorescent protein/beta 1C (758-805) chimera blocked growth in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145, consistent with a role for beta 1C as a growth inhibitor in vivo.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES

Integrins are a family of transmembrane cell adhesion receptors composed of alpha  and beta  subunits; 12 different alpha  subunits and 9 different beta  subunits have been identified to date (reviewed in Refs. 1 and 2). The beta 1 subunit pairs with alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, alpha 8, alpha 9, and alpha v to generate 10 different receptors that show distinct binding specificities for extracellular matrix proteins and other cell surface receptors. In addition to their role as adhesion receptors, integrins regulate cellular functions including cytoskeletal organization, cell growth, gene expression, survival, and migration. These effects are dependent on signaling events involving changes in protein phosphorylation, protein interactions, intracellular ion concentrations, and lipid metabolism. Integrin cytoplasmic domains lack intrinsic enzymatic activity but have been shown to activate a variety of signaling pathways including protein kinases (focal adhesion kinase, integrin-linked kinase, protein kinase C, and mitogen-activated protein kinase), lipid kinases, phospholipases, and small GTP-binding proteins (Ras, Rac, and Cdc 42) and to bind to cytoskeletal proteins including alpha -actinin, talin, filamin, and paxillin.

Four different beta 1 cytoplasmic domain isoforms have been identified (beta 1A, beta 1B, beta 1C, and beta 1D), and at least one is expressed in nearly every cell type (3-6). The beta 1A isoform is the primary isoform in most cells except for skeletal muscle, where beta 1D predominates (4, 5). These isoforms are generated by alternative splicing that occurs between the sixth and seventh exons (7). As a result, each of the beta 1 cytoplasmic domains contains a common 26-aa1 membrane proximal sequence and a unique COOH-terminal sequence. The potential significance of these splice variants is highlighted by the fact that many integrin-regulated signals are mediated by the beta  subunit cytoplasmic domain. For example, the beta 1A cytoplasmic domain has been shown to interact with the cytoskeletal proteins alpha -actinin, filamin, and talin, and sequences within the unique beta 1A domain are required for focal adhesion localization (8-15). Taken together, these observations suggest that alternative splicing within the beta 1 gene may have profound effects on receptor function.

We previously investigated the function of the beta 1C isoform in mouse 10T1/2 fibroblasts. We found that expression of beta 1C induced a potent inhibition of cell cycle progression, leading to arrest in late G1 (16). beta 1C failed to localize to focal adhesions, suggesting that it has altered interactions with cytoskeletal proteins (16). beta 1C also blocked the growth of Chinese hamster ovary cells (17). The functions of beta 1C in vivo are unknown, but beta 1C expression has been shown to correlate with growth arrest. It is up-regulated in human umbilical endothelial cells in response to treatment with tumor necrosis factor, which blocks the growth of these cells (17), and was found in quiescent prostate epithelial cells but was down-regulated in prostate carcinoma, suggesting that it could function as a tumor suppressor (18).

In this study, we used beta 1C chimeras to define the sequences necessary and sufficient for regulating growth. We identified a critical 18-aa region and showed that beta 1C sequences can function apart from the rest of the integrin, even in the absence of membrane targeting. We also found that the beta 1C-specific domain blocks DNA synthesis in endothelial and prostate carcinoma cells, suggesting that beta 1C protein may inhibit growth in vivo.

    MATERIALS AND METHODS

Cell Culture-- Mouse fibroblast C3H 10T1/2 cells were grown in Dulbecco's modified Eagle's medium low glucose (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies) and glutamine/penicillin/streptomycin (Life Technologies). The human prostate epithelial cell line DU145 was grown in Dulbecco's modified Eagle's medium high glucose supplemented with glutamine/penicillin/streptomycin, nonessential amino acids (Sigma), and 10% fetal bovine serum. The human endothelial cell line EV304 was grown in endothelial growth media (Clonetics) supplemented with 10% fetal bovine serum. C3H 10T1/2 cells were transiently transfected using LipofectAMINE (Life Technologies) as per the manufacturer's instructions. DU145 and EV304 cells were transiently transfected using Effectene (Qiagen) as per the manufacturer's instructions. DNAs for transfection were prepared using Plasmid Maxi Kits (Qiagen).

DNA Constructs-- IL2 receptor integrin chimeras were generated by fusing various integrin beta  subunit cytoplasmic domains to the extracellular/transmembrane domain of the IL2 receptor alpha  subunit (gp55) using the unique membrane proximal HindIII site. Chimeras were expressed under the control of the cytomegalovirus promoter in pCDNA3 expression vector (Invitrogen). Cytoplasmic domains and cytoplasmic domain fragments of the integrin beta 1A, beta 1C, beta 1D, and beta 5 subunits were generated by polymerase chain reaction amplification using the pBJbeta 1A, pBJbeta 1C, pCbeta 1D, and pBJbeta 5 cDNAs, respectively. The full-length beta 1B cytoplasmic domain was generated by polymerase chain reaction using overlapping primers and pBJbeta 1A cDNA. All fragments were subcloned as HindIII/XbaI fragments containing an in-frame NH2-terminal HindIII site and a COOH-terminal stop codon followed by a XbaI site. Construction of the IL2 receptor chimeras lacking a cytoplasmic domain (IL2R/TL) or containing the integrin beta 3 cytoplasmic domain (IL2R/beta 3) or the full-length beta 1D cytoplasmic domain (IL2R/beta 1D)2 has been described previously (19, 20). The GFP/beta 1C chimera was generated by polymerase chain reaction amplification of the beta 1C-specific domain 758-805 containing an in-frame NH2-terminal EcoRI site and a COOH-terminal stop codon followed by a SalI site and subcloned into the EcoRI/SalI sites of the GFP expression vector pEGFP-C1 (CLONTECH). Polymerase chain reaction primers were purchased from Life Technologies. All constructs were verified by DNA sequencing. The protein numbering for beta 1C is based on the mature protein such that the first residue of the beta 1C-specific domain is 758; other investigators have used a numbering system based on the immature protein such that the beta 1C-specific domain begins with 778 (17).

Flow Cytometry-- Cell growth in transiently transfected cells was measured by labeling cells with bromodeoxyuridine/fluorodeoxyuridine (BrdUrd; Amersham) for 16 h starting at 32 h after transfection. Labeled cells were then detached with trypsin/EDTA (Life Technologies), washed once with growth media and twice with 1% bovine serum albumin/PBS (nuclease- and protease-free; Calbiochem), and then fixed in cold 70% ethanol on ice for 30 min. Cellular DNA was denatured by treating fixed cells with 2 N HCL/0.5% Triton for 30 min and stopped by incubating cells in 0.1 M Na2B4O7, pH 8.5. Cells were then washed once with 0.5% Tween 20/1% bovine serum albumin/PBS (TBP). IL2 receptor chimera expression was determined by incubating cells with a phycoerythrin-conjugated anti-IL2 receptor antibody (PharMingen), and BrdUrd incorporation was determined by using a fluorescein isothiocyanate-conjugated anti-BrdUrd antibody (Becton Dickinson). Antibodies were diluted in TBP buffer, and labeling reactions were incubated at 37 °C for 30 min. Labeled cells were analyzed using a Becton Dickinson FACScan and CellQuest software. Approximately 100,000 cells were analyzed per construct in each experiment. BrdUrd incorporation was determined for different levels of chimera expression, with expression level gates set such that at least 1,000 cells were included in each gate. BrdUrd incorporation levels were determined, normalized against the IL2R/TL control, and expressed as the percentage of inhibition per mean expression level.

Immunofluorescence-- Transiently transfected cells were detached with trypsin/EDTA and replated in growth medium on glass coverslips 24 h after transfection. Cell growth was measured by labeling cells with BrdUrd for 5 h starting at 48 h after transfection. Labeled cells were fixed in 2% formaldehyde (EM Sciences)/PBS for 15 min at room temperature, extracted with 0.2% Triton/PBS for 10 min, treated with 0.1 unit/µl DNase I for 30 min at 37 °C, and blocked in 10% normal goat serum/PBS (Life Technologies). Primary and secondary antibodies were diluted in 10% normal goat serum/PBS, and labeling reactions were incubated for 30 min at 37 °C. In order to measure BrdUrd incorporation in cells expressing IL2 receptor chimeras, cells were probed with an anti-BrdUrd primary antibody (Calbiochem) followed by a fluorescein isothiocyanate-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch). Cells were then blocked with mouse IgG (Sigma) before labeling with a phycoerythrin-conjugated anti-IL2 receptor antibody (PharMingen). In order to measure BrdUrd incorporation in cells expressing GFP chimeras, cells were probed with an anti-BrdUrd primary antibody followed by a lissamine rhodamine sulfonyl chloride-conjugated goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch). Coverslips were mounted in Immunofluore mounting media (ICN) and examined using a Leitz diaplan fluorescence microscope. Fluorescent images were obtained using a Bio-Rad 1024 MRC Scanning Confocal Microscope.

    RESULTS

Localization of Integrin Cytoplasmic Domain-IL2 Receptor Chimeras-- To map the growth regulatory sequences in beta 1C, we generated a chimera containing the extracellular/transmembrane domain of the 55-kDa Tac subunit of the IL2 receptor fused to the cytoplasmic domain of beta 1C (IL2R/beta 1C). Similar chimeras have been used to investigate the signaling of other integrin beta  subunit cytoplasmic domains, including beta 1A, beta 1D, and beta 3 (19-24). These chimeras have been shown to mimic properties of wild type receptors, including focal adhesion localization and the activation of focal adhesion kinase phosphorylation, but in a ligand-independent manner (21). Mouse 10T1/2 fibroblasts were transiently transfected with IL2 receptor chimeras containing the beta 1A, beta 1B, beta 1C, and beta 1D cytoplasmic domains and stained for surface expression. A tail-less construct (TL) that did not contain any integrin sequences was used as a control. As shown in Fig. 1, IL2R/beta 1C (beta 1C) exhibited a diffuse staining pattern similar to the staining pattern of the tail-less IL2R chimera (TL). This result is consistent with the diffuse staining pattern observed for the full-length receptor (16). The IL2R/beta 1B chimera (beta 1B) also displayed a diffuse staining pattern similar to the wild type beta 1B receptor (25). In contrast to the beta 1C and beta 1B chimeras, the beta 1A and beta 1D chimeras were localized to focal adhesions, in agreement with previous reports (19, 20, 24).


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Fig. 1.   Localization of the IL2 receptor chimeras. Mouse 10T1/2 cells were transiently transfected with expression vectors coding for the IL2 receptor extracellular and transmembrane sequence lacking a cytoplasmic domain (TL) or fused to the cytoplasmic domains of the integrin beta 1 splice variants beta 1A, beta 1B, beta 1C, or beta 1D. Cells were fixed and stained for surface expression using a phycoerythrin-conjugated anti-IL2 receptor antibody. Bar, 10 µm.

Effects of IL2R Chimeras on DNA Synthesis-- We next tested the effects of the different chimeras on DNA synthesis. Transiently transfected cells were replated on glass coverslips after 24 h and incubated with the thymidine analogue BrdUrd for 5 h starting at 48 h after transfection. Cells were then fixed and double-stained for IL2R expression and BrdUrd incorporation. We have found that high levels of chimera expression induced cell retraction and rounding and inhibited cell adhesion or spreading,3 consistent with previous reports that these chimeras can function as dominant negative inhibitors for endogenous integrin function (20, 23, 26). Because the 10T1/2 fibroblasts are anchorage-dependent, effects on cell adhesion would result in nonspecific growth arrest. Therefore, to rule out this type of effect, only morphologically normal cells were scored for nuclear labeling with anti-BrdUrd. As shown in Fig. 2, the IL2R/beta 1C chimera blocked BrdUrd incorporation in >99% of the expressing cells. In contrast, the IL2R/beta 1A, beta 1B, and beta 1D chimeras showed varying but relatively modest effects on BrdUrd labeling. The beta 1A and beta 1B chimeras caused decreases in DNA synthesis, whereas the beta 1D chimera induced a modest increase compared with the IL2R/TL control (Fig. 2). We also examined chimeras containing the integrin beta 3 and beta 5 cytoplasmic domains, both of which localized to focal adhesions.3 As shown in Fig. 2, the beta 3 chimera induced an increase in cells labeled with BrdUrd, whereas the beta 5 chimera partially inhibited labeling. These results indicate that only the beta 1C cytoplasmic domain functions as a potent inhibitor, although the other beta  subunit cytoplasmic domains have detectable effects on growth. These data also indicate that the beta 1C cytoplasmic domain is sufficient to induce cell cycle arrest when separate from the rest of the integrin.


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Fig. 2.   Effect of IL2 receptor chimeras on cell growth. Mouse 10T1/2 fibroblasts were transiently transfected with vectors for IL2 receptor chimeras with no cytoplasmic domain (TL) or containing the cytoplasmic domains for the integrins beta 1A, beta 1B, beta 1C, beta 1D, beta 3, and beta 5. Transfected cells were replated on glass coverslips 24 h after transfection and labeled with BrdUrd for 5 h at 48 h after transfection. Cells were fixed and double-stained for IL2 receptor expression and BrdUrd incorporation. Cells positively stained with anti-IL2R were scored for BrdUrd labeling. 100 cells were scored per construct per experiment. Values are the means ± S.D. of three experiments.

Dose-Response Curves-- To obtain a more quantitative estimate of the dependence of growth inhibition on expression levels, dose-response curves for the beta 1A and beta 1C chimeras were generated. Starting at 30 h after transfection, cells were labeled with BrdUrd for 16 h and then detached, fixed, and stained for both BrdUrd incorporation and IL2R chimera expression. Cells were then assayed by flow cytometry. Using this protocol, the maximum growth inhibition was 45-50%; even cells that were detached and put into suspension at the beginning of the labeling period were inhibited by only 45%. Presumably, this result reflects the fact that a significant fraction of the cells have passed the restriction point and are already committed to or have entered S phase. The correlation between expression level for IL2R and BrdUrd staining is shown in Fig. 3. We found that both of the integrin chimeras induced growth inhibition, although at markedly different expression levels. The beta 1A chimera blocked growth at high expression levels, consistent with the idea that this chimera may function as dominant negative inhibitor for endogenous integrin function. In contrast, the beta 1C chimera was a distinctly more potent, maximally inhibiting DNA synthesis at a level of expression just above background.


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Fig. 3.   Dose-response analysis of IL2R/beta 1A and IL2R/beta 1C. Mouse 10T1/2 fibroblasts were transiently transfected with the IL2 receptor chimeras IL2R/TL, IL2R/beta 1A, or IL2R/beta 1C. Cells were labeled with BrdUrd for 16 h at 32-48 h after transfection, detached, fixed, and double-stained for IL2R chimera expression and BrdUrd incorporation. Approximately 106 labeled cells per construct were analyzed by flow cytometry. BrdUrd incorporation was determined for different levels of chimera expression and expressed as the percentage of inhibition per mean expression level relative to the IL2R/TL control. The results from one experiment are shown; similar results were seen in five independent experiments.

The results in Fig. 3 using fluorescence-activated cell-sorting analysis show significant inhibition of DNA synthesis at high levels of beta 1A expression, in disagreement with Fig. 2, in which DNA synthesis was only slightly decreased in beta 1A-expressing cells scored after plating on coverslips. One possible source of this discrepancy is that high expressors might be lost after replating and their influence might be further decreased because rounded cells were not scored. To investigate this question, cells transfected with beta 1A or beta 1C were left in the original dish or replated into a fresh tissue culture dish and then detached and analyzed for expression of the transfected integrins. Replating caused a substantial loss of the beta 1A high expressors from the population, whereas no decrease in beta 1C-expressing cells was observed.3 This result explains the difference between the two methods and supports the conclusion that growth inhibition by beta 1A is due to a dominant negative effect on cell adhesion, whereas inhibition by beta 1C is caused by a distinct mechanism.

Mapping Inhibitory beta 1C Sequences-- The beta 1C cytoplasmic domain (residues 732-805) is composed of a common region (732-757) shared with the other beta 1 splice variants and a unique region (758-805) specific to beta 1C. Hungerford et al. (27) showed that microinjection of a peptide derived from the common beta 1 region can block endogenous integrin function and induce programmed cell death in chicken fibroblasts in serum-free media. As shown in Fig. 4, deletion of the common region had no effect on growth inhibition by beta 1C, indicating that the beta 1C-specific domain was sufficient. We next asked whether the beta 1C-specific domain could block growth when expressed as a soluble protein in the absence of membrane targeting. A chimera was generated containing the beta 1C-specific domain (758-805) fused to the GFP. This chimera blocked growth in 93% of the expressing cells (Fig. 4B). Thus, growth inhibition by beta 1C occurs even when expressed as a soluble cytoplasmic protein.


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Fig. 4.   Effect of the beta 1C-specific domain (758-805) on cell growth. Mouse 10T1/2 fibroblasts were transiently transfected with various IL2 receptor chimeras (A) or GFP chimeras (B) and assayed for cell cycle progression. Transfected cells were replated on glass coverslips 24 h after transfection and labeled with BrdUrd for 5 h at 48 h after transfection. Cells were then fixed and analyzed for chimera expression and BrdUrd incorporation. Values are the means ± S.D. of three independent experiments.

In order to further define growth regulatory sequences, we generated a series of IL2R/beta 1C chimeras containing deletions within the beta 1C-specific domain (758-805). Deletion of the membrane-proximal 17 aa (Fig. 5, construct 2) had no effect on growth inhibition, whereas the deletion of an additional 8 aa (construct 3) led to a substantial increase in DNA synthesis. Expression of the COOH-terminal 13 aa alone (construct 4) had weak but reproducible growth-inhibitory activity. When we screened deletions from the COOH terminus of the beta 1C-specific domain, we found that deletion of the COOH-terminal 13 aa failed to increase DNA synthesis (constructs 2-4). This result was surprising, because we had previously reported that the deletion of this COOH-terminal 13 aa region (793-805) abolished growth inhibition by the intact receptor (16). Deletion of the next 10 residues (Fig. 5B, construct 5) almost completely eliminated growth inhibition. Taken together, these results identify an 18-aa region, 775-792, that is the major site of the growth-inhibitory activity. The COOH-terminal 13-aa region also has weak activity; curiously, it is required for growth inhibition by the intact receptor but not by the shorter beta 1C-specific domain expressed as a chimera with the IL2R. This sequence may therefore be important for the proper conformation of the intact receptor but not a shorter peptide.


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Fig. 5.   Effects of deletions within the beta 1C-specific sequence. Mouse 10T1/2 fibroblasts were transiently transfected with IL2 receptor chimeras containing various NH2-terminal (A) or COOH-terminal (B) deletions within the beta 1C-specific domain (758-805). Transfected cells were replated on glass coverslips 24 h after transfection and labeled with BrdUrd for 5 h at 48 h after transfection. Cells were than fixed and double-stained for IL2R chimera expression and BrdUrd incorporation. IL2 chimera expressors were scored for the number of cells labeled with BrdUrd, and the percentage of BrdUrd incorporation was normalized against the percentage of incorporation for IL2R/TL. 100 cells were scored per construct per experiment. Values are the means ± S.D. of three independent experiments.

Growth Inhibition in Human Cell Lines-- Previous reports suggested that beta 1C may function as a cell growth regulator: its expression is induced in human umbilical endothelial cells in response to tumor necrosis factor, which causes growth arrest, and beta 1C is down-regulated during the transition from normal, quiescent prostate epithelium to prostate carcinoma (17, 18). To test the ability of beta 1C to block growth in these cell types, GFP/beta 1C was transiently expressed in the human endothelial cell line EV304 and in the transformed prostate epithelial cell line DU145. Expressing cells were labeled with BrdUrd to assay cell cycle progression. As shown in Fig. 6, GFP/beta 1C induced growth arrest in both the EV304 cells and the DU145 cells, blocking BrdUrd labeling by 46.7% and 72.8%, respectively, compared with GFP controls. These results indicate that beta 1C can block cell growth in human cell lines relevant to its in vivo expression.


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Fig. 6.   Effect of GFP/beta 1C on the growth of endothelial cells and prostate epithelial cells. Human endothelial cells (EV304) (A) or transformed human prostate epithelial cells (DU145) (B) were transiently transfected with vector, GFP, or GFP/beta 1C constructs. Transfected cells were replated on glass coverslips 24 h after transfection and labeled with BrdUrd for 5 h at 48 h after transfection and then fixed and stained for BrdUrd incorporation. Total transfected cells (vector) or GFP-expressing cells (GFP; GFP/beta 1C) were scored for the percentage of cells labeled with BrdUrd. 100 cells were analyzed per construct per experiment. Values are the means ± S.D. from three independent experiments.


    DISCUSSION

We used chimeras of the beta 1C cytoplasmic domain with both the IL2 receptor and GFP to map growth regulatory sequences in integrin beta 1C. The data revealed that growth inhibition by beta 1C occurred at low expression levels and was mediated entirely by the beta 1C-specific domain. This domain functioned in the absence of the beta  subunit extracellular, transmembrane, and common cytoplasmic sequences, did not require the alpha  subunit, and was effective in the absence of membrane targeting. Additional deletion mapping showed that the primary activity resided within an 18-aa region (775-792). The beta 1C cytoplasmic domain also blocked growth when expressed in human endothelial cells and transformed prostate epithelial cells, suggesting that the endogenous protein may function in a similar capacity under physiological conditions. These conclusions are summarized in Fig. 7.


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Fig. 7.   Model of the beta 1C signaling domain. Deletion analysis indicates that the 18-aa region (775-792; box) within the beta 1C-specific domain is necessary and sufficient for inducing growth arrest. The COOH-terminal 13-aa region (793-805; underline) also induces partial growth arrest and is required for growth inhibition by the intact receptor (16). These results suggest that this region may contain a secondary effector region and may also be required for inducing the proper conformation within the full-length receptor. The region identified by Fornaro et al. (775-782; underline) (17) is also indicated. The protein sequences of four Alu-containing proteins that are homologous to beta 1C are shown below: an alternatively spliced variant of the neurofibromatosis 2 tumor suppressor gene (NF2), an alternatively spliced variant of the c-myb transcription factor (c-myb), the transformation-related protein (TRP), and the neuronal thread protein AD7c-NTP (NTP). The predicted protein sequence of an Alu-Sx element in the antisense orientation is also shown.

Other integrins had some effects on cell cycle progression. The beta 1A chimera decreased DNA synthesis at high levels, consistent with reports that it functions as a dominant negative inhibitor for endogenous integrin function in cell adhesion (20, 23, 26). Expression of beta 1B also decreased DNA synthesis, consistent with published data indicating that this isoform can function as a dominant negative inhibitor (28). We observed some stimulation of DNA synthesis by beta 1D, whereas Belkin and Retta (24) reported that the beta 1D chimera blocks cell growth when expressed in C2C12 myoblasts, suggesting that effects of beta 1D may be cell type-specific.

Two previous studies reported the effect of COOH-terminal deletions within the full-length beta 1C receptor on DNA synthesis (16, 17). We found that the COOH-terminal 13-aa region was required for growth inhibition by the full-length receptor in 10T1/2 cells, whereas, using a different protocol, Fornaro et al. (17) reported that a receptor lacking this region blocked growth in Chinese hamster ovary cells. The results reported here may help resolve this discrepancy by showing that the last 13 aa are not required for growth inhibition by a shorter IL2 receptor chimera that contains only the beta 1C-specific sequences. Thus, this region may be required for maintaining proper conformation in some contexts, but it does not appear to be essential for signaling.

Our findings and those of Fornaro et al. (17) both indicate that the 775-782 region is critical; however, we found that this region was not sufficient for growth inhibition but also required residues 783-792. By contrast, Fornaro et al. (17) observed growth inhibition in a construct lacking residues COOH-terminal to 782. This difference could be due to the difference in cell type or the distinct protocol used in their studies, in which expressing cells were analyzed after being captured by panning on anti-beta 1 antibodies. Alternatively, those studies used full-length beta 1C constructs that contained the common, membrane-proximal region. Dominant negative effects leading to growth inhibition could therefore complicate the interpretation of those results.

The beta 1C-specific exon is formed by an Alu sequence in the antisense orientation located between the sixth and seventh exon of the human beta 1 gene (3, 29). Alu sequences are members of the short interspersed nucleotide elements family of repetitive elements, and approximately 700,000 Alu elements are thought to be present per haploid human genome; thus, an average of more than 1 Alu element is predicted to be present in every gene (30). Many genes express Alu-containing sequences, and a few Alu-containing proteins have been reported previously (31). The function or significance of these elements is not known. We have mapped the beta 1C growth inhibitory domain to a region of beta 1C that overlaps a sequence that is conserved in other Alu-containing proteins including transformation-related protein and neuronal thread protein and alternatively spliced isoforms of neurofibromatosis 2 and c-myb (Fig. 7). The function of these proteins and their roles in growth control are not known. In addition to this conserved region, the beta 1C growth inhibitory domain also contains a TSR sequence that is not found in the other Alu-containing proteins (Fig. 7).

The design of cell growth inhibitors is one of the major strategies in the fight against cancer and other human diseases. We have identified a short peptide sequence that is sufficient to induce growth arrest in endothelial cells (EV304), in transformed prostate epithelial cells (DU145), and in the K562 transformed hematopoietic cell line (data not shown) as well as in rodent fibroblast cell lines. These results suggest that the identification of beta 1C-binding proteins and the characterization of beta 1C-mediated downstream signaling pathways could aid in the development of novel angiogenesis or tumor growth inhibitors.

    ACKNOWLEDGEMENTS

We thank Mark Ginsberg for generously providing us with the IL2R/beta 1D expression plasmid and Bette Cessna for administrative assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants P01 HL48728 (to M. A. S.), R01 GM47214 (to M. A. S.), and GM47157 (to Y. T.) and by National Institutes of Health Training Grant T32 HL07695.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger A research fellow of the American Heart Association, California Affiliate.

§ To whom correspondence should be addressed: Dept. of Vascular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-784-7137; Fax: 619-784-7360; E-mail: schwartz{at}scripps.edu.

2 D. Calderwood, C. Fenczik, and M. H. Ginsberg, manuscript in preparation.

3 J. Meredith and M. A. Schwartz, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: aa, amino acid(s); IL2, interleukin 2; IL2R, IL2 receptor; GFP, green fluorescent protein; BrdUrd, bromodeoxyuridine; PBS, phosphate-buffered saline.

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