Structure/Function Analysis of the Integrin/31 Subunit by Epitope Mapping

Monoclonal antibodies (mAbs) have been produced against the chicken/3, subunit that affect integrin functions, including ligand binding, ot subunit association, and regulation of ligand specificity. Epitope mapping of these antibodies was used to identify regions of the subunit involved in these functions. To accomplish this, we produced mouse/chicken chimeric 81 subunits and expressed them in mouse 3T3 cells. These chimeric subunits were fully functional with respect to heterodimer formation, cell surface expression, and cell adhesion. They differed in their ability to react with a panel of anti-chicken/~l mAbs. Epitopes were identified by a loss of antibody binding upon substitution of regions of the chicken/3, subunit by homologous regions of the mouse/31 subunit. The identification of the epitope was confirmed by a reciprocal exchange of chicken and mouse fit domains that resulted in the gain of the ability of the mouse subunit to interact with a particular anti-chicken/31 mAb. Using this approach, we found that the epitopes for one set of antibodies that block ligand binding mapped toward the amino terminal region of the/31 subunit. This region is homologous to a portion of the ligand-binding domain of the/~3 subunit. In addition, a second set of antibodies that either block ligand binding, alter ligand specificity, or induce a//3 subunit dissociation mapped to the cysteine rich repeats near the transmembrane domain of the molecule. These data are consistent with a model in which a portion of/31 ligand binding domain rests within the amino terminal 200 amino acids and a regulatory domain, that affects ligand binding through secondary changes in the structure of the molecule resides in a region of the subunit, possibly including the cysteine-rich repeats, nearer the transmembrane domain. The data also suggest the possibility that the ot subunit may exert an influence on ligand specificity by interacting with this regulatory domain of the/31 subunit. T HF. integrins represent a family of receptors involved in both cell-matrix and cell-cell adhesion (Albelda and Buck, 1990; Hemler, 1990; Hynes, 1992). They are heterodimers consisting of an ot subunit non-covalently associated with a /3 subunit. Currently, 14 different a subunits and 8 different/~ subunits (not including alternatively spliced forms) have been described. They join in different combinations to form the 20 integrins presently recognized in vertebrates. Each heterodimer forms a transmembrane complex, its extracellular domain interacting with a ligand and its cytoplasmic domain interacting with elements of the cytoskeleton. Integrin-ligand interactions also serve to generate intraceUular signals that result in cytoplasmic alkalinization (Schwartz et al., 1991), protein phosphorylation (Guan et al., 1991; Kornberg et al., 1991; Guan and Shalloway, 1992), changes in calcium influx (Ng-Sikorski et Please address all correspondence to Dr. Clayton A. Buck, Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. al., 1991; Pardi et al., 1989), and cell proliferation (Shimizu et al., 1990). In addition, integrin responses to second messages result in changes ofligand specificity or affinity (Shattil et al., 1985; Tanaka et al., 1992; Thornhill et al., 1991). Ligand binding and receptor activation are accompanied by conformational changes in integrins that result in new mAb binding sites and increased ligand affinity (Coller, 1985; Frelinger et al., 1990, 1991; Altieri and Edgington, 1988; Duet al., 1991). Thus integrins bind ligands, respond to intracellular and extracellular signals, coordinate cytoskeleton-membrane interactions, and transmit information into the cell resulting in changes in cell morphology and function (for review see Hynes, 1992). To identify regions of the Bt subunit that may be involved in several of these functions, we have mapped the epitopes of a panel of chicken fit-specific mAbs that interfere with a particular integrin function using a series of mouse/chicken chimeric subunits. The reagents employed in these studies include mAbs that perturb cell-substratum interactions, disrupt subunit association, or affect substratum specificity. © The Rockefeller University Press, 0021-9525/93/09/1361/11 $2.00 The Journal of Ceil Biology, Volume 122, Number 6, September 1993 1361-1371 1361 on July 9, 2017 jcb.rress.org D ow nladed fom Table L PCR Primers for Chimeric Plasmid Construction Chimeric construct Sequence Rest. Enzy. Site MC1 S 5'-AAGCCGAGGCCTGCCGACACCGACCCGAGACCC3' StuI AS 5'-AGCCAATTCGAAGTCTGAAGTAATCCTCCTCA-3' AsuII MC-2 S 5'-CAGACTTTCGAATTGGCTTTGGC3' AsuH AS 5'-TCACTAAGCTTCTGAACAAGGTG-3' HindM MC-3 S 5'-GTTCAG.~GCTTAGTGAAAATAATAT3' HindM AS 5'-ACTTGGGATCCGTGCACTTACAA-3' Band-If MC-5 S 5'-CAGACTTTCGAATTGGCTTTGGC-3' AsuH Mouse AS 5'-ACTTCATCTGTGCTACATTCAC-3' Chicken S 5'-GTGAATGTAGCACAGATGAAGT-3' AS 5'-TTTGGGATCCGTGCA3' BamHI PCR primers used in the construction of four chimeric chicken/mouse cDNAs are shown. For each construct the cloning restriction enzyme site within the primer is highlighted and the enzyme listed in the right column. The complementary sequences of the primer pair used for splice overlap extension in the construction of MC-5 are underlined. S, sense primer; AS, antisense primer. Among the antibodies that perturb cell substratum interactions are CSAT (Neff et al., 1982) and JG22 (Greve and Gottlieb, 1982), both of which have been extensively characterized. It is assumed that these antibodies interact with regions of the receptor involved in ligand binding. Another antibody, TASC (Neugebauer and Reichardt, 1991), has the ability to block the binding of neuronal cells to vitronectin, and also to stimulate their adherence to laminin. This antibody is assumed, therefore, to interact with regulatory domains of the integrin/3~ subunit. Our results demonstrate that ligand-binding activity is blocked by mAbs that recognize epitopes widely separated on a linear amino acid map, and that there exist both ligand binding and regulatory regions that are distinct from one another. Materials and Methods Chimeric Plasmid Construction We have previously described the construction and expression of the full length cDNA clone for chicken/~l, pCINT/~RV (Solowska et al., 1991). The chicken/31 cDNA was also cloned into the EcoRI site of the expression vector, pCMV-5 (Anderson et al., 1988), provided by Drs. Gary Cohen and Rosalyn Eisenberg of the University of Pennsylvania (Philadelphia, PA), that had been modified to eliminate the cloning sites between Kpn I and Sma I. The resulting expression plasmid was designated pCMV5CINT/~IRV. A 3.6-kb Hind HI cDNA fragment containing most ofthe coding region of the mouse/31 subunit in Bluescript (pBSM/~0 was kindly provided by Dr. V. Michael Hollers of Washington University (St. Louis, MO). PCR-directed mutagenesis and PCR-driven splice overlap extension (SOE) l (Horton et al., 1990) were used to generate five chimeric mouse/chicken ~1 expression plasmids, designated MC-1 through MC-5, as described below. PCR reactions were performed in an automatic thermal cycler (Perkin-Elmer Cetus Instrs., Norwalk, CT). Each 100/~1 reaction mixture included 10 mM Tris-HCl, pH 8.0, 1.5 mM MgCl2, 50 mM KC1, 0.1% gelatin, 10% DMSO, 0.06% 2-mercaptoethanol, 200 mM each dNTP and 2.5 U of Taq DNA polymerase (Perkin-Elmer Cetus Instrs.). Reactions were cycled 25 times through a program that included incubations at 94°C for 1 rain, 56°C for 1 min, and 72°C for 2 rain. The primers used in the construction of each of the mouse/chicken chimeric cDNAs are shown in Table I. Construction of MCI. Primers were designed to amplify the mouse E1 subunit from bp 1 to bp 627 and contained Stu I (sense primer) and Asu 1. Abbreviations used in this paper: RT-PCR, reverse transcription-PCR; SOE, splice overlap extension. II (antisense primer) sites at their 5' ends. Because pBSM/~I did not contaln 395 bp of the 5' coding region, we used reverse mmscription-PCR (RTPCR) to amplify this fragment. Total cellular RNA was isolated from 3"1"3 cells by sequential extraction in RNAzol (Biotecx, Houston, TX) and chloroform, followed by isopropanol precipitation. 5 #g of total RNA was subjected to RT-PCR using the cDNA Cycle Kit (Invitrogen, San Diego, CA) according to the manufacturer's directions. First strand synthesis was accomplished using random primers and reverse transcriptase. This strand was used as template in the standard PCR reaction. The RT-PCR product was gel purified using Gene Clean II glass beads (Bio !01) according to the manufacturer's directions and cut with Stu I and Asu II. pCINT~rRV was cut with Stu I and Asu 1I. The large piece of these digestion products was purified and ligated with the murine cDNA obtained above to yield the plasmid pMC1. Construction OfMC2. PCR primers were designed to amplify the region of mouse/~t from bp 627 to bp 1077 of the cDNA and contained Asu II (sense primer) and Hind Ill (antisense primer) restriction sites at their 5' ends. Amplified DNA product was separated on a 1.0% agarose gel, visualized by ethidium bromide, purified, and digested with Asu II and Hind M. pCINT~tRV was cut with Asu II and Hind m and the digestion products were separated on an agarose gel, and the larger piece was cut out and purified. This larger piece was then ligated with the amplified mouse ~1 cDNA fragment. The resulting plasmid was designated pMC2. Construction of MC3. Primers were designed to amplify the region of mouse ~t from bp 1077 to 1967 from the cDNA and contained Hind m (sense primer) and BamI-I I (antisense primer) sites near their 5' ends. PCR product was purified and cut with Hind M and BamH I. pCMV5CINT/~IRV cut with Hind m and BamH I was resolved on an agarose gel. The larger piece was purified and ligated with the mouse sequence obtained above. The resulting plasmid was designated pMC3. Construction of MC4. The plasmids pMC2 and pBSM/~I were digested with Tthlll I and Xho I and resolved on an agarose gel. The larger fragments from both digests were purified and ligated together. The resulting plasmid was designated pMC4. Construction OfMCS. PCR primers were designed to amplify the mouse ~1 subunit from bp 627 to 1631 using mouse cDNA template and included an Asu II site at the 5' end of the sense primer. Additional primers were designed to amplify the chicken ~1 subunit from bp 1642 to 1980 using the chicken cDNA as template and included a BamI-I I site at the 5' end of the antisense primer. The mouse/~1 antisense and the chicken ~1 sense primers corresponded to a region that was identical between the species and were complementary to each other. The amplified mouse and chicken cDNA fragments were mixed together and subjected to a further round of SOE in the presence of the mouse sense and chicken antisens¢ primers. The resulting spliced mouse/chicken cDNA was cut with Asu II and BamH I and gel purified. The coding sequence between Asu H and BamH I of pMC1 was cut out and replaced with the spliced cDNA to yield the plasmid pMC5. Confirmation of Chimeric cDNA Sequence. The chimeric nature of each construct was confirmed by PCR amplification using one mousespecific and one chicken-specific primer. Constructs were also sequenced across the chimeric junction by the dideoxy nucleotide method to further validate the nature of the insert. The Journal of Cell Biology, Volume 122, 1993 1362 on July 9, 2017 jcb.rress.org D ow nladed fom Transfection of 3T3 Cells NIH 3T3 cells maintained in DMEM with 10% FBS were transfected as described previously (Solowsks ct ai., 1989). Briefly, cells (105) plated the previous day in 100-ram dishes were cotranst~cted with 10/~g of each chimeric construct plasmid, 2 ~,g of pSV2-neo and 10/~g of salmon sperm DNA as a calcium phosphate precipitate. After two days, the cells were split 1:15 into DMEM with 10% FBS and 1 mg/ml Geneticin (00418, GIBCO BRL, Gaithersburg, MD). Approximately 2 wk later, G418 resistant clones were picked and screened for expression of the mouse/chicken chimeric B1 subunit by immunoblot assay using the chicken specific polyclonai antibody, Chickie (see below). The positive clones were further enriched by limited dilution subcloning and one clone of each construct was used for further characterization.