Correspondence to: Catherine J. Pallen, Institute for Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore. Tel:65-874-3742 Fax:65-779-1117
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Abstract |
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Glycosyl phosphatidylinositol (GPI)linked receptors and receptor protein tyrosine phosphatases (RPTPs), both play key roles in nervous system development, although the molecular mechanisms are largely unknown. Despite lacking a transmembrane domain, GPI receptors can recruit intracellular src family tyrosine kinases to receptor complexes. Few ligands for the extracellular regions of RPTPs are known, relegating most to the status of orphan receptors. We demonstrate that PTP, an RPTP that dephosphorylates and activates src family kinases, forms a novel membrane-spanning complex with the neuronal GPI-anchored receptor contactin. PTP
and contactin associate in a lateral (cis) complex mediated through the extracellular region of PTP
. This complex is stable to isolation from brain lysates or transfected cells through immunoprecipitation and to antibody-induced coclustering of PTP
and contactin within cells. This is the first demonstration of a receptor PTP in a cis configuration with another cell surface receptor, suggesting an additional mode for regulation of a PTP. The transmembrane and catalytic nature of PTP
indicate that it likely forms the transducing element of the complex, and we postulate that the role of contactin is to assemble a phosphorylation-competent system at the cell surface, conferring a dynamic signal transduction capability to the recognition element.
Key Words:
PTP, tyrosine phosphatase, glycosyl phosphatidylinositol, neural signal transduction
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Introduction |
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THE neural cell adhesion molecule contactin (F3/F11) contains Ig-like domains and FN-III repeats, and is attached to the cell surface through a glycosyl phosphatidylinositol (GPI)1 anchor (
As many signal transduction pathways are initiated through activation of intrinsic or associated receptor tyrosine kinase activity, the association of fyn with contactin may be central to contactin signaling. Several protein tyrosine phosphatases (PTPs) can activate src family kinases in various systems, suggesting a PTP to be a candidate component of a contactin signaling complex.
The transmembrane nature of the receptor-like PTPs (RPTPs) indicates that they transduce extracellular signals, although few such signals/ligands are known. The identification of extracellular ligands for RPTPs could provide critical insights into the specific functions and regulation of these enzymes. PTP is one such RPTP for which a ligand is sought. The short glycosylated extracellular region of PTP
, unlike many other RPTPs, lacks known adhesion motifs. It is linked via the transmembrane segment to the intracellular region that contains two homologous catalytic domains, both of which have intrinsic phosphatase activity (
indicate that it may, among other things, be involved in neuronal signaling. PTP
is particularly abundant in the brain and implicated in mediating neuronal differentiation, cell proliferation, and transformation, by its ability to activate the tyrosine kinase src (
also associates with, dephosphorylates, and activates brain fyn (
have reduced activities of brain src and fyn, demonstrating that PTP
is a positive physiological regulator of these kinases (
The shared and complementary features of PTP and contactin suggested that they may form a signaling complex. Both proteins are expressed in some of the same neuronal cell types such as migrating cerebellar granule cell neurons (
and fyn raises the possibility that PTP
provides a transmembrane link between contactin and fyn. Furthermore, the ability of PTP
to activate fyn indicates that PTP
could thereby transduce a signal originating from the extracellular engagement of contactin. We demonstrate that PTP
and contactin associate and define certain requirements for interaction. These findings indicate that contactin is a novel extracellular partner of PTP
and that this complex may regulate aspects of neuronal development.
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Materials and Methods |
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Expression Plasmids
Numbering of the human PTP amino acid sequence is according to
, VSVG-tagged PTP
, fyn, and CD45 have been described (
-neo, the PCR-amplified region of human src cDNA encoding the NH2-terminal myristylated sequence MGSNKSKPKDASQ was cloned into pGEM-T (Promega Corp.), and the reverse orientation was selected with the multiple cloning NotI site at the 5' end of the myristylation signal. A NotI-XhoI fragment was excised and cloned into pXJ41-neo, creating pXJ41-myr-neo. A PCR fragment of PTP
encoding the entire intracellular region (amino acids 148774), and flanked with engineered SalI sites, was inserted in-frame into XhoI-cut pXJ41-myr-neo. To construct pXJ41-PTP
/CD45-neo encoding the chimeric RPTP, a region of the VSVGPTP
cDNA encoding the signal peptide and amino acids 1121 of PTP
was amplified by PCR using forward and reverse primers with engineered NotI and BglII sites, respectively. This NotI-BglII fragment was inserted into a pXJ41-neo intermediate vector. A CD45 cDNA fragment encoding 35 extracellular juxtamembrane amino acids, the transmembrane, and the entire intracellular regions was released from pXJ41-CD45-Hy with BglII and inserted into the intermediate vector.
Cell Culture and Transient Transfections
COS-1 cells were maintained and transiently transfected as described (
COS-7 cells cultured on 1 ml polylysine or alcian bluecoated glass 12-mm-diam coverslips were processed for immunocytochemistry 2448 h after transfection. Images were taken using a Zeiss Axiovert 100M equipped with a Hamamatsu C5810 3 color CCD cooled camera. Images were processed directly with Adobe Photoshop.
Antibody-mediated Copatching of Contactin and PTP in Transfected Cells
Copatching of contactin or VSVG-PTP was performed on COS-7 cells 2448 h after transfection as described (
(see below) were used complementary to the mAbs, together with goat antirabbit antibodies labeled with FITC or RITC (Tago).
Western Blots and Immunoprecipitation
Six embryonic chick brains (15-d-old) were mechanically suspended in 30 ml of ice-cold PBS buffer, filtered through a nylon mesh, homogenized by 10 strokes in a glass Dounce homogenizer, and washed three times in PBS. Chick brain cells or transfected COS cells were lysed in 10 mM Tris-Cl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% Brij-96, 20 µg/ml aprotinin, and 2 mM PMSF, and the lysates were clarified by centrifugation. Antibodies toward VSVG (Sigma Chemical Co.), contactin (4D1) ( (antiserum 2205, raised against PTP
-D1), and NCAM (Chemicon International, Inc.) were used for immunoprecipitation and/or immunoblotting.
Phosphatase Assays
The phosphatase activity of 5 µl immunoprecipitate toward 2 µM phosphotyrosyl-RR-src peptide was measured at 30°C for 15 min as described (
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Results |
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PTP and Contactin Are Associated in Chick Brain
Contactin and PTP immunoprecipitates from embryonic chick brain lysates were probed for the presence of contactin and PTP
. PTP
was present in anticontactin immunoprecipitates (Figure 1 A), and contactin was present in PTP
immunoprecipitates, but was not precipitated by preimmune serum (Figure 1 B). Thus, PTP
and contactin exist in a complex in brain lysates. To check the specificity of the association of PTP
and contactin, we examined the interaction of PTP
with NCAM, another fyn-associated (
was not present (Figure 1 C). Likewise, NCAM could not be detected in anti-PTP
immunoprecipitates prepared from these lysates (Figure 1 D) using the same anti-PTP
antiserum as employed with the chick brain lysates (raised to the species conserved intracellular D1 region of PTP
).
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Association of Ectopically Expressed PTP and Contactin
To investigate the molecular basis of the association between PTP and contactin, we used a transient expression system where the interaction of different forms could be manipulated. PTP
and contactin coimmunoprecipitated with one another from COS cells coexpressing PTP
(tagged in its extracellular region with an epitope of VSVG to facilitate immunoprecipitation [
alone or with contactin contained comparable levels of phosphatase activity, whereas virtually no activity was detectable in anti-VSVG immunoprecipitates from cells expressing contactin alone. Anticontactin immunoprecipitates from coexpressing cells contained about a fivefold higher phosphatase activity than those from cells expressing contactin or PTP
alone. These results indicate that the contactinPTP
complexes are functionally active. The levels of PTP
protein and phosphatase activity were much lower in anticontactin immunoprecipitates than in anti-VSVG immunoprecipitates from the coexpressing cells, likely because only a portion of the expressed PTP
associates with contactin.
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Similar experiments were carried out with contactin and CD45, a receptor-like PTP with structural similarity to PTP. No coimmunoprecipitation of contactin and CD45 from coexpressing cells was detected (Figure 2 C), indicating that the interaction of PTP
and contactin is specific and not merely due to heterologous expression.
Colocalization and Coclustering of Contactin and PTP
In situ localization of contactin and PTP in cotransfected COS cells revealed a similar distribution for both proteins within the plane of the plasma membrane (Figure 3A and Figure B). In contrast, control transfections of contactin together with the RPTP CD45, gave a completely different pattern of distribution to that of contactin (data not shown), indicating that they do not associate in the same cellular complexes.
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We examined whether an enforced redistribution of either contactin or PTP, induced by incubating the live cells with antibodies to the extracellular domains of these molecules, would lead to coclustering of the respective partner. Clustering of PTP
(Figure 3 E) caused contactin to redistribute to closely match the PTP
pattern (Figure 3 F). The close similarity of these two patterns supports the efficacy of clustering via the free-standing VSVG tag on the NH2-terminal of PTP
, leading to little or no interference with the interactions between PTP
and contactin. Coclusters of contactin and PTP
could also be induced by 4D1 mAb specific for contactin (Figure 3C and Figure D). Clusters of various sizes were induced in individual cells, with the PTP
localization largely matching the contactin pattern.
The Extracellular Region of PTP Is Required for Association with Contactin
To identify the region of PTP involved in the interaction with contactin, we generated a membrane-associated intracellular form of PTP
by replacing its extracellular and transmembrane regions with the myristylation signal of src (myr-PTP
). Expressed myr-PTP
was associated with the membrane fraction (data not shown), however, contactin only associated with wild-type PTP
and not with myr-PTP
(Figure 4 A). Thus, contactin does not interact with the PTP
lacking the extracellular and transmembrane regions.
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Since CD45 does not associate with contactin, we created a PTP/CD45 hybrid molecule where most of the extracellular region of CD45 was replaced with that of PTP
. PTP
or the PTP
-CD45 hybrid was coexpressed with contactin. Anti-VSVGimmunoprecipitated PTP
-CD45 hybrid and PTP
were complexed with contactin (Figure 4 B), demonstrating that contactin associates with the extracellular region of PTP
. Furthermore, the transmembrane region of PTP
is not specifically involved in the association with contactin since it can be replaced with that of CD45.
N-linked Glycosylation Is Not Required for the Association of PTP and Contactin
The mature PTP protein contains both N- and O-linked oligosaccharides (
and contactin were cultured with tunicamycin, an inhibitor of N-linked glycosylation, faster migrating, less diffuse forms of PTP
and contactin were detected on SDS-PAGE (Figure 4 C), which is consistent with a loss of N-linked oligosaccharides. Nevertheless, contactin was present in anti-VSVG immunoprecipitates from cells treated with or without tunicamycin (Figure 4 C, lanes 7 and 8), indicating that the association of contactin and PTP
occurs independently of N-linked glycosylation of either protein.
PTP and Contactin Associate In Cis but Not In Trans
Two experiments were carried out to address the question of whether PTP and contactin associate in a cis or trans conformation. First, anticontactin precipitates were prepared from lysates of COS cells expressing either PTP
or contactin, or from cells coexpressing both PTP
and contactin (Figure 5 A, lanes 13), as well as from another sample made by mixing lysates from the cells expressing either contactin or PTP
(Figure 5 A, lane 4). Anticontactin immunoprecipitates prepared from coexpressing cells contained PTP
, but those from mixed cell lysates did not (Figure 5 A, bottom, lanes 7 and 8). The lack of detectable association of PTP
and contactin in the mixed lysates suggests that interaction cannot take place in a trans conformation. Still, this may require a particular presentation of these cell surface molecules in growing cells that cannot form in solubilized cell lysates. Therefore, contactin-expressing cells were trypsinized 24 h after transfection and replated in dishes containing PTP
-expressing cells (these were not trypsinized for replating because this resulted in a large decrease in PTP
expression). After 24 h of coculture, the cells were lysed and immunoprecipitates were prepared. As a positive control for contactinPTP
association, PTP
- and contactin-cotransfected cells were cultured for 48 h, harvested, and processed the same way. PTP
and contactin coimmunoprecipitated from cotransfected cells (Figure 5 B, top, lanes 3 and 5), but not from cocultured cells (Figure 5 B, top, lanes 4 and 6). Thus, even when cells expressing contactin are cultured together with other cells expressing PTP
, no association in trans of these two receptor proteins occurs.
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Discussion |
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We demonstrate that a receptor protein tyrosine phosphatase forms a membrane-spanning complex with a neuronal GPI-anchored receptor. PTP and contactin associate with one another in a lateral (cis) forming complex mediated through the extracellular region of PTP
. This complex is sufficiently stable to permit its isolation from brain lysates or transfected cells through immunoprecipitation of either component, and to permit coclustering of PTP
with contactin upon antibody-induced clustering of contactin within cells and vice versa. Our findings that contactinPTP
complexes form in cotransfected cells, but not upon coculture or in mixed lysates of PTP
-expressing cells and contactin-expressing cells, provides compelling evidence that PTP
and contactin can only associate within the same cell; thus, forming a receptor complex rather than a ligandreceptor pair.
This is the first identification of an extracellular partner and potential regulator of PTP. As contactin lacks any intracellular region and is tethered to the external face of the plasma membrane through a GPI linkage, PTP
could, thus, link an extracellular contactin-mediated signal to an intracellular response. Fyn is complexed with contactin and is transiently activated upon aggregation of contactin (
and is dephosphorylated and activated by PTP
(
and contactin suggests that PTP
might act as an intermediary molecule in a tripartite complex of contactin, PTP
, and fyn, in accord with its proposed role as a transducer.
and contactinfyn complexes brings PTP
into proximity with fyn, allowing the subsequent dephosphorylation and activation of fyn. In view of the constitutive activity of PTP
(and other RPTPs), such a contactin-regulated access of PTP
to its substrate provides an attractive mechanism for mediating RPTP activity and the action of GPI-anchored receptors as modulators in larger signaling complexes.
Contactin interacts with multiple ligands and the identification of PTP as a novel contactin-associated protein extends the number and the nature of possible contactin-containing receptor complexes. Contactin is found in regions of active neuronal migration or outgrowth in the developing brain and in areas of synaptic development and activity (
An intriguing possibility arising from this study is that contactin acts as an adapter to bring together two RPTPs, namely RPTP/ß and PTP
. The NH2-terminal carbonic anhydrase-like region of the transmembrane and secreted extracellular forms of glial cell RPTP
/ß associates in trans with contactin, and in doing so promotes neurite growth (
functions as a signaling component of a RPTP
/ßbound contactinPTP
complex, this would represent a novel mode of RPTP interaction and regulation.
The proposed signaling through a contactinPTP complex represents a new paradigm of receptor-mediated tyrosine kinase activation. Receptors with intrinsic tyrosine kinase activity or directly associated with active nonreceptor tyrosine kinases have been well documented. Contactin, lacking the intracellular region required for either of these mechanisms, may utilize an associated RPTP, PTP
, to effect intracellular activation of tyrosine kinases. This is reminiscent of the recent finding that the GPI-anchored cell surface receptors GDNFR-
and NTNR-
form functional coreceptor complexes with the transmembrane tyrosine kinase Ret (
. The components of a novel signal transduction pathway, thus, have been identified and can now be tested for function and physiological relevance to aspects of neuronal development.
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Footnotes |
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L. Zeng, L. D'Alessandri, and M.B. Kalousek contributed equally to this work.
M.B. Kalousek's present address is Laves-Arzneimittel GmbH, CH-6247 Schötz, Switzerland.
1 Abbreviations used in this paper: FN-III, fibronectin type III; GPI, glycosyl phosphatidylinositol; PTP, protein tyrosine phosphatase; RPTP, receptor protein tyrosine phosphatase; VSVG, vaccinia stomatitis virus glycoprotein.
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Acknowledgements |
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We thank G. Koretzky for the CD45 cDNA, P. Bello for the pp60c-src cDNA, V. Bhandari, J.E. Walker, and K.L. Lim for construction of pXJ41-PTP/CD45-neo, pXJ41-contactin-neo, and pXJ41-myr-PTP
-neo, and C. Cagnoli for help with preparation of antiPTP
antibodies.
This work was supported by the Krebsforschung Schweiz and the Swiss National Science Foundation (to L. Vaughan) and the National Science and Technology Board of Singapore (to C.J. Pallen).
Submitted: 14 September 1999
Revised: 1 October 1999
Accepted: 4 October 1999
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References |
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