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Volume 270, Number 2, Issue of January 13, 1995 pp. 715-719
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of the Protein 4.1 Binding Interface on Glycophorin C and p55, a Homologue of the Drosophila discs-large Tumor Suppressor Protein (*)

(Received for publication, August 17, 1994; and in revised form, October 28, 1994)

Shirin M. Marfatia (1),  Robert A. Lue (2),  Daniel Branton (2),  Athar H. Chishti (1)(§)

From the  (1)Department of Biomedical Research, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135 and (2)Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.

INTRODUCTION

Protein 4.1 is an 80-kDa peripheral membrane phosphoprotein that plays a pivotal role in regulating erythroid shape and membrane skeleton mechanical properties(1) . In many individuals suffering from hereditary elliptocytosis, primary defects in erythroid protein 4.1 cause aberrant morphology and hemolysis(2) . An explanation of these effects will require an understanding of how protein 4.1 binds to other membrane and cytoskeletal proteins(3, 4, 5, 6) . Several studies have shown that an N-terminal 30-kDa domain of protein 4.1 contains the membrane attachment site that interacts with transmembrane proteins(7, 8, 9) . We recently showed that this 30-kDa domain of protein 4.1 binds to both glycophorin C and p55(10) . Further evidence for this ternary complex comes from the study of subjects with either protein 4.1(-) hereditary elliptocytosis or the Leach phenotype where erythrocytes lack glycophorin C(11, 12) . The erythrocytes of these individuals display aberrant elliptocytic morphology, and exhibit a concomitant loss of p55 along with protein 4.1 and glycophorin C(12) .

Human erythrocyte glycophorin C is a transmembrane glycoprotein that is widely expressed in many non-erythroid cells(13) . The fact that erythrocytes lacking glycophorin C have an elliptocytic shape suggests that glycophorin C plays a role in the regulation of discoid morphology of normal red blood cells(14) . The regulation of erythroid membrane deformability and mechanical stability by glycophorin C is believed to be mediated by the binding of its cytoplasmic domain to protein 4.1(1, 10) .

p55 is a palmitoylated erythrocyte membrane protein whose sequence and domain organization identify it to be a member of a family of proteins now termed membrane-associated guanylate kinase homologues (MAGUKs)(^1)(15) . This family includes the Drosophila discs-large tumor suppressor protein, Dlg (16) and its human homologue, Hdlg(34) , the rat synaptic protein, PSD-95 or SAP90(17, 18) , and the tight junction proteins, Z0-1 and Z0-2 (19, 20, 21) . The primary structure of p55 can be subdivided into three distinct domains: 1) an N-terminal domain that includes a single incomplete copy of the discs-large homologous region (DHR). In the Drosophila discs-large tumor suppressor protein and other MAGUKs, there are three copies of the DHR domain(15) . The function of this domain is not known; 2) a central src homology 3 (SH3) domain which may mediate specific protein-protein interactions; and 3) a C-terminal domain that shows significant homology to the guanylate kinases(15, 22) . The function of this domain is not known.

Here, we localize the sites in glycophorin C and p55 that bind the 30-kDa domain of protein 4.1. In glycophorin C, a 12-amino acid segment within the cytoplasmic domain binds the 30-kDa domain of protein 4.1. In p55, a novel charged motif flanked by the SH3 and guanylate kinase domains binds the 30-kDa domain of protein 4.1. A similarly charged motif is found in both the Drosophila discs-large tumor suppressor protein and its human homologue. The significance of these findings is discussed in terms of the membrane localization of MAGUKs in both erythroid and non-erythroid cells.

EXPERIMENTAL PROCEDURES

Glycophorin C Peptides

The entire cytoplasmic domain of glycophorin C contains 47 of glycophorin's 128 amino acid residues. Peptides P23 and P24 were synthesized by standard methods, and coupled to biotin at their amino termini. Peptides P12m, P12c, and P12a were coupled to biotin via an inert spacer sequence, SGSG (see Fig. 1). An irrelevant peptide with the sequence NELKKKASLF was included to determine the extent of nonspecific binding to the beads. The synthetic peptides were purified by analytical reverse phase high pressure liquid chromatography, and the quality of purified peptides was confirmed by amino acid analysis and mass spectrometry.

Figure 1: Synthetic peptides of human erythroid glycophorin C. The peptide P12a (not shown) corresponds to the peptide P12m except that the amino acids RHK were replaced by AAA. The presence of the spacer sequence SGSG was found to be necessary to reduce the nonspecific binding of short peptides.


N-terminal, I-Labeled, 30-kDa Domain of Protein 4.1

Protein 4.1 was isolated using the procedure of Ohanian and Gratzer (23) which yields pure protein, free of any contaminating protein kinases. Purified protein 4.1 was digested with alpha-chymotrypsin at an enzyme to substrate ratio of 1:25(7) . The digestion was carried out on ice for 90 min in 10 mM Tris-HCl, pH 8.0. After quenching the protease activity with 4.0 mM diisopropyl fluorophosphate, the digested protein was fractionated on a Mono Q column using a linear gradient of 20-500 mM NaCl. The purified 30-kDa domain of protein 4.1 was then radiolabeled with I-labeled Bolton-Hunter reagent as described elsewhere (see Fig. 2)(24) .

Figure 2: Isolation of the 30-kDa domain of protein 4.1. Purified protein 4.1 from human erythrocyte membranes (lane 1); purified 30-kDa domain (lane 2); autoradiograph of the I-labeled 30-kDa domain (lane 3). The details of the isolation procedure are described under ``Experimental Procedures.''


p55 Recombinant Constructs

Five cDNA constructs termed D1, D2, D3, D4, and D5 were produced containing various domains of the human erythroid p55 (see Fig. 4Fig. 5Fig. 6). The cDNA constructs were amplified using the polymerase chain reaction with the following set of primers. All primers contained the BamHI adapter sequence to facilitate subsequent cloning of the polymerase chain reaction products into the plasmid vector.

Figure 4: Determination of the protein 4.1 binding site on human erythroid p55. A, the cDNA constructs containing various domains of p55 were produced as glutathione S-transferase fusion proteins. B, binding of the I-labeled 30-kDa domain of protein 4.1 to the GST-p55 fusion proteins as detected by the blot-overlay assay and autoradiography.


Figure 5: Binding of the I-labeled 30-kDa domain of protein 4.1 to the p55 GST-fusion protein D5. The 39-amino acid sequence of p55, as shown in Fig. 6, was produced as glutathione S-transferase fusion protein (D5). The binding of the I-labeled 30-kDa domain was detected by a blot-overlay assay. Lanes 1 and 3, Ponceau S stain; and lanes 2 and 4, (autoradiography). Lanes 1 and 2, GST; and lanes 3 and 4, (GST-39 amino acids). Note that the GST-fusion protein in lane 3 is degraded producing a truncated protein. The truncated fusion protein does not bind to the I-labeled 30-kDa domain of protein 4.1.


Figure 6: Location and alignment of the protein 4.1 binding region of erythroid p55. A, the 39-amino acid region located between the SH3 and guanylate kinase domains of human p55 was used to make the construct D5. The D5 fusion protein was used in the blot-overlay assay shown in Fig. 5. B, a comparison of the protein 4.1 binding region of p55 with similar sequences found in Dlg and Hdlg. Hdlg is the human homologue of the Drosophila tumor suppressor protein Dlg(16, 34) . The triangle in the Hdlg sequence shows the beginning of the fusion protein which binds to protein 4.1(34) . It therefore appears that the conserved cluster of C-terminal lysine residues in MAGUKs may be critical for the binding of protein 4.1.


1) Construct D1, primers p55-1 (sense) and p55-7 (antisense); 2) construct D2, primers p55-1 (sense) and p55-5 (antisense); 3) construct D3, primers p55-4 (sense) and p55-2 (antisense; 4) construct D4, primers p55-3 (sense) and p55-2 (antisense); 5) construct D5, primers p55-8 (sense) and p55-9 (antisense). In each case, the polymerase chain reaction product was ligated into the pGEX-2T vector which was expressed in Escherichia coli strains 71/18 or DH5alpha. The glutathione S-transferase fusion proteins were then purified as described previously(10) . All of the cDNA constructs were confirmed by nucleotide sequence analysis, and the identity of the recombinant proteins was established using polyclonal antibodies against p55 and its synthetic peptides.

Blot Overlay Assay

GST-p55 fusion proteins were transferred to nitrocellulose after SDS-polyacrylamide gel electrophoresis. The nitrocellulose blots were blocked with TBS-Tween buffer (20 mM Tris-HCl, pH 7.6 + 150 mM NaCl + 0.1% Tween 20 + 0.02% sodium azide) for 1 h, followed by an overnight incubation in the blocking buffer (TBS-Tween + 3% bovine serum albumin). The blots were incubated for 24 h at 4 °C with the I-labeled 30 kDa of protein 4.1 in the binding buffer (5.0 mM sodium phosphate, pH 7.6, 1.0 mM 2-mercaptoethanol, 0.5 mM EDTA, 120 mM KCl, 0.02% sodium azide and 1.0 mg/ml bovine serum albumin). The bound radioactivity was detected by autoradiography.

Sedimentation Assay

Biotinylated glycophorin C peptides were immobilized to the streptavidin agarose beads in the binding buffer for 2 h at 4 °C. The beads with bound peptides were extensively washed with the binding buffer(10) , and blocked with 1.0 mg/ml of d-biotin in order to reduce nonspecific binding. The beads were incubated with the I-labeled 30-kDa domain of protein 4.1 in the binding buffer containing 0.5% Nonidet P-40. The amount of radioactivity sedimented with the beads was measured in a counter. For competition experiments, molar excess of competing molecules was added prior to the addition of the I-labeled 30-kDa domain of protein 4.1 in the assay mixture. In all binding experiments, control beads with either no bound peptide or with an irrelevant peptide were used to account for the nonspecific binding. The binding of the I-labeled 30-kDa domain of protein 4.1 to the GST-p55 fusion proteins was carried out as described previously(10) .

ELISA

The purified 30-kDa domain of protein 4.1 was adsorbed to the plastic surface (Immulon 2 plate, Dynatech Labs. Inc.) for 2 h. The plate was blocked with the blocking buffer for 3 h at room temperature. The biotinylated peptides were dissolved in the blocking buffer (20.0 µg in each well) and incubated overnight in the ELISA plate at 4 °C. The plate was washed extensively with the blocking buffer, and the amount of bound peptide was measured at 405 nm using the streptavidin-alkaline phosphatase and p-nitrophenyl phosphate as substrate.

RESULTS

To determine the protein 4.1 binding site on glycophorin C, peptides were synthesized corresponding to the defined segments of the cytoplasmic domain of human erythrocyte glycophorin C (Fig. 1). The cytoplasmic domain of glycophorin C contains 47 amino acids(25) . Peptide P23 consists of 23 amino acids including residue 82-104 of the cytoplasmic domain of glycophorin C. This segment of glycophorin C is proximal to the inner face of the erythroid plasma membrane. The P24 peptide, which lies distal to the plasma membrane, contains the remaining 24 amino acids (residue 105-128) of the cytoplasmic domain of glycophorin C. Biotinylated peptides were conjugated to the streptavidin agarose beads and used to quantify the binding of the I-labeled 30-kDa domain of protein 4.1. The 30-kDa domain was produced after proteolytic digestion of purified protein 4.1, and its purity was examined by gel electrophoresis (Fig. 2). The I-labeled 30-kDa domain of protein 4.1 specifically binds to P23, the peptide proximal to the plasma membrane (Table 1). Binding was completely inhibited in the presence of a molar excess of the unlabeled 30-kDa domain of protein 4.1. In contrast, no binding of the I-labeled 30-kDa domain was detected with either P24 or an irrelevant peptide derived from dematin. These results were confirmed by an ELISA which was designed to measure the binding of peptides in solution to the immobilized 30-kDa domain of protein 4.1 (Table 1).

To further define the protein 4.1 binding site within the P23 peptide, two peptides were synthesized corresponding to the 23 amino acids of the P23 peptide (Fig. 1). Peptide P12 m consists of N-terminal 12 amino acids (residue 82-93), and the peptide P12c consists of the C-terminal 12 amino acids (residue 94-105). To determine the binding of peptides P12m and P12c to the 30-kDa domain of protein 4.1, a binding inhibition assay was designed (Fig. 3). In this assay, the binding of P23 peptide to the I-labeled 30-kDa domain was measured in the presence of either the P12m or P12c peptide. The peptide P12m quantitatively inhibited the binding of P23 peptide to the I-labeled 30-kDa domain of protein 4.1 (Fig. 3). Half-maximal inhibition of P23 binding was achieved with 20 µM P12m. In contrast, no inhibition was observed with P12c (Fig. 3). These results show that the 30-kDa domain of protein 4.1 binds to glycophorin C in a region defined by the amino acids 82-93 (RYMYRHKGTYHT). Since this segment of the cytoplasmic domain of glycophorin C contains a cluster of positively charged residues i.e. RHK, we synthesized another peptide P12a where the RHK cluster was replaced by AAA. A comparison of the binding of peptides P12a and P12m to the 30-kDa domain of protein 4.1 indicated that the substitution of alanine (RYMYAAAGTYHT) for RHK completely abolished the association of the 30-kDa domain of protein 4.1 with peptide P12a (data not shown).

Figure 3: Effects of the peptides P12c and P12m on the binding of the I-labeled 30-kDa domain of protein 4.1 to the peptide P23. The binding of the I-labeled 30-kDa domain to peptide P23 was measured using a sedimentation assay in the presence of increasing amounts of peptides P12c and P12m. The half-maximal inhibition of binding was achieved at 20 µM concentration of the peptide P12m.


To identify the binding site for protein 4.1 on p55, defined cDNA constructs of human erythroid p55 were expressed in bacteria as glutathione S-transferase fusion proteins (Fig. 4A). The binding of the I-labeled 30-kDa domain of protein 4.1 to these constructs was measured by a blot overlay assay. The p55 constructs D1, D2, and D3 did not bind to the 30-kDa domain of protein 4.1 (Fig. 4B). The I-labeled 30-kDa domain of protein 4.1 bound specifically to the D4 construct which contains the guanylate kinase domain, the linker region, and the SH3 motif of p55 (Fig. 4A). The p55 constructs which contained either the guanylate kinase domain (D3) or the SH3 motif in isolation did not bind to the I-labeled 30-kDa domain of protein 4.1 (Fig. 4A, data not shown).

The results obtained by the blot overlay assay (Fig. 4B) were quantified using a sedimentation assay (Table 2). Again, the I-labeled 30-kDa domain of protein 4.1 specifically bound to the construct D4. The binding capacity of the D4 was almost half that of the full-length p55, suggesting that the N-terminal undefined domain may be required to reconstitute the full binding capacity of intact p55. Alternatively, the undefined domain may contain a low affinity protein 4.1 binding site which we could not detect using the D1 construct under the given binding conditions. These results strongly suggest that the protein 4.1 binds to p55 within the region located between the SH3 and guanylate kinase domains.

To confirm that the protein 4.1 binding site is located between the SH3 and guanylate kinase domains of p55, a GST fusion protein was produced that contained the 39 amino acids of this region (see Fig. 6, construct D5). The I-labeled 30-kDa domain of protein 4.1 specifically bound to the GST fusion protein (Fig. 5, lane 4), and this binding was completely abolished in the presence of a 10-fold molar excess of the unlabeled 30-kDa domain. It is of interest to note that the partial degradation of the GST fusion protein produced a higher mobility band, as shown in Fig. 5(lane 3), and this truncated fusion protein did not bind to 30-kDa domain of protein 4.1 (Fig. 5). Since both intact and truncated fusion proteins are recognized by anti-peptide polyclonal antibodies raised against the N-terminal half of the binding sequence (not shown), the degradation must have occurred at the C-terminal end of the fusion protein. The lack of protein 4.1 binding to the truncated fusion protein therefore suggests that residues located in the C-terminal half of the 39-amino acid region may be necessary for the binding of p55 to protein 4.1 (Fig. 6). The C-terminal half of the binding region is characterized by a cluster of lysine residues, which is also present in other MAGUKs that contain the conserved region, suggesting that a positively charged surface may mediate the binding of MAGUKs to protein 4.1.

DISCUSSION

Our identification of the sequence RYMYRHKGTYHT as the binding site in glycophorin C for protein 4.1 is in agreement with the observations of Jons and Drenkhahn (26) showing that a positively charged sequence of band 3 mediates its binding to the N-terminal domain of protein 4.1. In the same study, it was shown that the purified protein 4.1 failed to bind to the stripped erythrocyte membrane vesicles in the presence of a peptide IRRRY. This observation again suggested the participation of positively charged residues in the binding of protein 4.1 to the membrane vesicles which contain both band 3 and glycophorins(26) . While our studies were in progress, Hemming et.al. (27) showed that intact protein 4.1 binds to a 17-amino acid segment located within the cytoplasmic domain of glycophorin C. This 17-amino acid segment includes the 12-amino acid segment which we have identified in this study as the binding site for the 30-kDa domain of protein 4.1.

The determination of the protein 4.1 binding site on glycophorin C begins to explain how protein 4.1 and some of its homologues may interact with the plasma membrane via their conserved N-terminal 30-kDa domains. This result is consistent with the observation that ezrin, a member of the protein 4.1 superfamily, binds to the inner face of the fibroblast membrane via its N-terminal 30-kDa domain(28) . Recently, it has been shown that ezrin, moesin, and radixin associate with CD44, a 140-kDa integral membrane protein of broad distribution(29) , and that ezrin precisely colocalizes with CD43 in the cleavage furrow of dividing leukocytes(30) . Although CD44, CD43, and glycophorin C do not share any significant sequence similarity, all three are heavily glycosylated membrane proteins(29) , and may represent alternative ways by which 4.1-related proteins associate with the membrane. Among red cell glycophorins, only glycophorin C is expressed in non-erythroid cells(13) , and we have found abundant expression of glycophorin C mRNA in human brain (data not shown). Whether other members of the protein 4.1 superfamily including protein tyrosine phosphatases PTP-MEG and PTP H1, and the brain tumor suppressor protein merlin/schwannomin, use a mechanism mediated by protein 4.1 to interact with the plasma membrane remains to be determined(31, 32, 33) .

The results shown in Fig. 4Fig. 5Fig. 6identify a novel sequence located between the SH3 motif and the guanylate kinase domain of p55 as the binding site for the 30-kDa domain of protein 4.1. A characteristic of this 39-amino acid sequence is the presence of a cluster of lysine residues located in the C-terminal half of the protein (Fig. 6). The identification of the protein 4.1 binding site on human erythroid p55 suggests a mechanism by which members of the p55 superfamily may interact with the membrane cytoskeleton via protein 4.1. These results are consistent with the observation that p55 and its non-erythroid homologues are associated with the plasma membrane(22, 34) .

The positively charged character and location of the protein 4.1 binding motif of p55 is conserved in both Dlg and Hdlg. The Drosophila protein is localized at the septate junctions (16) (Fig. 6B) as is a Drosophila homologue of protein 4.1(35) , and we have demonstrated that both Hdlg and protein 4.1 localize at regions of cell-cell contact in the human breast carcinoma cell line, MCF-7(34) . Thus, protein 4.1 may provide a membrane localization site for Dlg homologues (35) and other related MAGUKs. Similar, polybasic domains of Ras and nuclear lamins have previously been shown to direct these proteins to the plasma and nuclear membranes, respectively(36, 37) . In fact, immunoreactive isoforms of protein 4.1 have been identified in the nuclei of several eukaryotic cells(38) . Whether the polybasic domains of p55 and its homologues mediate the targeting of these proteins to their respective membrane locales will be of considerable significance. Although our results identify the respective binding sites in vitro, it will now be important to use heterologous expression studies in eukaryotic cells to demonstrate these interactions in vivo.

FOOTNOTES

*
This work was supported by the National Institutes of Health Grants HL51445, HL37462 (to A. H. C.), and HL17411 (to D. B.). 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.

§
To whom correspondence should be addressed: Dept. of Biomedical Research, ACH4, St. Elizabeth's Medical Center, 736 Cambridge St., Boston, MA 02135. Tel. 617-789-3118; Fax: 617-789-3111.

(^1)
The abbreviations used are: MAGUKs, membrane-associated guanylate kinase homologues; GST, glutathione S-transferase; SH3, src homology 3; Dlg, Drosophila discs-large tumor suppressor protein; Hdlg, human homologue of the discs-large tumor suppressor protein; ELISA, enzyme-linked immunosorbent assay.

ACKNOWLEDGEMENTS

The acronym MAGUK was originally suggested by Drs. D. Woods and P. Bryant of the University of California, Irvine. We thank Dr. A. Azim of the Department of Biomedical Research for helpful discussions and Dr. Aida Metzenberg for providing anti-peptide antibodies against p55.

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