Epitope-Specific Antibodies to the β1C Integrin Cytoplasmic Domain Variant

Experimental and Molecular Pathology 70, 275–280 (2001) doi:10.1006/exmp.2001.2369, available online at http://www.idealibrary.com on

Epitope-Specific Antibodies to the ␤1C Integrin Cytoplasmic Domain Variant

Mara Fornaro, Mariarosaria Lovecchio,1 Powell Jose, Duo-Qi Zheng, Loredana Moro,1 and Lucia R. Languino2 Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520

Received January 2, 2001

The ␤1C integrin is an alternatively spliced variant of the ␤1 subunit that contains a unique 48-amino-acid sequence in its cytoplasmic domain. We have shown previously that ␤1C is a potent inhibitor of cell proliferation and that in vivo its expression is downregulated in prostate and breast carcinoma. In this study, we describe a panel of specific monoclonal antibodies that react with the ␤1C cytodomain. We show by immunoblot analysis that the newly generated monoclonal antibodies specifically recognize the ␤1C cytodomain expressed as glutathione Stransferase fusion protein. The specificity of the antibodies to ␤1C was confirmed in competition studies by immunoblotting using ␤1C-specific synthetic peptides. These monoclonal antibodies reacted, in enzymelinked immunosorbent assays, with the ␤1C 785–808 peptide but failed to bind the ␤1C 778–794, ␤1C 805–825, or ␤1A 765–798 peptides. Thus, the epitope recognized by the antibodies is located within the Q795 –F804 ␤1C cytoplasmic sequence; this region overlaps the previously described Q795 –Q802 domain necessary for ␤1C to inhibit cell proliferation. To our knowledge, these are the first monoclonal antibodies specific for a ␤1 cytoplasmic isoform. The monoclonal antibodies described here will be useful tools for dissecting functional differences, among ␤1 integrin variants, as well as for the study of the role of ␤1C in prostate and breast epithelial cell proliferation. 䉷 2001 Academic Press Key Words: alternative spliced variants; integrin cytoplasmic domain; prostate cancer cells; breast cancer cells; cell proliferation.

INTRODUCTION

Integrins are a large family of transmembrane receptors composed of an ␣ and a ␤ subunit that have been shown to regulate cell growth, survival, and differentiation, in addition to cell adhesion to the extracellular matrix (Hynes, 1992; Ruoslahti and Reed, 1994). It is well established that the cytoplasmic domain of the ␤ subunit is required for integrins to modulate many cellular functions as well as to trigger signaling events that result in protein phosphorylation (Fornaro and Languino, 1997; Hemler et al., 1995; Wei et al., 1998) and interactions with intracellular proteins (Hemler, 1988). Therefore, structural differences in the amino acid sequence of the ␤ subunit cytodomain can contribute to the specificity of integrin signaling. The ␤1C integrin is an alternatively spliced variant of the ␤1 subfamily that contains a unique 48-amino-acid sequence in its cytoplasmic domain (Languino and Ruoslahti, 1992). The ␤1 integrin subunits ␤1C and ␤1A that contain variant cytoplasmic domains differentially affect cell proliferation. It has been shown previously that either full-length ␤1C or its cytoplasmic domain inhibits prostate cancer epithelial cell (Fornaro et al., 1998; Meredith et al., 1999), endothelial cell (Meredith et al., 1999), and fibroblast (Fornaro et al., 1995; Meredith et al., 1995, 1999) proliferation, whereas ␤1A promotes it. In vivo, ␤1C is expressed in nonproliferative,

1 These authors are on a leave of absence from the Center of Study on Mitochondria and Energy Metabolism, Consiglio Nazionale delle Ricerche, Bari, Italy. 2 To whom requests for reprints should be addressed at the Department of Pathology, Yale University School of Medicine, P.O. Box 208023, 310 Cedar Street, New Haven, CT 06520. Fax: (203) 7371455. E-mail: [email protected].

0014-4800/01 $35.00 Copyright 䉷 2001 by Academic Press All rights of reproduction in any form reserved.

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276 differentiated epithelium; its expression is selectively downregulated in prostatic adenocarcinoma and inversely correlates with markers of cell proliferation in breast carcinoma (Fornaro et al., 1996, 1998, 1999; Manzotti et al., 2000). Moreover, in a recent study we elucidated ␤1C-mediated downstream signaling pathways that control cell proliferation and survival. Specifically, we have shown that at variance with ␤1A, ␤1C strongly inhibits cell proliferation by preventing Ras/MAP kinase pathway activation (Fornaro et al., 2000). By deletion analysis, we reported earlier an 8amino acid region (Q795 –Q802) within the ␤1C cytoplasmic domain that is necessary and sufficient to inhibit cell proliferation (Fornaro et al., 1995). We describe here the characterization of a panel of monoclonal antibodies (mAbs) that specifically recognize the ␤1C Q795 –F804 sequence. To our knowledge, these are the first mAbs specific for a ␤1 cytoplasmic isoform. These epitopespecific antibodies will be useful for dissecting functional differences among ␤1 integrin variants and will help in elucidating ␤1C downstream effectors and their role in prostate and breast epithelial cell proliferation.

MATERIALS AND METHODS

Reagents and Antibodies Rabbit antibodies specific for the ␤1C subunit cytoplasmic domain were affinity-purified as described previously (Fornaro et al., 1996). The following antibodies were used: mouse mAb K20 to human ␤1 integrin (Coulter– Immunotech, Miami, FL); rabbit affinity-purified antibody to ERK1 and 2 (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit antiserum to ␤1A integrin (Chemicon International, Inc., Temecula, CA); goat affinity-purified antibody to glutathione S-transferase (GST; Amersham Pharmacia Biotech Inc., Piscataway, NJ). Nonimmune mouse Ig (mIg) was purchased from Sigma Chemical Co. (St. Louis, MO). ␤1C- and ␤1A-specific synthetic peptides used for this study were ␤1C 778–794 (kkSLSVAQPGVQWCDISSL), ␤1C 785–808 (kkVQWCDISSLQLTSRFQQFSCLS), ␤1C 805– 825 (kkSCLSLPSTWDYRVKILFIRVP), ␤1C 795–812 (kkQPLTSRFQQFSCLSLPST), ␤1C 778–825 (kkSLSVAQPGVQWCDISSLQLTSRFQQFSCLSLPSTWDYRVKILFIRVP), and ␤1A 765–798 (KFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK). A peptide derived from the ␤3 integrin cytoplasmic domain sequence [762–788 (RAKWDTANNPLYKEATSTFTNITYRGT)] was used as a control

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in enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis. Generation of ␤1C-Specific mAbs The ␤1C-specific mAbs were generated in BALB/c mice (Cocalico Biologicals Inc., Reamstown, PA) using, as immunogens, synthetic peptides [778–825 (kkSLSVAQPGVQWCDISSLQLTSRFQQFSCLSLPSTWDYRVKILFIRVP) and 795–812 (kkQPLTSRFQQFSCLSLPST)] from the deduced sequence of ␤1C (Languino and Ruoslahti, 1992). Plasmids and Recombinant Proteins The GST– ␤1C fusion protein consisting of amino acids 778–825 of the human ␤1C -specific cytodomain fused to GST was generated as follows: a 146-bp cDNA fragment corresponding to nucleotides 2435 to 2581 from the cytoplasmic domain of human ␤ 1C was generated by polymerase chain reaction (PCR) as described (Languino and Ruoslahti, 1992). Primers contained BamHI (5⬘CGGGATCCTCTCTCTCTGTCGC-3⬘) and EcoRI (5⬘CGGAATTCTTACGGCACTCTT-3⬘) sites at their 5⬘ ends (restriction sites are underlined). The amplified product was subcloned into BamHI and EcoRI sites in the pGEX–2T plasmid (Amersham Pharmacia Biotech Inc.). The fragment generated by PCR was sequenced by the dideoxy chaintermination reaction using Sequenase 2.0 (United States Biochemical, Cleveland, OH). Either GST or the GST– ␤1C fusion protein expression in transformed JM105 Escherichia coli was induced using 0.1 mM isopropyl-1-thio-␤ -D-galactopyranoside (American Bioanalytical, Natick, MA) for 1 h at 37⬚C. Fusion proteins were isolated by affinity chromatography on glutathione–Sepharose 4B according to the manufacturer’s instructions (Amersham Pharmacia Biotech Inc.). Immunoblotting Ten micrograms of purified GST or GST– ␤1C fusion proteins were separated by 15% SDS–PAGE and transferred to PVDF membranes (Millipore, Bedford, MA) as described previously (Fornaro et al., 1996). Membranes were then blocked in Tris-buffered saline (TBS; 20 mM Tris, pH 7.5, 150 mM NaCl) containing 0.3% Tween 20 (American Bioanalytical) and 5% nonfat dry milk for 3 h at room temperature. Immunostaining was performed using either a mAb to ␤1C or a control culture supernatant (1:10 dilution of culture supernatant in blocking buffer) for 1 h at room temperature. Membranes were then washed three times in TBS containing

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0.3% Tween 20 (TBS-T) and incubated with horseradish peroxidase-conjugated affinity-purified antibody to mouse Ig (Amersham Pharmacia Biotech Inc.) in blocking buffer for 1 h at room temperature. After three washes in TBS-T, proteins were visualized using the enhanced chemiluminescence system (ECL; NEN, Boston, MA). Goat affinity-purified antibody to GST (5 ␮g/ml) was used to control for protein loading. The specificity of the mAb reactivity for ␤1C was confirmed for mAb BA6 in immunoblotting by incubating the BA6 culture supernatant with 30 ␮g/ml of either a mixture of three ␤1C-specific peptides (␤1C 778–794, ␤1C 785–808, and ␤1C 805–825) or a negative control ␤3derived peptide. ELISA Epitope mapping of the mAbs to ␤1C was performed by ELISA as described previously (Fornaro et al., 1996) using 96-well microtiter ELISA plates coated with 20 ␮g/ml synthetic peptides containing ␤1C cytoplasmic domain sequences. A synthetic ␤1A- or a ␤3-derived peptide was used as control.

FIG. 2. MAb specificity for the ␤1C cytodomain. (A and B) Ten micrograms of either GST or GST– ␤1C fusion protein was separated by 15% SDS–PAGE and transferred to PVDF membranes. (A) The GST– ␤1C fusion protein was detected by immunoblotting using BA6, mAb to ␤1C (1:10 dilution of culture supernatant; left panel). ␤1C immunostaining was specifically inhibited by incubating BA6 with 30 ␮g/ml of a mixture of three ␤1C-specific peptides containing overlapping sequences of the ␤1C cytoplasmic domain (␤1C 778–794, ␤1C 785–808, and ␤1C 805–825; middle panel) but not with 30 ␮g/ml of a control ␤3-derived peptide (right panel). (B) Membranes used for the competition study described for A were reblotted with 0.1 ␮g/ml affinity-purified polyclonal antibody either to ␤1C (left panel) or to ERK1/2 as a negative control (right panel). Proteins were visualized by ECL. WB, Western blotting.

FIG. 1. Characterization of the newly generated mAbs to ␤1C by immunoblot analysis. A representative mAb is shown. Ten micrograms of either GST (lane 1) or GST– ␤1C (lane 2) fusion protein was separated by 15% SDS–PAGE and transferred to PVDF membranes. The GST– ␤1C fusion protein was detected by immunoblotting using either BE1, mAb to ␤1C, or nonimmune Ig as a negative control (1:10 dilution of culture supernatant; top and middle panels, respectively). Membranes were reblotted using 5 ␮g/ml affinity-purified antibody to GST (bottom panel) to detect GST-containing proteins. Proteins were visualized by ECL.

RESULTS AND DISCUSSION Antibody Specificity

␤1C-specific mAbs were generated in BALB/c mice immunized with a mixture of two synthetic peptides containing overlapping sequences in the ␤1C 778–825 cytoplasmic domain. The immunoreactivity of the mAbs to the ␤1C cytoplasmic domain was assessed by immunoblotting using a

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FIG. 3. Epitope mapping of mAbs to ␤1C. Ninety-six-well microtiter ELISA plates were coated with 20 ␮g/ml synthetic peptides containing either ␤1C or ␤3 (A and B) or ␤1A (B) cytoplasmic domain sequences. The dilution of the mAbs used (BA6, mAb to ␤1C, and AH11, negative control Ab) was either 1:100 or 1:10. In A, rabbit antiserum to ␤1C (polyclonal Ab) and normal rabbit serum (NRS) were used at 1:1000 dilution. In B, rabbit antiserum to ␤1A (polyclonal Ab) and NRS were used at 1:2000 dilution. Absorbance was quantitated at 405 nm.

MONOCLONAL ANTIBODIES TO THE ␤1C INTEGRIN VARIANT

FIG. 4. The amino acid sequence of the epitope recognized by the newly generated mAbs to ␤1C. The epitope mapping results shown in this study indicate that the Q795 –F804 amino acid sequence (box) within the ␤1C cytoplasmic domain is recognized by the newly generated mAbs. The 8-amino-acid region (Q795 –Q802) shown to be necessary and sufficient to inhibit cell proliferation (Fornaro et al., 1995) is in bold. The mAbs that recognize the ␤1C Q795 –F804 amino acid sequence are indicated between brackets below the epitope sequence.

GST recombinant protein containing the ␤1C S778 –P825 sequence. Seven mAbs (BA6, BF10, BC8, BC5, BD5, AC1, and BE1) that specifically recognized the GST– ␤1C fusion protein but not GST alone (Fig. 1 and not shown) were selected for further characterization. The specificity of the reactivity of the mAbs to ␤1C was confirmed by immunoblotting analysis in competition studies using ␤1C-specific peptides. As shown in Fig. 2A, incubation of BA6, mAb to ␤1C, with a mixture of three ␤1C-specific peptides (␤1C 778–794, ␤1C 785–808, and ␤1C 805–825), but not with a control ␤3-derived peptide, inhibited BA6 reactivity with the GST– ␤1C fusion protein. Membranes used for competition studies were reblotted with affinity-purified polyclonal antibody to ␤1C to control for GST– ␤1C fusion protein loading (Fig. 2B). Three of the seven selected mAbs (BA6, BF10, and BC8) specifically recognized in immunoblotting the human native ␤1C immunoprecipitated from detergent extracts generated from CHO-␤1C stable cell transfectants (Fornaro et al., 2000) using K20, mAb to human ␤1 integrin (data not shown). The results show that the newly generated mAbs specifically recognize ␤1C cytoplasmic domain sequences. Epitope Mapping In ELISA, the seven selected mAbs recognized the G785 – S ␤1C sequence but failed to react with two different ␤1Cspecific peptides (S778 –L794 and S805 –P825), with a control ␤3-derived peptide (Fig. 3A), or with a synthetic peptide containing ␤1A cytoplasmic domain sequences (Fig. 3B). Therefore, the newly generated mAbs recognize a specific epitope (Q795 –F804) within the ␤1C cytodomain (Fig. 4). In conclusion, we have generated a panel of mAbs that recognize the Q795 –F804 sequence within the ␤1C cytoplasmic domain. This region overlaps the previously described Q795 – Q802 domain in ␤1C that inhibits cell proliferation (Fig. 4). 808

279 These epitope-specific antibodies will be used to study both ␤1C-specific interactions with intracellular proteins and ␤1C downstream signaling pathways, thus bringing new insights into the role of ␤1 integrins in prostate and breast epithelial cell proliferation. Furthermore, these mAbs will be a useful tool for studying the expression of specific ␤1 integrin variants in human benign and malignant tissues.

ACKNOWLEDGMENTS

We thank Dr. Dave Wright for helpful discussion regarding the synthesis of ␤1C peptides. We also thank Ms. N. Bennett for helping with preparation of the manuscript. This work was supported by National Institutes of Health Grants CA-71870 and DK-52670 (to L.R.L.), by the Army PCRP Grant DAMD17-98-1-8506 (to L.R.L. and M.F.), and by fellowships from ASI (ARS-99-97) and Consiglio Nazionale delle Ricerche (bando n. 203.04.17) to M.R.L. and L.M., respectively.

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