©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Homophilic Interactions Mediated by Receptor Tyrosine Phosphatases and
A CRITICAL ROLE FOR THE NOVEL EXTRACELLULAR MAM DOMAIN (*)

Gerben C. M. Zondag (1), Gregory M. Koningstein (1), Ying-Ping Jiang (2), Jan Sap (2), Wouter H. Moolenaar (1)(§), Martijn F. B. G. Gebbink (1)

From the (1)Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands and the (2)Department of Pharmacology, New York University Medical Center, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The receptor-like protein tyrosine phosphatases (RPTP) µ and RPTP have a modular ectodomain consisting of four fibronectin type III-like repeats, a single Ig-like domain, and a newly identified N-terminal MAM domain. The function of the latter module, which comprises about 160 amino acids and is found in diverse transmembrane proteins, is not known. We previously reported that both RPTPµ and RPTP can mediate homophilic cell interactions when expressed in insect cells. Here we show that despite their striking structural similarity, RPTPµ and RPTP fail to interact in a heterophilic manner. To examine the role of the MAM domain in homophilic binding, we expressed a mutant RPTPµ lacking the MAM domain in insect Sf9 cells. Truncated RPTPµ is properly expressed at the cell surface but fails to promote cell-cell adhesion. Homophilic cell adhesion is fully restored in a chimeric RPTPµ molecule containing the MAM domain of RPTP. However, this chimeric RPTPµ does not interact with either RPTPµ or RPTP. These results indicate that the MAM domain of RPTPµ and RPTP is essential for homophilic cell-cell interaction and helps determine the specificity of these interactions.


INTRODUCTION

Receptor-like protein tyrosine phosphatases (RPTPs)()are transmembrane proteins that are thought to transduce external signals by dephosphorylating phosphotyrosine residues on cytosolic substrates. As such, they can be considered as the counterparts of the receptor tyrosine kinases. Intracellularly, RPTPs contain one or two conserved catalytic domains, but the extracellular domains show great structural diversity. Based on this diversity, the RPTPs were originally classified into four subtypes(1, 2) . Later, a fifth subtype was identified that contains an N-terminal domain homologous to carbonic anhydrase(3) . The ``type II'' RPTPs are characterized by an ectodomain containing one to three Ig domains and several FN III-like repeats, which resembles the structure of cell adhesion molecules of the Ig superfamily.

We and others recently showed that two closely related members of the type II RPTPs, RPTPµ and RPTP, can mediate homophilic cell-cell interaction when expressed in non-adherent insect cells(4, 5, 6) , suggesting that these receptors serve a normal physiological function in cell-to-cell signaling. We also showed that the intrinsic catalytic activity is not required for homophilic binding.

To date, little is known about the structural features of RPTPµ and RPTP that are important for cell-cell interaction. In addition to an Ig domain and four FN III-like repeats, the ectodomain of both receptors contains a recently identified N-terminal MAM domain of unknown function(7) . This MAM domain (meprin/A5/µ), which comprises about 160 amino acids including four conserved cysteine residues, is also present in the unrelated transmembrane proteins meprin and A5 glycoprotein(7) .

In the present study we addressed two major questions. First, given their structural similarity, can RPTPµ and RPTP interact with each other? In fact, some molecules of the Ig superfamily can mediate both homophilic and heterophilic interactions(8) . Second, what is the role of the MAM domain in mediating cell-cell interactions? Our results show that despite their structural similarity, RPTPµ and RPTP fail to interact with each other, indicating a high degree of binding specificity for both receptors. Furthermore, we show that the MAM domain is essential for RPTPµ-mediated homophilic binding and confers binding specificity.


EXPERIMENTAL PROCEDURES

Plasmid Construction

Cloning of full-length human RPTPµ and the C-terminally truncated ExJ mutant have been described (4). Plasmid pVL-RPTP was constructed by cloning a 3.5-kb HpaI-EcoRI fragment from pK(9) into the SmaI and EcoRI sites of pVL1393 (PharMingen), followed by insertion of the 1.1-kb EcoRI fragment from pK containing the remaining C-terminal RPTP sequences. Full-length cDNA coding for mouse RPTPµ was cloned from pMT2 mFL1 (10) into pVL1392 using NotI-XbaI digestion to generate pVL-mFLµ. The MAM domain of RPTPµ was deleted by a two-step PCR amplification, which preserved the Kozak and signal sequences. With pBS-hFL (4) serving as template, primers M1 (5` GGTGGCCGCGTCCATCTG 3`) and M2 (5` GTGAGGAGTCCTGCCACCTGAGAACGTCTC 3`) were used to amplify the sequence upstream of the MAM domain and primers M3 (5` TTCTCAGGTGGCAGGACTCCTCACTTCCTG 3`) and M4 (5` CTTTCCAGCATCTCGTTTGG 3`) to amplify the downstream sequence up to the BstEII site at position 705. Both PCR products were mixed and used as a template in a second PCR using primers M1 and M4. The resulting product lacking the MAM domain sequences was digested with SacI/BstEII and cloned into pBS-hFL digested with the same enzymes. The 2.3-kb BstEII fragment, which was lost from the coding region in the last step, was cloned back, resulting in pBS-FLMAM. By replacing the 2.5-kb BglII fragments of pVL-ExJ with the 2.0-kb BamHI-BglII fragment of pBS-hFLMAM the baculotransfer plasmid pVL-ExJMAM was generated. Exchange of the RPTPµ MAM domain with that of RPTP was also approached by a two-step PCR procedure. The location of the fusion between RPTP and RPTPµ sequences was chosen in the conserved region Pro-His-Phe-Leu-Arg directly following the MAM domain at amino acid positions 195-199 in RPTP and 186-190 in RPTPµ. Primers S1 (5` CGGAGATCTAACCGCCATGGATGTGG 3`) and S2 (5` TTCTGAATTCGCAGGAAATGAGGAGATTTATCG 3`) were used on RPTP cDNA to amplify a 610-bp fragment ranging from the start codon up to and including the RPTP MAM domain. Using primers S3 (5` CTCCTCATTTCCTGCGAATTCAGAATGTGG 3`) and S4 (5` CCTTCTAGAATTCTTTAACTACCAACTC 3`) a 348-bp fragment was amplified from the RPTPµ cDNA (bp 553-850) containing sequences downstream of the MAM domain. Both fragments were mixed and amplified in a second PCR using primers S1 and S4 to yield a hybrid product. This hybrid fragment was cloned into a BglII-XbaI-digested pVL1392 vector resulting in pVL-MAM-µIg. An internal BstEII site in the pVL1392 vector was used to remove a 3.8-kb BstEII fragment from pVL-MAM-µIg containing the polyhedrin promoter region fused to the amplified product. This fragment was subsequently cloned into pVL-ExJ digested with BstEII. The resulting chimeric construct was named pVL-ExJMAM. The cDNA constructs were sequenced to verify the desired mutations.

Cell Culture, Infections, and Recombinant Baculovirus Generation

The Spodoptera frugiperda cell line Sf9 was propagated in supplemented Grace's insect medium with 10% fetal calf serum, 50 IU/ml penicillin, and 50 µg/ml streptomycin (Life Technologies, Inc.). Generation of baculovirus encoding full-length human RPTPµ and C-terminally truncated RPTPµ (ExJ, lacking both catalytic domains) has been described(4) . Recombinant baculoviruses encoding the full-length mouse RPTPµ and RPTP, the deletion mutant ExJMAM, and the chimeric ExJMAM protein were generated with the corresponding pVL constructs using the BaculoGold transfection kit (PharMingen) according to the manufacturer's instructions. For infection, subconfluent monolayers of Sf9 cells were inoculated in a small volume of recombinant baculovirus with a multiplicity of infection of 10 or higher. After a 1-h incubation at 26 °C, fresh medium was added, and cells were further incubated for 2 days at 26 °C.

Antibodies

Monoclonal antibody 3G4 is directed to the fibronectin type III-like repeats of human RPTPµ(4) . Polyclonal serum 116 is directed to part of the MAM domain of RPTP(6) . Monoclonal antibody 4B7 is part of a panel of monoclonal antibodies, directed to the extracellular domain of human RPTPµ.()

Protein Analysis

Two days after infection, Sf9 cells were collected and lysed on ice in Nonidet P-40 lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Nonidet P-40, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 2.5 µg/ml aprotinin) for 20 min. After pelleting of nuclei and cell debris, the supernatant was mixed with 4 sample buffer (250 mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 20% -mercaptoethanol) and denatured for 5 min at 95 °C. Total lysate was separated on 7.5% SDS-polyacrylamide (11) and transferred onto nitrocellulose(12) . After blocking in 5% nonfat dry milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20), blots were probed with first antibody, washed in TBST, incubated with peroxidase-conjugated second antibody, and washed again. Blots were developed using enhanced chemiluminescence (ECL, Amersham Corp.).

Cell Aggregation Assays

Two days after infection with the appropriate recombinant baculovirus, infected Sf9 cells were harvested and resuspended in 30-mm plastic dishes at 10 cells/ml of Grace's medium with 10% FCS and antibiotics. Dishes were rotated on an orbital shaker for 2 h at room temperature to allow cell aggregation. For mixing experiments cells were incubated for 60 min at 26 °C with either 50 µM 5-(and 6-)-carboxy-2`,7`-dichlorofluorescein diacetate succinimidyl ester (CFDA, Molecular Probes Inc.) or 20 µM DiI (1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate, Molecular Probes Inc.). After labeling cells were washed twice in 1 ml of Grace's medium with 10% FCS, mixed, and resuspended in dishes and allowed to aggregate with gentle rotation. Aggregate formation and composition were examined by confocal microscopy on a Bio-Rad model MRC-600 instrument.

Immunofluorescence Microscopy

Approximately 4 10 Sf9 cells were harvested 2 days after viral infection, washed once, and resuspended in 0.5 ml of cold Grace's medium supplemented with 10% FCS, antibiotics, and 0.02% NaN. Purified monoclonal antibody 4B7 was added to 1 µg/ml, and cells were incubated for 60 min at 4 °C after which they were washed once in 1 ml of cold medium. Cells were then resuspended in 200 µl of fresh medium and incubated with fluorescein isothiocyanate (FITC)-conjugated goat F(ab`) anti-mouse IgG (Zymed Laboratories Inc.) for 30 min at 4 °C. After two subsequent washes in 1 ml of medium, fluorescence was examined by confocal microscopy. Recombinant baculovirus encoding a chimeric EGFR/RPTPµ receptor (4) in which the extracellular domain of RPTPµ is replaced by that of the EGF receptor was used for control staining.

Sequence Alignments

DNA and protein sequences were retrieved from the SWISSPROT and GenBank/EMBL data bases using the Genetic Computer Group software package(13) . The similarity between RPTPµ and RPTP was calculated for each domain separately, using the GCG program BESTFIT.


RESULTS AND DISCUSSION

Cell-Cell Interaction Mediated by RPTPµ and RPTP

The predicted structures of RPTPµ and RPTP are remarkably similar, with individual domains being conserved in both position and sequence (Fig. 1A). The extreme N terminus of both RPTPµ and RPTP contains the MAM domain, sharing 68.6% similarity, followed by a single Ig-like domain and four fibronectin type III-like repeats. The high structural similarity between RPTPµ and RPTP suggests that both receptors may interact in a heterophilic manner. To test this possibility we used the baculovirus-Sf9 insect cell system for assaying cell-cell interaction, since Sf9 cells are normally non-adherent. Recombinant baculovirus was generated containing the full-length mouse RPTP cDNA. To exclude the possibility that the difference in species plays a role in possible heterophilic interactions between RPTPµ and RPTP, we also generated recombinant baculovirus encoding the mouse homolog of RPTPµ. As expected, cells infected with either RPTPµ or RPTP aggregate into large clusters. To study heterophilic interactions, Sf9 cells expressing mouse RPTPµ or RPTP were fluorescently stained with CFDA (green) or DiI (red), respectively. Both cell populations were mixed, resuspended, and then allowed to aggregate. After 2 h of gentle agitation, distinct cell clusters of either green RPTPµ cells or red RPTP cells are visible (Fig. 1B, upperpanel). There is no sign of mixed cell aggregates consisting of both green and red cells. On the other hand, when cells expressing RPTPµ were stained with either CFDA or DiI and then mixed, all cell aggregates consist of a mixture of green and red cells (Fig. 1B, lowerpanel). Hybrid aggregates were also observed when human and mouse RPTPµ-expressing cells were mixed. It thus appears that RPTPµ and RPTP cannot undergo heterophilic interactions, implying that the homophilic binding properties of these receptors are highly specific.


Figure 1: Cell-cell interaction mediated by RPTPµ and RPTP. A, schematic diagram showing the modular structure of RPTPµ and RPTP. Amino acid similarities between corresponding domains were calculated using the BESTFIT program and are given in percentage. B, mixed cell aggregation assays showing homophilic but no heterophilic interaction. Upper panel, Sf9 cells expressing mouse RPTPµ (stained green with CFDA) and RPTP-expressing cells (stained red with DiI) were 1:1 mixed and then resuspended and allowed to aggregate. Note the lack of heterophilic interaction between green and red cells. Lower panel, control experiment in which RPTPµ-expressing Sf9 cells were stained either green or red, 1:1 mixed, and then resuspended and allowed to aggregate. After 2 h of gentle rotation, cell aggregation was analyzed by confocal microscopy. Yellowcolor (lowerpanel) results from the superposition of red and green fluorescence.



Role of the MAM Domain in Cell-Cell Interaction

The MAM domain is present not only in RPTPµ and RPTP (9, 10) but also in the unrelated transmembrane proteins meprin and Xenopus A5 antigen(7) . The MAM domain is about 160 amino acids long and is characterized by four conserved cysteine residues, possibly involved in disulfide bridging, and two conserved regions with no similarity to known proteins. Furthermore, the MAM domain sequence contains several aromatic and hydrophobic residues at conserved positions and predicts the presence of a -sheet(7) . Besides an extracellular modular structure, proteins bearing the MAM domain have seemingly little in common. Meprins are zinc-dependent metalloproteases (14, 15) and the A5 protein is believed to be involved in neurite outgrowth or axonal guidance(16, 17) , whereas RPTPµ and RPTP are thought to signal cell-cell interaction.

To investigate the importance of the MAM domain in mediating homophilic interactions, we deleted the entire MAM domain (157 amino acids including three potential N-glycosylation sites) from the C-terminally truncated RPTPµ construct, termed ExJ; the ExJ protein, which lacks both catalytic domains, has adhesive properties indistinguishable from those of full-length RPTPµ(4) . MAM-deficient ExJ was termed ExJMAM (Fig. 2A). To ensure proper transport to the cell surface, the signal peptide sequences were preserved. As predicted, expression of the ExJMAM construct in Sf9 cells results in a protein of about 115 kDa (Fig. 2B). Correct surface expression of the ExJMAM protein was confirmed by immunofluorescence using a monoclonal antibody against the RPTPµ ectodomain (Fig. 2C). However, the deletion mutant failed to mediate any detectable cell aggregation as shown in Fig. 3A. Moreover, cells expressing ExJMAM did not interact with cells expressing full-length RPTPµ. These results suggest a critical role for the MAM domain in mediating homophilic binding and indicate that the Ig- and FN III-like domains are not sufficient for homophilic binding.


Figure 2: Expression of various forms of RPTPµ and RPTP and cell aggregation in Sf9 cells. A, schematic diagram showing the structure of the various constructs used. The extent of Sf9 cell adhesion was measured microscopically as described under ``Experimental Procedures'' and in Ref. 4 (+, full aggregation; -, no aggregation). PTP, protein tyrosine phosphatase domain; B, expression of the various constructs in Sf9 cells. Cells infected with recombinant baculovirus were lysed 48 h after infection, and total protein was analyzed by Western blotting. The extracellular domain of both human (h) and mouse (m) RPTPµ was detected using monoclonal antibody 3G4 against the FN III-like repeats; the MAM domain of mouse RPTP was detected using polyclonal antibody 116. The predicted molecular masses for full-length RPTPµ and RPTP are 195 and 140 kDa for ExJ and chimeric ExJMAM and 115 kDa for truncated ExJMAM. Additional bands observed with ExJ, mouse RPTPµ and RPTP probably represent breakdown products. The positions of molecular mass markers are indicated. C, immunofluorescence analysis of cell surface expression. Sf9 cells expressing full-length RPTPµ (upperpanel), ExJMAM (middlepanel), and the EGFR/RPTPµ chimera (lowerpanel) were incubated with monoclonal antibody 4B7 to the RPTPµ ectodomain, stained with FITC-conjugated second antibody, and then visualized by confocal microscopy. Sf9 cells infected with the EGFR/RPTPµ chimera (lowerpanel) served as a control for specificity of staining.




Figure 3: Role of the MAM domain in aggregation. A, Sf9 cells expressing the ExJMAM protein (stained green with CFDA) were 1:1 mixed with cells expressing full-length RPTPµ (stained red with DiI) and then resuspended and allowed to aggregate for 2 h. Cell aggregation was monitored by confocal microscopy. Note that ExJMAM does not mediate any homophilic interactions nor does it interact heterophilically with full-length RPTPµ. B, two populations of ExJMAM-expressing Sf9 cells were stained either green or red, 1:1 mixed, and allowed to aggregate. The yellowcolor results from the superposition of green and red fluorescence. C, mixed aggregation assay using a 1:1 mixture of cells expressing chimeric ExJMAM (stained green) and cells expressing full-length RPTPµ (stained red). D, mixed aggregation assays using a 1:1 mixture of cells expressing ExJMAM (stained green) and cells expressing full-length RPTP (stained red).



Characterization of a Chimeric RPTPµ/RPTP Molecule

We next sought to replace the MAM domain of RPTPµ with that of RPTP to examine if aggregation could be restored and to assign a functional role to the MAM domain. A chimeric cDNA was constructed in which the signal peptide and MAM domain of the C-terminally truncated ExJ construct were replaced by those of RPTP (ExJMAM, Fig. 2A). As expected, the size of the chimeric ExJMAM protein is similar to that of the ExJ protein (Fig. 2B). Immunofluorescence analysis revealed that the ExJMAM protein was present on the cell surface of infected cells (not shown). When cells expressing the chimeric protein were stained fluorescently green or red and allowed to aggregate, large clusters consisting of both green and red cells were readily observed (Fig. 3B). In other words, the MAM domain of RPTP can functionally substitute for that of RPTPµ. Thus, the MAM domains of RPTPµ and RPTP can be considered as independent modules that are essential for homophilic binding.

Having established that the chimeric ExJMAM receptor mediates homophilic interaction, we next examined the binding specificity of the chimeric molecule toward either RPTPµ or RPTP. Cells expressing the chimeric ExJMAM protein and those expressing expressing full-length RPTPµ were stained, mixed, and allowed to interact. After 2 h of mixing by gentle rotation, aggregates had formed, which consisted exclusively of cells expressing either chimeric ExJMAM or full-length RPTPµ, with no sign of heterophilic interaction (Fig. 3C). We also mixed cells expressing chimeric ExJMAM with cells expressing full-length RPTP. This also resulted in sorting out of cell clusters expressing either the chimera or RPTP, with again no sign of cross-interaction (Fig. 3D). This suggests that the MAM domain plays a critical role in determining the specificity of homophilic binding. Furthermore, it follows that the MAM domain is necessary but not sufficient to mediate homophilic binding. Additional interactions in the Ig- and/or FN III-like domains must therefore also participate in homophilic binding.

Concluding Remarks

In conclusion, we have shown that (i) RPTPµ and its close relative RPTP interact in a homophilic but not heterophilic manner and (ii) the MAM domain is necessary but not sufficient for mediating homophilic interaction. That both receptors fail to bind to each other suggests that cell-cell interactions mediated by RPTPµ and RPTP are highly specific and strictly homophilic in nature. As the tissue distribution of both receptors appears to be different (i.e. RPTPµ expression is highest in lung while RPTP is predominantly expressed in kidney(9, 10) ), it seems likely that RPTPµ and RPTP mediate similar homophilic interactions (and subsequent signaling events) in different cell types.

Our results with the MAM deletion mutants imply that the MAM domain can be considered as an independent module that can be exchanged between related receptors without loss of primary function. Based on our results obtained with the chimeric receptor, we propose that the MAM domain may have a ``fine tuning'' function by contributing to the specificity of homophilic binding. Deletion of the MAM domain abolishes homophilic interaction indicating that the Ig-like and/or FN III-like domains are not sufficient for mediating homophilic binding. This is substantiated by the lack of interaction between the MAM deletion mutant and full-length RPTPµ (Fig. 3A).

While this paper was in preparation, Brady-Kalnay and Tonks (18) suggested that the Ig domain of RPTPµ is both necessary and sufficient for homophilic binding, with no apparent role for the MAM domain, as assessed under non-physiological conditions using ``Covaspheres'' coated with various soluble fragments of the RPTPµ ectodomain. However, as the authors themselves point out(18) , their in vitro results do not exclude the possibility that the MAM domain is critical for homophilic binding in vivo. The apparent discrepancy with our findings is likely due to the very different assay conditions used in both studies (coated beads versus intact cells). Furthermore, the protein fragments used by Brady-Kalnay and Tonks (18) are expressed in either Escherichia coli or insect cells using cDNA constructs that lack appropriate signal peptide sequences; this implies that the encoded proteins may well be misfolded or otherwise inappropriately processed.

The present results strongly suggest that the MAM domain has a general role in mediating protein-protein interaction. Thus, in meprins the MAM domain may mediate the observed dimerization or oligomerization of meprin subunits(15, 19) . The MAM-containing A5 protein, when transfected into L-cells, can promote neurite outgrowth of A5-expressing neurons but not of A5-deficient neurons(17) , again consistent with the MAM domain being involved in homophilic binding. Monoclonal antibodies against the MAM domain should help to further elucidate its biological function and importance. Such studies are currently under way.


FOOTNOTES

*
This work was supported by the Dutch Cancer Society. 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. Tel.: 31-20-512-1971; Fax: 31-20-512-1989.

The abbreviations used are: RPTP, receptor-like protein tyrosine phosphatase; bp, base pair(s); kb, kilobase(s); PCR, polymerase chain reaction; FCS, fetal calf serum; CFDA, 5-(and 6-)-carboxyl-2`,7`-dichlorofluorescein diacetate succinimidyl ester; DiI, 1,1`-dioctadecyl-3,3,3`,3`-tetramethylindocarbocyanine perchlorate; FITC, fluorescein isothiocyanate; FN III, fibronectin type III; EGFR, epidermal growth factor receptor.

M. Gebbink, G. Zondag, G. Koningstein, E. Feiken, R. Wubbolts, and W. Moolenaar, submitted for publication.


ACKNOWLEDGEMENTS

We thank Lauran Oomen for assistance with confocal microscopy and image processing.


REFERENCES
  1. Fischer, E. H., Charbonneau, H., and Tonks, N. K.(1991) Science253, 401-406 [Medline] [Order article via Infotrieve]
  2. Charbonneau, H., and Tonks, N. K.(1992) Annu. Rev. Cell Biol.8, 463-493 [CrossRef]
  3. Barnea, G., Silvennoinen, O., Shaanan, B., Honegger, A. M., Canoll, P. D., D'Eustachio, P., Morse, B., Levy, J. B., LaForgia, S., Huebner, K., Musacchio, J. M., Sap, J., and Schlessinger, J.(1993) Mol. Cell. Biol.13, 1497-1506 [Abstract]
  4. Gebbink, M. F. B. G., Zondag, G. C. M., Wubbolts, R. W., Beijersbergen, R. L., van Etten, I., and Moolenaar, W. H. (1993a) J. Biol. Chem.268, 16101-16104 [Abstract/Free Full Text]
  5. Brady-Kalnay, S. M., Flint, A. J., and Tonks, N. K.(1993) J. Cell Biol.122, 961-972 [Abstract]
  6. Sap, J., Jiang, Y. P., Friedlander, D., Grumet, M., and Schlessinger, J.(1994) Mol. Cell. Biol.14, 1-9 [Abstract]
  7. Beckmann, G., and Bork, P.(1993) Trends Biochem. Sci.18, 40-41 [CrossRef][Medline] [Order article via Infotrieve]
  8. Sonderegger, P., and Rathjen, F. G.(1992) J. Cell Biol.119, 1387-1394 [Medline] [Order article via Infotrieve]
  9. Jiang, Y.-P., Wang, H., D'Eustachio, J. M., Musacchio, J. M., Schlessinger, J., and Sap, J.(1993) Mol. Cell. Biol.13, 2942-2951 [Abstract]
  10. Gebbink, M. F. B. G., van Etten, I., Hateboer, G., Suijkerbuijk, R., Beijersbergen, R. L., Geurts van Kessel, A., and Moolenaar, W. H. (1991) FEBS Lett.290, 123-130 [CrossRef][Medline] [Order article via Infotrieve]
  11. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  12. Towbin, H., Staehelin, T., and Gordon, J.(1979) Proc. Natl. Acad. Sci. U. S. A.76, 4350-4354 [Abstract]
  13. Devereux, J. P., Haeberli, P., and Smithies O.(1989) Nucleic Acids Res.12, 387-396 [Abstract]
  14. Dumermuth, E., and Sterchi, E. E.(1991) J. Biol. Chem.266, 21381-21385 [Abstract/Free Full Text]
  15. Jiang, W., Gorbea, C. M., Flannery, A. V., Beynon, R. J., Grant, G. A., and Bond, J. S.(1992) J. Biol. Chem.267, 9185-9193 [Abstract/Free Full Text]
  16. Takagi, S., Hirata, T., Agata, K., Mochii, M., Eguchi, G., and Fujisawa, H.(1991) Neuron7, 295-307 [Medline] [Order article via Infotrieve]
  17. Hirata, T., Takagi, S., and Fujisawa, H.(1993) Neurosci. Res.17, 159-169 [Medline] [Order article via Infotrieve]
  18. Brady-Kalnay, S. M., and Tonks, N. K.(1994) J. Biol. Chem.269, 28472-28477 [Abstract/Free Full Text]
  19. Craig, S. S., Reckelhoff, J. F., and Bond, J. S.(1987) Am. J. Physiol.253, C535-C540

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