The Human WD Repeat Protein WAIT-1 Specifically Interacts with the Cytoplasmic Tails of β7-Integrins*

Integrins of the β7 subfamily, α4β7 and αEβ7, contribute to lymphocyte homing and to the development of protective or autoreactive immune responses at mucosal sites. The β subunits of integrins are considered important for regulation of stimulated cell adhesion and adhesion-dependent signal transduction. Using a yeast interaction trap screen, a human WD repeat protein, termed WAIT-1, was isolated that interacts with the integrin β7 cytoplasmic tail and is homologous to mouse EED andDrosophila ESC proteins. WAIT-1 also binds to the cytoplasmic domains of α4 and αE but not to those of integrin β1, β2, and αL subunits. Association of WAIT-1 and β7-integrin was confirmed by coprecipitation from transiently transfected 293 cells. The binding site for WAIT-1 was mapped to a short membrane-proximal region of the β7 cytoplasmic tail with Tyr-735 being of critical importance. Northern blot analysis revealed multiple WAIT-1-related transcripts with differential expression in circulating leukocytes, tissue-resident cells of diverse origin, and lymphoid malignancies. These results suggest that WAIT-1, together with the recently identified RACK1, may define a novel subfamily of WD repeat proteins that interact with distinct subsets of integrin cytoplasmic tails and may act as specific regulators of integrin function.

␤7-Integrins are considered important for the development and function of gut-associated lymphoid tissues. Interaction of ␣4␤7 with MAdCAM-1 allows for tissue-specific migration of circulating lymphocytes into the lamina propria and Peyer's patches of the gut (10,11), whereas ␣E␤7 may retain intraepi-thelial lymphocytes within the gut epithelium through binding of E-cadherin on epithelial cells (12,13). Lack of ␤7-integrins severely impairs the development of the gut immune system, as Peyer's patches are absent or hypoplastic, and fewer intraepithelial lymphocytes are detected in ␤7-integrin-deficient mice (14). Moreover, gut-homing ␣4␤7 ϩ CD4 T cells specifically harbor cellular memory for intestinal antigens, suggesting that ␣4␤7 helps to target and segregate intestinal versus systemic immune responses (15).
Integrins of the ␤7 family are involved in the development and/or progression of diseases such as colitis (16,17), diabetic insulitis (18), and lymphoid malignancies. Integrin ␣4␤7 has been implicated in the mucosal localization of malignant lymphomatous polyposis, a gastrointestinal variant of the mantle cell lymphoma (19), the homing of lymphocytes to the thymus of AKR mice resulting in thymic lymphoma (20), and is specifically expressed on mucosa-associated T and B cell non-Hodgkin's lymphomas (21). Analysis of skin biopsies from patients with mycosis fungoides revealed that expression of integrin ␣E␤7 correlates with epidermotropism of infiltrating T cells and the stage of the disease (22,23).
Together, these findings indicate that ␤7-integrins play an important role for physiological functions and pathological alterations of the immune system. The cytoplasmic tails of the ␤7 subunit are critical for integrin function because they regulate receptor avidity (24) and signaling (25). In this study, we applied a yeast interaction trap screening to identify cytoplasmic interactors for the human integrin ␤7 subunit. We describe the molecular cloning and characterization of the human WD repeat protein WAIT-1, which specifically interacts with the cytoplasmic tails of ␤7-integrin ␣ and ␤ subunits and is thus likely to be involved in regulation of either receptor avidity or signaling.

EXPERIMENTAL PROCEDURES
Antibodies and Cell Lines-The rat-anti-murine integrin ␤7 antibody M301 was kindly provided by Dr. P. Kilshaw (AFRC Institute of Animal Physiology and Genetics Research, Babraham, Great Britain). Polyclonal rabbit anti-lexA serum was purchased from Invitrogen. To generate antibodies specific for the mouse and human integrin ␣4 subunit, rabbits were immunized with the cytoplasmic peptide sequence NH 2 -RRDSWSYINSKSNDD-COOH (Eurogentech, Seraing, Belgium). Horseradish peroxidase-conjugated antisera directed against rat, rabbit, or human Ig were purchased from Dianova (Hamburg, Germany) or Bio-Rad (Munich, Germany).
The human lymphoma cell lines Jurkat and Ramos as well as 293 cells were obtained from ATCC (Rockville, MD). The C3H/He B cell lymphoma 38C13 expresses integrin ␣4 subunits but lacks detectable cell surface protein and RNA transcripts for integrin ␤1 and ␤7 subunits. 38C13 lymphoma cells were transduced by retroviruses containing the ␤7 subunit or control vector generating cell lines 38-␤7 and 38-LXSN (26).
Yeast Interactor Screening-The screening procedure for isolation of proteins interacting with the human integrin ␤7 cytoplasmic domain was essentially performed as described (28). The plex202-␤7 expression vector was transformed into yeast strain EGY48 bearing the JK103 lacZ reporter plasmid and used for screening of a Jurkat cDNA library (27). cDNAs from positive yeast clones were isolated and transformed into Escherichia coli KC8 (28) for further analysis.
The 5Ј-end of WAIT-1 was obtained by nested RACE-PCR using a splenocyte cDNA template (CLONTECH). Primers used were AP1 and AP2 (CLONTECH) for the 5Ј-end and 5Ј-CCAGCTACAGCCAGCAGAG-GATGG-3Ј and 5Ј-GGCTCGTATTGCTATCATAGGTCC-3Ј for the 3Јend. In two independent experiments, the nested PCR resulted in a single cDNA fragment of identical nucleotide sequence. Following addition of the peptide MDYKDDDDKGG to the N terminus of WAIT-1, the 5Ј cDNA fragment isolated by nested RACE-PCR was used to generate the full-length protein. The primary structure of tagged WAIT-1 was confirmed by nucleotide sequence analysis.
Precipitation of ␤7-Integrins with WAIT-1-GST Fusion Protein-Amino acids 24 -427 of WAIT-1 were subcloned into the pGEX-4T-1 expression vector (Pharmacia, Freiburg, Germany) generating a Cterminal fusion with glutathione S-transferase (GST). GST fused to amino acids 463-511 of the murine zinc finger transcription factor Egr-1 was used as control. Both constructs were verified by doublestranded nucleotide sequencing. GST fusion proteins were then expressed in E. coli DH5␣ following induction with isopropyl ␤-D-thiogalactopyranoside. Bacteria were lysed by sonication in buffer containing 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, 1 mM EDTA, 50 mM Tris, pH 8.0. Fusion proteins were purified from lysates by adsorption to glutathione-conjugated Sepharose and used as a matrix for precipitation after vigorous washing in lysis buffer.
38-LXSN and 38-␤7 cells were washed with phosphate-buffered saline and lysed in buffer containing 1% Triton X-100, 0.5% SDS, 100 mM NaCl, 1 mM EDTA, 10 mM Tris, pH 8.0, and protease inhibitors on ice for 30 min. The soluble fraction of cell lysates was incubated with the WAIT-1-GST or EGR-1-GST matrices for 3 h at 4°C. After vigorous washing with lysis, bound proteins were separated on a 7.5% SDS polyacrylamide gel under non-reducing conditions. Proteins were blotted onto a nitrocellulose membrane, and filters were probed with anti-␤7-integrin antibody M301 followed by peroxidase-conjugated goat antiserum against mouse Ig (Bio-Rad) and chemoluminescence detection using the ECL reagent (Amersham, Braunschweig, Germany).
Association of WAIT-1 and ␤7-Integrin in Vivo-cDNA clones encoding the full-length murine ␣4 or ␤7 subunits were subcloned into the expression vectors pRK5 (Pharmingen, Hamburg, Germany) or pSR␣ (29), respectively. Full-length WAIT-1 was fused to the C terminus of domains 2 and 3 of the human IgG1 constant region and subcloned into the pCDM7 vector generating the WAIT-Fc fusion protein. 293 cells were transiently transfected with pRK5-␣4, pSR␣-␤7, and pCDM7-WAIT-Fc expression plasmids as described (30). In control transfections, plasmid pCDM7-WAIT-Fc was replaced by the pCDM7-Fc vector driving cytosolic expression of domains 2 and 3 of the human IgG1 constant region (control Fc fragment). Transfected 293 cells were lysed in buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 2 mM MnCl 2 , 25 mM Tris, pH 7.5, and protease inhibitors at 4°C for 30 min. Lysates were incubated overnight with 25 l of sedimented protein A-Sepharose CL4B (Pharmacia), and adsorbed proteins were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting.
Northern Blot Analysis-Total RNA from Jurkat and Ramos cells was separated on formaldehyde-containing agarose gels and blotted onto a nitrocellulose membrane. The Northern blot containing mRNA of various human tissues was purchased from CLONTECH. cDNA probes spanning the coding region of wait-1 or ␤-actin (CLONTECH) were labeled with [␣-32 P]dCTP (Amersham) using the high prime kit (Boehringer, Mannheim, Germany) and used for hybridization according to the manufacturer's instructions (CLONTECH).

Molecular Cloning of WAIT-1, a Human WD Repeat Protein
Interacting with ␤7-Integrin Cytoplasmic Domains-The complete 52-amino acid cytoplasmic tail of the human integrin ␤7 subunit was fused in frame with the lexA DNA binding domain and used as a bait protein for yeast interaction trap screening of a Jurkat cDNA library. The integrin ␤7 bait protein was tested in yeast to exclude intrinsic activation of reporter genes, and expression was demonstrated by Western blot analysis of yeast lysates using polyclonal rabbit antibodies against lexA or the human integrin ␤7 cytoplasmic tail (data not shown). The interaction trap screen revealed 19 reproducibly positive cDNA clones as determined by their ability to allow growth of the auxotrophic yeast strain EGY 48 on leucine-deficient medium and indigo blue staining of yeast in the presence of X-gal. Restriction enzyme analysis and nucleotide sequencing indicated that 14 out of these clones represented three independent cDNAs derived from a single mRNA transcript (Fig. 1A). Because no transcriptional start site was found in these clones, a nested RACE-PCR was applied to isolate the 5Ј-end of the putative ␤7 interactor. In two independent RACE-PCR experiments, a single cDNA fragment was isolated containing an additional 5Ј sequence. The resulting full-length cDNA contained an ATG start codon in a translation initiation environment (31) and an open reading frame of 1281 nucleotides encoding 427 amino acids. The overall length of the cDNA including the 5Ј-and 3Ј-untranslated regions was 1528 base pairs. Considering the addition of a poly(A) tail, the cDNA size is consistent with a major 1.7-kb mRNA transcript detected in Jurkat and Ramos lymphoma cell lines (Fig. 1C). Interaction with the integrin ␤7 cytoplasmic domain was confirmed with the full-length protein (data not shown).
A search for protein motifs in the deduced amino acid sequence revealed homology of amino acids 238 -248 with a repeat motif found in the regulator of chromosome condensation-1, RCC1 (32). Homology with a repeat motif from the ␤ subunit of heterotrimeric G proteins (33) was identified at position 192-206. In addition, five WD repeat motifs as defined by Neer et al. (33) were detected in the protein sequence (Fig.  1B). The ␤7-integrin interactor isolated by the yeast interaction trap assay therefore represents a member of the WD repeat protein family and was termed WAIT-1 (WD protein associating with integrin cytoplasmic tails-1).
Homology search of nucleic acid and protein data bases demonstrated that WAIT-1 represents the human homologue of the murine eed gene that was recently identified by positional cloning (34,35). WAIT-1 and EED proteins were 100% identical, and the similarity of the cDNA sequences was 92% (Fig. 2). Moreover, wait-1 is also related to the Drosophila esc gene, which was proposed to play a role in the repression of hox gene transcription (36). The overall identity of WAIT-1 and ESC was 51% at the amino acid level and 54% at the DNA level ( Fig. 2B and data not shown).
Coprecipitation of WAIT-1 with ␤7-Integrin-To confirm the interaction of WAIT-1 with the integrin ␤7 cytoplasmic tail, WAIT-1 was expressed as GST fusion protein and used for coprecipitation experiments. GST fused to the C-terminal activation domain of the murine zinc finger transcription factor Egr-1 (GST-Egr-1) served as a negative control. Equal amounts of fusion proteins were bound to glutathione-Sepharose, and lysates of 38-LXSN or 38-␤7 cells, which exclusively differ in the expression of the integrin ␤7 subunit (26), were sequentially incubated with GST-Egr-1 and GST-WAIT-1. The results depicted in Fig. 3 demonstrate that proteins of approximately 90 and 110 kDa corresponding to the precursor and mature forms of ␤7 were precipitated by GST-WAIT-1 but not GST-Egr-1 from 38-␤7 cell lysates. It should be noted that WAIT-1 interaction with the integrin ␤7 cytoplasmic domain was stable in highly stringent detergent conditions (1% Triton X-100 and 0.5% SDS). In contrast, specific protein bands were not detectable when lysates of 38-LXSN cells were precipitated with the GST-WAIT-1 matrix (Fig. 3).
To demonstrate the association of WAIT-1 and ␤7-integrin in vivo, murine ␣4and ␤7-integrin subunits and the WAIT-Fc or control Fc proteins were transiently expressed in 293 cells. The results in Fig. 4A demonstrate that ␤7-integrin was coprecipitated with WAIT-Fc but not with the control Fc fragment. Reprobing of the filter with antibodies against human IgG revealed similar expression of WAIT-Fc and Fc proteins (Fig.  4A). In addition, Western blot analysis of total cell lysates indicated comparable expression levels of ␣4 and ␤7 subunits in 293 cells transfected with either WAIT-Fc or the control Fc construct (Fig. 4B). Together, these data confirm the results of the yeast interaction trap assays and directly demonstrate the association of WAIT-1 with integrin ␤7 both in vitro and in vivo.
Specificity of WAIT-1 Interaction for Cytoplasmic Tails of ␤7-Integrins-To determine whether association with WAIT-1 is specific for the integrin ␤7 chain, cytoplasmic domains of various integrin subunits and unrelated surface receptors were fused to the lexA DNA binding domain and analyzed for interaction with a WAIT-1-B42 prey construct. Expression of the bait proteins in yeast was confirmed by Western blot analysis using a rabbit polyclonal antiserum against lexA (data not shown). The results in Table I demonstrate that, in addition to ␤7, the cytoplasmic tails of ␣4 and ␣E subunits, which both may form heterodimers with ␤7, interact with WAIT-1. In contrast, the cytoplasmic domains of integrin subunits ␤1, ␤2, and ␣L, and of the structurally unrelated surface receptors CD4 and CD8, did not associate with WAIT-1 in yeast (Table I). Intrinsic ability of the bait proteins to activate reporter genes in yeast was excluded by control assays using the SEC7 domain of cytohesin-1 as a prey construct. Consistent with previous results (27), the SEC7 domain of cytohesin-1 interacted with the cytoplasmic tail of the integrin ␤2 chain but not with those of other proteins (Table I). Together, these results indicate that WAIT-1 specifically interacts with both the ␣ and ␤ subunit cytoplasmic tails of ␤7-integrins.
WAIT-1 Interacts with the Membrane-proximal Region of the ␤7 Cytoplasmic Tail-To determine the binding site of WAIT-1 in the integrin ␤7 cytoplasmic tail, recombinant PCR was used to generate various deletion and amino acid substitution mutants as fusion proteins with lexA. The primary structure of the mutants was confirmed by nucleic acid sequencing, and expression in yeast was confirmed by Western blot analysis using polyclonal antiserum against lexA (data not shown). Interaction in yeast of ␤7 cytoplasmic tail mutants was examined with WAIT-1 or the SEC7 domain of cytohesin-1 fused to the B42 transactivator. The results presented in Table II demonstrate that WAIT-1 interacted with the N-terminal fragment of the ␤7-integrin cytoplasmic domain spanning amino acids 729 -751, but not with the C-terminal portion. Further deletional analysis showed that the membrane proximal nine amino acids of the ␤7 cytoplasmic tail were sufficient to mediate association with WAIT-1 (Table II). In contrast, no interaction was seen with a 14-amino acid segment located C-terminal of the active peptide (Table II). Sequence comparison of integrin ␤ subunits revealed that a tyrosine residue at position 735 is unique to the membrane-proximal region of ␤7 (Fig. 4). To test a potential role of Tyr-735 for the interaction of WAIT-1 and ␤7-integrin, Tyr-735 was exchanged for phenylalanine or alanine. As shown in Table II, WAIT-1 efficiently interacted with the Y735F mutant, whereas interaction was completely abrogated by the Y735A mutation. These data therefore demonstrate specific interaction of the human WD repeat protein WAIT-1 with the membrane-proximal portion of the ␤7 subunit cytoplasmic tail, with Tyr-735 being of critical importance. Interestingly, tyrosine residues located at a homologous position are present in the WAIT-1-interacting cytoplasmic domains of ␣4 and ␣E but not in the ␣L cytoplasmic tail that fails to bind WAIT-1 (Fig. 5).
Distribution of WAIT-1 mRNA in Human Tissues-Expres-sion of WAIT-1 in various human tissues was analyzed by Northern blotting using the entire coding region of wait-1 as a probe. Except for peripheral blood leukocytes, predominant mRNA transcripts of 1.7 and 2.2 kb and low levels of 2.7-and 3.2-kb mRNA species were detected in all tissues examined (Fig. 6). In peripheral blood leukocytes, however, the 2.7-and

␤7-Integrin Interacting WD Repeat Protein
3.2-kb transcripts were predominant, and the 1.7-and 2.2-kb forms were expressed at low levels (Fig. 6). Equal loading of mRNAs from most tissues was demonstrated by rehybridization of the filter with a ␤-actin probe (Fig. 6). In addition, it should be noted that in the human T lymphoma line Jurkat and in Ramos B lymphoma cells, the 1.7-kb transcript of wait-1 was predominantly expressed (Fig. 1B). These results therefore suggest that expression of distinct WAIT-1 mRNA species may depend on the type of tissue or the activation state of cells. DISCUSSION Surface receptors of the integrin family are engaged in regulated cell adhesion and ligand-induced signal transduction events with the cytoplasmic tails of ␣ and ␤ subunits being of critical importance. Using a yeast interaction trap system, we have isolated a human WD repeat protein, WAIT-1, that is homologous to the mouse EED and the Drosophila ESC proteins and specifically interacts with the cytoplasmic domains of integrin ␤7, ␣4, and ␣E subunits. A tyrosine residue uniquely found in the membrane-proximal segment of the ␤7 cytoplasmic tail seems to be important for WAIT-1 interaction. Together with a previous report showing that the WD repeat protein RACK1 associates with various integrin ␤ chains (37), the results presented here suggest the existence of a subfamily of integrin-binding WD repeat proteins.
Several interactors of integrin cytoplasmic tails have been discovered, which may either link integrins to the cytoskeleton or may exert regulatory functions for integrin adhesion and signaling. Putative regulatory proteins recently identified include cytohesin-1, p27 BBP , ILK, ␤3-endonexin, ICAP-1, and RACK1, which interact with various ␤ subunits including ␤1, ␤2, ␤3, ␤4, and ␤5 (27, 37-40) as well as CIB, which was reported to associate with the ␣IIb cytoplasmic tail (41). It is interesting to note, however, that none of the integrin cytoplas-mic tail-associated proteins was reported to recognize both ␣ and ␤ subunits. In contrast, we demonstrate that WAIT-1 interacts not only with the ␤7 cytoplasmic tail but also with those of ␣4 and ␣E subunits, which form heterodimers with the ␤7 chain. These results suggest a distinct structural basis of WAIT-1 association with integrins and are consistent with WAIT-1 binding to ␤7-integrins being stabilized by interactions with both ␣ and ␤ chain cytoplasmic domain sequences.
Deletion of the entire cytoplasmic domain of the murine ␤7 subunit results in highly activated ligand binding by the ␣4␤7integrin (24). In contrast, deletion of a 34-amino acid C-terminal fragment of the ␤7 cytoplasmic tail abrogated adhesion of 38C13 B lymphoma cells to ␣4␤7-integrin ligands (24). These results indicated that the membrane-proximal segment of the ␤7 cytoplasmic domain contains a negative regulatory element for ␣4␤7-mediated cell adhesion. In the present study, mutational analysis was used to demonstrate that a short sequence of nine membrane-proximal amino acids in the ␤7 cytoplasmic tail is sufficient for WAIT-1 interaction. Moreover, a tyrosine residue at position 735 that is unique to the ␤7 subunit was shown to be important. It is therefore tempting to speculate that association with WAIT-1 may function to prevent or downregulate ligand recognition of ␤7-integrins. WAIT-1 binding to ␤7-integrins may either retain the integrin in a low affinity conformation or prevent binding of integrin-activating proteins.
The murine WAIT-1 homologue EED was proposed to function as a transcriptional regulator of homeobox genes based on developmental abnormalities of mutant mice (35). A similar function has been suggested for the Drosophila esc gene (36). Consistent with a function in gene regulation, the EED protein was found to suppress the activity of a synthetic promoter when fused to the yeast transcription factor Gal4 (42) and to  (34,43). It is therefore conceivable that WAIT-1 may not only associate with membrane-bound ␤7-integrins but, under certain conditions, may also localize to the nucleus. The association with integrins and the potential subcellular distribution of WAIT-1 are therefore reminiscent of ␤3-endonexin and p27 BBP proteins, which were reported to interact with the integrin ␤3 and ␤4 cytoplasmic tails, respectively, and to be localized partly to the nucleus (40,44). It will be interesting to determine whether WAIT-1 shuttles between membrane-associated ␤7-integrins and the nucleus, thereby linking cell adhesion and signal transduction or regulation of gene expression. Consistent with a role of ␤7-integrins as signaling molecules, it was shown that ligation of ␣4␤7-integrin on B and T lymphocytes induces tyrosine phosphorylation of several substrates including focal adhesion kinase, lck, fyn, and mitogen-activated protein kinases (25,45).
Northern blot analysis of various human tissues indicated ubiquitous distribution of WAIT-1 mRNA transcripts, whereas ␤7-integrin expression is largely limited to leukocytes. These observations suggest alternate functions of WAIT-1 that are not related to its interaction with ␤7-integrin cytoplasmic domains. Involvement of WAIT-1 in multiple cellular functions is also suggested by findings showing that murine EED mutations are embryonic lethal (35), whereas ␤7 knockout mice are viable (14). Similar observations have been reported for cytohesin-1, which interacts with the cytoplasmic domain of leukocyte ␤2-integrins. Cytohesin-1 also shows a broad tissue distribution that only partially overlaps with the expression of integrin ␤2 (27).
Analysis of WAIT-1 tissue distribution revealed multiple mRNA species of 1.7, 2.2, 2.7, and 3.2 kb that hybridize with the WAIT-1 probe. Different WAIT-1-related mRNA species detected in human tissues may indicate the existence of alternatively spliced WAIT-1 variants or a family of WAIT-1-related WD-repeat proteins. In addition, the presence of alternative polyadenylation sites or alternate transcriptional start sites due to regulation of WAIT-1 by distinct promoters may also account for some of the differences in transcript size. In circulating leukocytes, dominant mRNA transcripts of 2.7 and 3.2 kb were observed, whereas in other tissues including spleen transcripts of 1.7 and 2.2 kb predominated. It is therefore conceivable that alternate forms of WAIT-1 are expressed in circulating and tissue-resident leukocytes. Consistent with this possibility, MAdCAM-1, the major ligand of ␣4␤7-integrin, is expressed on endothelial cells of mucosal tissues and in the marginal sinus of spleen (46,47) and is therefore selectively exposed to circulating leukocytes. Moreover, the 2.2-kb mRNA transcript may conform to the recently described 67-kDa form of EED that starts from an unusual GTG initiation codon upstream of the WAIT-1 translational start site and contains an additional WD repeat (42). In contrast to peripheral blood leukocytes, cultured T and B lymphoma cells predominantly express the 1.7-kb WAIT-1 transcript. Integrin ␣4␤7 adhesionrelated functions of normal leukocytes and lymphoma cells differ, however, because both cell types bind MAdCAM-1 in vitro, but only normal leukocytes efficiently migrate to mucosal   I WAIT-1 specifically interacts with cytoplasmic tails of ␤7-integrins Cytoplasmic domains of various human integrin subunits and of CD4 and CD8 were fused to the lexA DNA binding domain, and interaction with WAIT-1 or the SEC7 domain of cytohesin-1 linked to the transcriptional activator B42 was tested by induction of ␤-galactosidase activity in yeast. Expression of bait constructs was verified by Western blot analysis of yeast protein extracts using a polyclonal antiserum against lexA.

Cytoplasmic tail
Interactor WAIT-1 Cytohesin-1/SEC7 Ϫ Ϫ ␤7-Integrin Interacting WD Repeat Protein sites in vivo (48). It will therefore be interesting to address the question whether alternate forms of WAIT-1 exist that may differ in regulating ␤7-integrin functions in circulating, tissueresident, and transformed leukocytes. II Mapping of the WAIT-1 binding site in the integrin ␤7 cytoplasmic tail Mutants of the integrin ␤7 cytoplasmic tail were fused to the lexA DNA binding domain, and interaction with WAIT-1 or the SEC7 domain of cytohesin-1 linked to the transcriptional activator B42 was tested by induction of ␤-galactosidase activity in yeast. Expression of bait constructs was verified by Western blot analysis of yeast protein extracts using a polyclonal antiserum against lexA. NT, N-terminal segment; CT, C-terminal segment; NT-MP, membrane-proximal region of NT; NT-MD, membrane-distal region of NT.  5. Amino acid sequence alignment of human integrin cytoplasmic tails. Sequence comparison reveals that Tyr-735 is unique to the membrane-proximal region of the ␤7 cytoplasmic tail. Interestingly, a tyrosine residue located at a similar position was found in ␣4 and ␣E but not ␣L subunit cytoplasmic domains. The presence of this tyrosine residue correlates with the WAIT-1 interaction.