©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Membrane-proximal Scavenger Receptor Cysteine-rich Domain of CD6 Contains the Activated Leukocyte Cell Adhesion Molecule Binding Site (*)

(Received for publication, June 5, 1995)

Gena S. Whitney(§)(¶) Gary C. Starling (§) Michael A. Bowen Brett Modrell Anthony W. Siadak Alejandro Aruffo

From the Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Binding studies with a CD6 immunoglobulin fusion protein (CD6 Rg) resulted in the identification and cloning of a CD6 ligand. This ligand was found to be a member of the immunoglobulin supergene family and was named ALCAM (activated leukocyte cell adhesion molecule). Cell adhesion assays showed that CD6-ALCAM interactions mediate thymocyte-thymic epithelium cell binding. ALCAM is also expressed by activated leukocytes and neurons and may be involved in interactions between T cells and activated leukocytes and between cells of the immune and nervous systems, respectively. Herein we describe the preparation of domain-specific murine CD6 Rg fusion proteins and show that the membrane-proximal SRCR (scavenger receptor cysteine-rich) domain of CD6 contains the ALCAM binding site. We also show that mAbs which bind to this domain preferentially block CD6-ALCAM binding. These results demonstrate that the membrane-proximal SRCR domain of CD6 is necessary for CD6 binding to ALCAM and provide the first direct evidence for the interaction of an SRCR domain with a ligand.


INTRODUCTION

CD6 is a type I cell surface protein and a member of the SRCR (^1)family of proteins(1) . The extracellular domain of the mature CD6 protein is composed of three SRCR domains followed by a short 33-amino-acid stalk region. This extracellular domain is anchored to the cell membrane via a short hydrophobic domain followed by a cytoplasmic domain of variable length (2) . Cloning of cDNAs encoding the murine homologue of CD6 (mCD6) and comparison of its predicted amino sequence with that of the human clone revealed the possible existence of multiple CD6 isoforms which result from the variable splicing of exons encoding the cytoplasmic domain (3) . Within the SRCR family of proteins, CD6 is most closely related to CD5(1) . These two proteins share a similar extracellular domain organization and are both expressed by thymocytes, T cells, B cell CLLs, and a subset of normal B cells.

Antibody cross-linking studies have shown that CD6 can function as an accessory molecule in T cell activation. For example, the anti-CD6 mAb 2H1 can induce T cell activation in conjunction with phorbol 12-myristate 13-acetate or with the anti-CD2 mAb T11(3)(4) . On T cells, ligation of CD6 with the anti-CD6 mAb G3-5 causes an increase in CD3-induced cytoplasmic calcium concentration (5) while CD6 cross-linking with the anti-CD6 mAb T12 has been shown to enhance the activation mediated by suboptimal doses of anti-CD3 mAbs(6) . Additional evidence for the role of CD6 in T cell activation comes from studies showing that CD6 becomes hyperphosphorylated on Ser and Thr residues (7, 8, 9) and phosphorylated on Tyr residues (10) following T cell activation. This last observation suggests that the cytoplasmic domain of CD6 may provide a membrane anchor for interaction with SH2-containing proteins involved in intracellular signaling.

Studies using CD6 Rg, an immunoglobulin fusion protein consisting of the extracellular domains of CD6 fused to human IgG(1) constant domains, resulted in the identification of cells which express a CD6 ligand(11) . The identification of these cells allowed the characterization (11, 12) and cloning (13) of a human CD6 ligand. This CD6 ligand, which is recognized by mAb J4-81(14) , was named ALCAM. ALCAM is a member of the immunoglobulin supergene family and appears to be the human homologue of the chicken neural adhesion molecule BEN/SC-1/DM-GRASP (15, 16, 17) and the rat protein KG-CAM(18) . ALCAM is a type I membrane protein composed of two amino-terminal V-set domains followed by three C2-set domains, a hydrophobic transmembrane domain, and a short cytoplasmic anchor sequence. ALCAM is expressed by thymic epithelial (TE) cells (11, 12) and transiently on activated leukocytes (13) . In addition, ALCAM is expressed by neurons in the brain(12) . Cell adhesion assays demonstrated that CD6-ALCAM interactions are in part responsible for mediating thymocyte binding to TE cells(13) .

Herein we present the results of experiments designed to determine whether binding to ALCAM can be localized to a single domain of CD6. Two independent approaches were used: first, a series of CD6 Rg fusion proteins containing different CD6 extracellular domains, alone or in combinations, were used in cell and protein binding assays; second, mAbs directed against different CD6 extracellular domains were used to block CD6 ALCAM binding and provided independent evidence for which CD6 domain(s) is responsible for ALCAM binding. The results obtained from both of these experimental approaches indicated that the membrane-proximal SRCR domain of CD6 contains the ALCAM binding site.


MATERIALS AND METHODS

Cell Lines, Tissue Culture, and Antibodies

The human T cell line HPB-ALL was obtained from the American Type Culture Collection (Rockville, MD) and maintained in culture in Iscove's modified Dulbecco's medium + 10% fetal bovine serum. The preparation and characterization of the rat anti-mCD6 mAbs M6-1A.1, M6-3A.1, and M6-3B.1 mAbs will be described elsewhere. (^2)All three mAbs bound to murine thymocytes as determined by flow cytometry analysis. Analysis of which domains contained the epitope recognized by each mAb was determined by examining the binding pattern of the mAbs on the mCD6 Rg fusion proteins by ELISA. M6-1A.1 bound to mCD6 Rg, mCD6D1-2 Rg, and mCD6D1 Rg but did not bind mCD6D2-S Rg, mCD6D2 Rg, or mCD6D3 Rg. M6-3A.1 and M6-3B.1 each bound mCD6 Rg, mCD6D2-3 Rg, and mCD6D3 Rg but not mCD6D1-2 Rg, mCD6D1 Rg, or mCD6D2 Rg. We therefore concluded that M6-1A.1 recognizes mCD6 SRCR-D1 and that both M6-3A.1 and M6-3B.1 recognize mCD6 SRCR-D3.

Murine CD6 Rg Constructs

Complementary DNA fragments encoding individual or groups of mCD6 extracellular domains were obtained by polymerase chain reaction. The mCD6 fusion protein contains bases 119-1181 of the mCD6 cDNA (3) fused to a cDNA fragment encoding the hinge, CH2, and CH3 domains of human IgG1(19) . The mCD6D1, mCD6D2, mCD6D3, mCD6D1-2, mCD6D2-3, mCD6D1-3, mCD6D2-S, and mCD6D3-S fusions contain mCD6 bases 119-468, 469-775, 776-1081, 119-775, 469-1081, 119-1081, 469-1181, and 776-1181, respectively. To ensure that no mutations were introduced by the procedure, all chimeric genes were sequenced. In all cases, the amino-terminal secretory signal sequence used in these chimeric constructs was derived from the human CD5 gene. The fusion proteins were produced by transient expression in COS cells and were purified by adsorption and elution from a protein A-Sepharose column. Protein concentrations were determined using a Bradford dye-binding procedure (Bio-Rad) and verified by SDS-polyacrylamide gel electrophoresis.

Binding Assays

Goat anti-mouse IgG (4 µg/ml, Jackson Laboratories, West Grove, PA) was coated on 96-well plastic dishes overnight at room temperature. After washing to remove excess antibody, ALCAM Rg (2 µg/ml) was added. The ALCAM Rg fusion protein used for these studies contains a murine IgG domain. Dilutions of mCD6 Rg fusion proteins (7.5 ng/ml-1.0 µg/ml) and control CD7 Rg were added and allowed to bind to the immobilized ALCAM Rg. After washing, the bound mCD6 Rg and CD7 Rg fusion proteins were detected with horseradish peroxidase-conjugated donkey anti-human IgG (Jackson Laboratories). Absorbance readings were taken at 450 nm and 630 nm. Standard deviations were calculated on the basis of three independent binding measurements.

HPB-ALL cells (5 10^5) were labeled with 20 µg/ml purified fusion protein for 1 h at 4 °C. Cells were washed and subsequently labeled with fluorescein isothiocyanate-goat anti-human IgG (Tago, Burlingame, CA) for 30 min at 4 °C and washed. The level of fusion protein binding was determined by flow cytometry (FACScan, Becton-Dickinson).

Antibody Blocking Experiments

The ability of the anti-mCD6 mAbs to inhibit the binding of ALCAM Rg to mCD6 was examined using murine thymocytes obtained from C57BL/6 mice. In each assay, 5 10^5 cells were incubated with 50 µl of conditioned media obtained from each of the hybridomas producing the anti-mCD6 mAbs or from a hybridoma producing a rat anti-human gp39 mAb as a negative control for 30 min at 4 °C. Cells were washed and stained with 20 µg/ml human ALCAM Rg for 30 min at 4 °C. Cells were then washed and labeled with fluorescein isothiocyanate-donkey anti-human IgG (Jackson Laboratories) with minimal cross-reactivity to rat and bovine IgG. The cells were then washed and subsequently analyzed by flow cytometry using a FACScan.


RESULTS

Mapping of the CD6 ALCAM Binding Site

The extracellular region of mature CD6 can be divided into four domains, three consecutive SRCR domains (D1-D3) followed by a 33-amino-acid membrane proximal ``stalk'' (S) domain. To determine which of these domains contains the ALCAM binding site, we prepared six different mCD6 immunoglobulin fusion proteins (Rg, recombinant globulins) which contain different mCD6 domains alone or in combinations: mCD6 Rg which contains the complete extracellular domain of CD6 or mCD6D1 Rg, mCD6D2 Rg, mCD6D3-S Rg, CD6D1-2 Rg, and CD6D2-S Rg (Fig. 1A). These mCD6 Rg fusion proteins were prepared by transient expression in COS cells and purified by adsorption to and elution from a protein A-Sepharose column (Fig. 1B).


Figure 1: Murine CD6 Rgs. A, line drawing representations of the chimeric genes encoding the mCD6 immunoglobulin fusion proteins used in this study. The SRCR domains in the extracellular domain of mCD6 are represented by boxes which are labeled D1, D2, and D3. The box which represents the membrane-proximal 33-amino-acid stalk domain is labeled S. The human IgG(1) constant domains including hinge, CH2, and CH3 are represented by a bold line. The name of each of the mCD6 Rg fusion proteins is shown to the right. B, radiolabeled fusion proteins were purified as described under ``Materials and Methods'' and electrophoresed under reducing conditions. The electrophoretic mobility of molecular mass standards in kDa is shown to the left.



We had previously shown that hCD6 Rg, an immunoglobulin fusion protein containing the extracellular domain of human CD6, was capable of binding to murine cells lines(11) . Here we show that mCD6 Rg is capable of binding ALCAM expressed on the human T cell line HPB-ALL (Fig. 2A) or to ALCAM Rg, an immunoglobulin fusion protein which contains the extracellular domain of human ALCAM (Fig. 2B)(13) . The ability of mCD6 to bind to human ALCAM allowed us to use the mCD6 Rg fusion proteins described above in two different binding assays to identify the mCD6 extracellular subdomain(s) responsible for ALCAM binding. In the first assay, the binding of mCD6 Rg to HPB-ALL cells was compared with that of the different mCD6 Rg domain fusion proteins by flow cytometry. As shown in Fig. 2A, the binding of mCD6D2-S Rg and mCD6D3-S Rg to the HPB-ALL cells was comparable to that of mCD6 Rg. On the other hand, mCD6D1 Rg, mCD6D2 Rg, and mCD6D1-2 Rg as well as our control fusion protein mCD7 Rg failed to bind to the HBP-ALL cells. Similar results were obtained when the ability of the mCD6 Rg fusion proteins to bind to purified ALCAM Rg was examined by ELISA. As shown in Fig. 2B, fusion proteins containing CD6 SRCR-D3 and S domains bound ALCAM Rg. In addition, similar results were obtained when the ability of the mCD6 Rg fusion proteins to bind to COS cells transfected with a plasmid containing a cDNA encoding ALCAM was examined (data not shown). These results suggest that the ALCAM binding site in CD6 is contained in the membrane-proximal region of the protein which contains the SRCR-D3 and S domains of CD6.


Figure 2: Binding of the mCD6 Rgs to HPB-ALL cells or ALCAM Rg. A, flow cytometry profiles of the binding of the mCD6 Rgs (mCD6 Rg, mCD6D1-2 Rg, mCD6D2-S Rg, mCD6D3-S Rg, mCD6D1 Rg, mCD6D2 Rg, mCD6D1-3 Rg, mCD6D2-3 Rg, and mCD6D3 Rg (dark profiles) and the control fusion protein, CD7 Rg (empty profile), to HPB-ALL cells. Mean channel fluorescence values for each mCD6 Rg are noted on each histogram. The mean channel fluorescence value for CD7 Rg was 2.4. These binding profiles are a representative set selected from a group of three independent binding assays. B, binding of increasing concentrations of the mCD6 Rg fusion proteins listed in A of this figure legend and the control CD7 Rg fusion protein to ALCAM Rg immobilized in the wells of a 96-well plastic dish. Standard deviations were calculated on the basis of three binding measurements.



To further examine the contribution of the CD6 S domain to CD6-ALCAM binding, we prepared three additional mCD6 Rg fusion proteins: mCD6D1-3 Rg, mCD6D2-3 Rg, and mCD6D3 Rg (Fig. 1). HPB-ALL (FACS) and ALCAM Rg (ELISA) binding studies with this set of fusion proteins showed that the membrane-proximal SRCR domain of CD6 alone can bind ALCAM (Fig. 2). Taken together these data suggest that the CD6 SRCR-D3 domain is necessary for CD6-ALCAM binding.

Blocking of CD6-ALCAM Interactions by Domain-specific Anti-CD6 Antibodies

Recently we prepared a number of rat anti-mCD6 mAbs. The ability of these mAbs to bind to the different CD6 Rg domain constructs was examined.^2 These studies allowed the identification of domain-specific anti-mCD6 mAbs. Three anti-mCD6 mAbs were selected for blocking studies. One of these antibodies recognizes the CD6 SRCR-D1 (M6-1A.1), the other two (M6-3A.1 and M6-3B.1) recognize CD6 SRCR-D3. As shown in Fig. 3, mAb M6-3A.1 completely blocked the ability of ALCAM Rg to bind murine thymocytes expressing CD6. MAb M6-3B.1 was able to partially block while M6-1A.1 blocked only very weakly or not at all the binding of ALCAM Rg to murine thymocytes. As expected, an isotype-matched rat anti-human gp39 mAb used as a control was unable to block the binding of ALCAM Rg to murine thymocytes (Fig. 3). Inhibition of ALCAM binding to mCD6 by domain 3 but not domain 1 antibodies was confirmed by ELISA (data not shown). These results provide further evidence that the third SRCR domain of CD6 contains the ALCAM binding site.


Figure 3: Blocking of the CD6-ALCAM interaction by domain-specific mAbs. Flow cytometry profiles showing the binding of ALCAM Rg to murine thymocytes which had been pretreated with hybridoma supernatants containing the rat anti-mCD6 mAbs M6-1A.1, M6-3A.1, and M6-3B.1 or an isotype-matched rat anti-human gp39 mAb (dark profile) compared to binding at the negative control, CD7Rg (empty profiles). These profiles are a representative set derived from a group of two independent blocking assays.




DISCUSSION

Using immunoglobulin fusion proteins containing single or multiple extracellular domains of mCD6, we have mapped the ALCAM binding site to the membrane-proximal SRCR domain of CD6. Fusion proteins containing this domain are capable of binding ALCAM, and mAbs directed against this CD6 domain were found to block CD6-ALCAM binding. Taken together these results demonstrate that mCD6 SRCR-D3 is necessary for CD6 binding to ALCAM. However, these results do not rule out the possible contribution of other CD6 domains in CD6-ALCAM binding. Indeed we observed that some of the truncated fusion proteins containing SRCR-D3, such as mCD6D3 Rg and mCD6D1-3 Rg, bound ALCAM Rg less efficiently than the others (Fig. 2B. This might be the result of a loss of residues in other CD6 domains which directly contribute to ALCAM binding and/or due to structural perturbations of SRCR-D3 caused by the truncation of adjacent domains. In this regard, we also observed that mCD63-S Rg bound ALCAM as well as mCD6 Rg. This suggests that the S domain may play a more important role in ALCAM binding than SRCR-D1 or -D2 by either contributing directly to ALCAM binding, playing a structural role in facilitating CD6-ALCAM interactions, and/or by providing an appropriate spacer between the SRCR domain and the Ig portion of the fusion protein which allows efficient binding. These findings leave open the possibility that the other extracellular domains of mCD6, mCD6 SRCR-D1, and -D2, have other ligands.

Indirect evidence for additional CD6 ligands is provided by immunoprecipitation studies of CD6 binding proteins from HBL-100 cells (11) . These studies showed that CD6 Rg bound to three proteins, only one of which had a molecular weight consistent with the predicted molecular weight of ALCAM(11, 13) . Also, anti-CD6 mAbs which bind to different CD6 epitopes have different functional characteristics(4, 6, 20) . This has led to the suggestion that CD6 has multiple ligands which might differentially regulate CD6 function(20) . There are a number of examples of leukocyte receptors which have multiple ligands. Perhaps one of the best studied is CD2 which has been found to interact with CD48, CD58, and CD59(21, 22) . The CD6 fusion proteins described here may be useful for the identification of the biological function of the other CD6 subdomains. Binding studies with mCD6D1-2 Rg on different cell types are ongoing to explore the possibility that the SRCR-D1 and -D2 extracellular subdomains of CD6 interact with other ligands.

Ligands for other members of the SRCR family of proteins have been reported. In particular, CD5 has been reported to bind CD72(23, 24) , the type I macrophage scavenger receptor binds to acetylated low density lipoproteins(25) , 90K binds to MAC-2(26) , and cyclophilin-C (27) , complement factor I, binds to the activated complement proteins C3b and C4b(28) , the SPERACT receptor binds to the peptide SPERACT (29) , and MARCO binds Escherichia coli and other bacteria (30) . In some cases, these receptor-ligand interactions do not involve the SRCR domain(s). This is exemplified by the interaction between the type I macrophage scavenger receptor and acetylated low density lipoprotein. In this case, receptor-ligand binding is not mediated through the SRCR domain(25) . In other cases, indirect evidence for the role of the SRCR domain in receptor-ligand binding has been reported. As an example, in studies of the E. coli binding function of MARCO, polyclonal antiserum raised against sequences found in the SRCR domain of MARCO was found to inhibit binding to E. coli(30) . Presently it is not known if this effect is due to direct blocking of the ligand binding site or steric hindrance. However, in most cases, the role of the SRCR domain(s) in receptor-ligand interactions has not been examined. Our finding that CD6 SRCR-D3 binds ALCAM provides the first direct demonstration that an SRCR domain can participate in binding interactions. Future work defining the role of other SRCR domains in receptor-ligand binding will be required to determine the range of molecular interactions mediated by this well conserved protein domain.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The contribution of the first two authors should be considered equal.

To whom correspondence and reprint requests should be addressed: Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Ave., Seattle, WA 98121. Tel.: 206-728-4800; Fax: 206-727-3602.

^1
The abbreviations used are: SRCR, scavenger receptor cysteine-rich; ALCAM, activated leukocyte cell adhesion molecule; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay.

^2
G. C. Starling, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Vicki McDonald, Marcia Gordon, Gary Carlton, and Nicola Tinari for their assistance, Jeff Ledbetter for critical review of this manuscript, and Debby Baxter for help in its preparation.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.