PTPα and Contactin Are Associated in Chick Brain
Contactin and PTPα immunoprecipitates from embryonic chick brain lysates were probed for the presence of contactin and PTPα. PTPα was present in anticontactin immunoprecipitates ( A), and contactin was present in PTPα immunoprecipitates, but was not precipitated by preimmune serum ( B). Thus, PTPα and contactin exist in a complex in brain lysates. To check the specificity of the association of PTPα and contactin, we examined the interaction of PTPα with NCAM, another fyn-associated ( Beggs et al. 1997
) cell adhesion molecule highly expressed in the brain. The 120- and 140-kD NCAM isoforms were detected in anti-NCAM immunoprecipitates from mouse brains, but PTPα was not present ( C). Likewise, NCAM could not be detected in anti-PTPα immunoprecipitates prepared from these lysates ( D) using the same anti-PTPα antiserum as employed with the chick brain lysates (raised to the species conserved intracellular D1 region of PTPα).
Figure 1 PTPα associates with contactin in chick brain. Embryonic chick (A and B) or adult mouse (C and D) brain lysates (WCL) and immunoprecipitates made with anticontactin antibody (A), anti–NCAM antibody (C), or affinity-purified anti–PTPα (more ...)
Association of Ectopically Expressed PTPα and Contactin
To investigate the molecular basis of the association between PTPα and contactin, we used a transient expression system where the interaction of different forms could be manipulated. PTPα and contactin coimmunoprecipitated with one another from COS cells coexpressing PTPα (tagged in its extracellular region with an epitope of VSVG to facilitate immunoprecipitation [ Bhandari et al. 1998
]) and contactin ( A). In other experiments, the immunocomplexes were assayed for PTP activity ( B). Anti-VSVG immunoprecipitates from cells expressing PTPα 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 PTPα alone. These results indicate that the contactin–PTPα complexes are functionally active. The levels of PTPα 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 PTPα associates with contactin.
Figure 2 Association of ectopically expressed PTPα and contactin. COS-1 cells were transfected with VSVG-PTPα (α), contactin (cont), or VSVG-PTPα and contactin (α/cont) cDNAs. (A) Whole cell lysates (WCL), and anti-VSVG (more ...)
Similar experiments were carried out with contactin and CD45, a receptor-like PTP with structural similarity to PTPα. No coimmunoprecipitation of contactin and CD45 from coexpressing cells was detected ( C), indicating that the interaction of PTPα and contactin is specific and not merely due to heterologous expression.
Colocalization and Coclustering of Contactin and PTPα
In situ localization of contactin and PTPα in cotransfected COS cells revealed a similar distribution for both proteins within the plane of the plasma membrane ( and ). 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.
Figure 3 Ectopically expressed PTPα and contactin colocalize. (A and B) COS-7 cells cotransfected with contactin and VSVG-tagged PTPα cDNAs were fixed and incubated with a mouse anti–VSVG antibody and with a rabbit anticontactin antibody, (more ...)
We examined whether an enforced redistribution of either contactin or PTPα, 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 PTPα ( E) caused contactin to redistribute to closely match the PTPα pattern ( F). The close similarity of these two patterns supports the efficacy of clustering via the free-standing VSVG tag on the NH2-terminal of PTPα, leading to little or no interference with the interactions between PTPα and contactin. Coclusters of contactin and PTPα could also be induced by 4D1 mAb specific for contactin ( and ). Clusters of various sizes were induced in individual cells, with the PTPα localization largely matching the contactin pattern.
The Extracellular Region of PTPα Is Required for Association with Contactin
To identify the region of PTPα involved in the interaction with contactin, we generated a membrane-associated intracellular form of PTPα by replacing its extracellular and transmembrane regions with the myristylation signal of src (myr-PTPα). Expressed myr-PTPα was associated with the membrane fraction (data not shown), however, contactin only associated with wild-type PTPα and not with myr-PTPα ( A). Thus, contactin does not interact with the PTPα lacking the extracellular and transmembrane regions.
Figure 4 The extracellular region of PTPα is required for association with contactin. (A) Lysates (WCL) and anti-PTPα immunoprecipitates (IP) from COS-1 cells transiently expressing contactin (cont), myr-PTPα/contactin (myr-α/cont), (more ...)
Since CD45 does not associate with contactin, we created a PTPα/CD45 hybrid molecule where most of the extracellular region of CD45 was replaced with that of PTPα. PTPα or the PTPα-CD45 hybrid was coexpressed with contactin. Anti-VSVG–immunoprecipitated PTPα-CD45 hybrid and PTPα were complexed with contactin ( B), demonstrating that contactin associates with the extracellular region of PTPα. Furthermore, the transmembrane region of PTPα 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 PTPα and Contactin
The mature PTPα protein contains both N- and O-linked oligosaccharides ( Daum et al. 1994
). Chick contactin has nine potential sites for N-linked glycosylation ( Ranscht and Dours 1988
; Brümmendorf et al. 1989
). When COS cells coexpressing PTPα and contactin were cultured with tunicamycin, an inhibitor of N-linked glycosylation, faster migrating, less diffuse forms of PTPα and contactin were detected on SDS-PAGE ( 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 ( C, lanes 7 and 8), indicating that the association of contactin and PTPα occurs independently of N-linked glycosylation of either protein.
PTPα and Contactin Associate In Cis but Not In Trans
Two experiments were carried out to address the question of whether PTPα and contactin associate in a cis or trans conformation. First, anticontactin precipitates were prepared from lysates of COS cells expressing either PTPα or contactin, or from cells coexpressing both PTPα and contactin ( A, lanes 1–3), as well as from another sample made by mixing lysates from the cells expressing either contactin or PTPα ( A, lane 4). Anticontactin immunoprecipitates prepared from coexpressing cells contained PTPα, but those from mixed cell lysates did not ( A, bottom, lanes 7 and 8). The lack of detectable association of PTPα 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 PTPα-expressing cells (these were not trypsinized for replating because this resulted in a large decrease in PTPα expression). After 24 h of coculture, the cells were lysed and immunoprecipitates were prepared. As a positive control for contactin–PTPα association, PTPα- and contactin-cotransfected cells were cultured for 48 h, harvested, and processed the same way. PTPα and contactin coimmunoprecipitated from cotransfected cells ( B, top, lanes 3 and 5), but not from cocultured cells ( B, top, lanes 4 and 6). Thus, even when cells expressing contactin are cultured together with other cells expressing PTPα, no association in trans of these two receptor proteins occurs.
Figure 5 PTPα and contactin do not associate in a trans configuration. (A) COS cells were transfected with VSVG-PTPα (α), contactin (cont), or VSVG-PTPα and contactin cDNAs (α/cont). Whole cell lysates (WCL) (lanes 1–3), (more ...)