Abstract. RPTPIx is a transmembrane protein tyrosine phosphatase with an adhesion molecule-like ectodomain. It has recently been shown that RPTPIx mediates homophilic interactions when expressed in insect cells. In this study, we have examined how RPTPIx may function as a cell contact receptor in mink lung epithelial cells, which express RPTPIx endogenously, as well as in transfected 3T3 cells. We find that RPTPI x has a relatively short half-life (3--4 hours) and undergoes posttranslational cleavage into two noncovalently associated subunits, with both cleaved and uncleaved molecules being present on the cell surface (roughly at a 1:1 ratio); shedding of the ectodomain subunit is observed in exponentially growing cells. Immunofluorescence analysis reveals that surface expression of RPTPIx is restricted to regions of tight cell-cell contact.RPTPIx surface expression increases significantly with increasing cell density. This density-induced upregulation of RPTPIx is independent of its catalytic activity and is also observed when transcription is driven by a constitutive promoter, indicating that modulation of RPTPIx surface expression occurs posttranscriptionally. Based on our results, we propose the following model of RPTPIx function: In the absence of cell--cell contact, newly synthesized RPTPIx molecules are rapidly cleared from the cell surface. Cell-cell contact causes RPTPIx to be trapped at the surface through homophilic binding, resulting in accumulation of RPTPIx at intercellular contact regions. This contact-induced clustering of RPTPIx may then lead to tyrosine dephosphorylation of intracellular substrates at cell-cell contacts.ECZl'ToR-like protein tyrosine phosphatases (receptor PTPs) 1 represent a new family of transmembrane proteins that are thought to transduce external signals by dephosphorylating phosphotyrosine residues on intracellular substrates (for review see Walton and Dixon, 1993). Although the receptor PTPs are growing in number and diversity, little is known about the nature of their extracellular ligands and their normal intracellular activities. It is also unknown where at the cell surface receptor PTPs are localized and how delivery and localization to the cell surface is regulated.We have recently cloned and characterized a mammalian receptor PTP, termed RPTPIx, which has an ectodomain similar to both the Ig-like and fibronectin type III-like domains of cell adhesion molecules (Gebbink et al., 1991).

The expression pattern of tumor necrosis factor-alpha (TNF-alpha) mRNA and protein was examined in vivo in experimental mouse skin wounds by in situ hybridization and immunohistochemistry. TNF-alpha mRNA and protein is detected in a distinct layer of mainly neutrophils subadjacent to the would clot. The layer of TNF-alpha-positive cells extends from the margin of the advancing epithelial outgrowth to the opposing one. By in situ hybridization the TNF-alpha mRNA is detectable 12 h after wounding; the signal peaks after 72 h and remains visible up to at least 120 h after wounding. TNF-alpha mRNA could not be detected in the normal skin or in 5-hour-old wounds. Immunohistochemical staining for TNF-alpha and macrophages on adjacent sections confirms that the main part of TNF-alpha-positive cells are polymorphonuclear neutrophils and shows that most of the cells located just beneath the layer of TNF-alpha-positive neutrophils are macrophages with weak TNF-alpha immunoreactivity. The data reported here show that neutrophils serve as an important source of TNF-alpha during healing of mouse skin wounds. We suggest that this specific expression of TNF-alpha is related to the process of re-epithelialization.

RPTP is a receptor-like protein-tyrosine phosphatase (RPTP) whose ectodomain mediates homotypic cellcell interactions. The intracellular part of RPTP contains a relatively long juxtamembrane domain (158 amino acids; aa) and two conserved phosphatase domains (C1 and C2). The membrane-proximal C1 domain is responsible for the catalytic activity of RPTP, whereas the membrane-distal C2 domain serves an unknown function. The regulation of RPTP activity remains poorly understood, although dimerization has been proposed as a general mechanism of inactivation. Using the yeast two-hybrid system, we find that the C1 domain binds to an N-terminal noncatalytic region in RPTP, termed JM (aa 803-955), consisting of a large part of the juxtamembrane domain (120 aa) and a small part of the C1 domain (33 aa). When co-expressed in COS cells, the JM polypeptide binds to both the C1 and the C2 domain. Strikingly, the isolated JM polypeptide fails to interact with either full-length RPTP or with truncated versions of RPTP that contain the JM region, consistent with the JM-C1 and JM-C2 interactions being intramolecular rather than intermolecular. Furthermore, we find that large part of the juxtamembrane domain (aa 814 -922) is essential for C1 to be catalytically active. Our findings suggest a model in which RPTP activity is regulated by the juxtamembrane domain undergoing intramolecular interactions with both the C1 and C2 domain. Protein-tyrosine phosphatases (PTPs)1 play important roles in signal transduction pathways regulated by tyrosine phosphorylation. Members of the superfamily of PTPs use the same catalytic mechanism and are broadly classified into transmembrane or receptor-like PTPs (RPTPs) and intracellular, nonreceptor PTPs (reviewed in Refs. 1 and 2). Members of the RPTP subfamily are type I membrane proteins consisting of a variable ectodomain, a single membrane-spanning region, and in most cases, two conserved intracellular phosphatase domains. The RPTPs are further classified according to the structure of their ectodomains (reviewed in Refs. 3 and 4). The large variety in ectodomain structure suggests the existence of an equal number of putative ligands, yet in most cases the corresponding ligands have not been identified.RPTP is the prototype member of a subfamily of RPTPs that mediate homophilic cell-cell interactions via their ectodomains and, hence, are thought to play a role in cell adhesionmediated processes (5-8). The ectodomain of RPTP shows similarities with that of cell-cell adhesion molecules and consists of an N-terminal "MAM" domain, which is critical for mediating cell-cell adhesion (9), followed by an Ig-like domain and four fibronectin type III repeats (10). Its intracellular part consists of a juxtamembrane domain of 158 amino acids (aa), which is relatively long compared with that in other RPTPs, and two tandem phosphatase domains referred to as C1 and C2. As in most other RPTPs, the membrane-proximal C1 domain of RPTP is catalytically active, whereas the membranedistal C2 domain shows no activity, a...

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Connexin-43(Cx43)-based gap junctional communication is transiently inhibited by certain G protein-coupled receptor agonists, including lysophosphatidic acid, endothelin and thrombin. Our previous studies have implicated the c-Src protein tyrosine kinase in mediating closure of Cx43 based gap junctions. Pervanadate, an inhibitor of protein tyrosine phosphatases, mimics activated Src in inhibiting Cx43 gap junctional communication, apparently by promoting tyrosine phosphorylation of the Cx43 C-terminal tail. However, the identity of the protein tyrosine phosphatase(s) that may normally prevent Src-induced gap junction closure is unknown. Receptor-like protein tyrosine phosphatases that mediate homotypic cell-cell interaction are attractive candidates. Here we show that receptor protein tyrosine phosphatase mu (RPTPmu) interacts with Cx43 in diverse cell systems. We find that the first catalytic domain of RPTPmu binds to Cx43. Our results support a model in which RPTPmu, or a closely related protein tyrosine phosphatase, interacts with the regulatory C-terminal tail of Cx43 to prevent Src-mediated closure of Cx43 gap junctional channels.