Abstract
The receptor-type protein tyrosine phosphatase PTP mu comprises an extracellular segment containing a MAM domain, an immunoglobulin domain and four fibronectin type III repeats, a transmembrane segment, and two intracellular PTP domains. We have previously shown that PTP mu binds homophilically, i.e. PTP mu on the surface of one cell binds to PTP mu on an apposing cell, and that the extracellular segment alone is sufficient for homophilic binding. In this study we report that in MvLu cells PTP mu is proteolytically processed into two noncovalently associated fragments, one comprising most of the extracellular segment (approximately 100 kDa) and the other containing predominantly the transmembrane and intracellular portions (approximately 100 kDa). We have also identified the homophilic binding site within the extracellular segment. We have generated, expressed, and purified various fragments of the extracellular segment of PTP mu and have used fluorescent beads (Covaspheres) coated with these fragments in three binding assays: (i) measurement of bead aggregation, (ii) binding of beads to surfaces of dishes coated with purified PTP mu, or (iii) binding to MvLu cells. Only beads coated with recombinant fragments that contained the immunoglobulin domain underwent aggregation or bound to surfaces displaying PTP mu, suggesting that neither the MAM domain nor the fibronectin type III repeats bound homophilically in these assays. The fragment containing the Ig domain alone bound as well as any other Ig domain-containing fragment, suggesting that the Ig domain is both necessary and sufficient for homophilic binding under these conditions.
Highlights
PTPp,suggesting that neither thMeAM domain nor the grins
MATERIALS AND METHODS Plasmid andBaculovirus Construction-Polymerase chain reaction coated with purified preparations(5 &well) of fragments of the extracellular segment of PTPp which were adsorbed to 8-well chambers (Nunc, Naperville, IL)for 30 min, remaining unbound sites were was used to amplifcyDNA corresponding to three fragments; thMe AM domain, the Igdomain, and IgFN containing the Ig domain and two FN I11 repeats, which were ligated into pAcSG-His-NT
IgFN)bound to PTPpACD,PPp-extra, and MIF(Table[11]).These their extracellular segments that are suggestive of a role in data show that theIg domain Covaspheres bound well to cell-cell adhesion (3, 4)
Summary
Plasmid andBaculovirus Construction-Polymerase chain reaction coated with purified preparations(5 &well) of fragments of the extracellular segment of PTPp which were adsorbed to 8-well chambers (Nunc, Naperville, IL)for 30 min, remaining unbound sites were was used to amplifcyDNA corresponding to three fragments; thMe AM domain (amino acid2s0-184), the Igdomain (amino acid1s85-289), and IgFN containing the Ig domain and two FN I11 repeats (amino acids 185-434), which were ligated into pAcSG-His-NT PTPp-linked Covaspheres (20 pl) were added to the dishes ina final volume of 300 pl of phosphate-buffered saline. PTPp-ACD has been previously described(11).The various fragmentsof human PTPpwere expressed inSf9cells from recombinant baculoviruses a s described previously (11).Other PTPp recombinant fragments were madeas GST fusion proteins in E. coli. PTPpextra has been described (ll),and MIF which expresses theMAM, Ig subconfluent MvLu cells in multiwell chambersas described previously (11). The MvLu cells were incubated for 30 min with the appropriate antibodies to either the MAM domain (50 p1 of negative control or BK2 ascites) or the Ig doma(1in[00] pl of preimmune or immune serum CS33H8) prior tothe additionof 20 p1 ofPTPp-linked Covaspheres
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