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We have isolated cDNA clones encoding the Xenopus homologue of receptor-type protein tyrosine phosphatase beta (RPTPbeta), and identified 13 forms of the mRNA provably generated by alternative splicing. All the conceptual translates have a carbonic anhydrase-like domain, a fibronectin type III-like repeat and a spacer in the extracellular segment. Eleven of them (designated XRPTPbeta.1-XRPTPbeta.11) also have highly conserved two intracellular PTP domains, whereas the other two variants (sXRPTPbeta.1 and sXRPTPbeta.2) have neither transmembrane nor cytoplasmic segment. There are five peptides that can be inserted in various combinations into the spacer region. Northern and Western blot analyses show central nervous system-specific expression of the XRPTPbeta mRNAs and proteins. Chondroitinase ABC treatment of the brain and spinal cord extracts results in separation of six protein bands on the Western blot, in association with a decrease in the size of major bands, indicating that the major XRPTPbeta variants are chondroitin sulfate proteoglycans. The results of these as well as reverse-transcribed polymerase chain reaction analyses suggest that the amounts of different XRPTPbeta variants are regulated in tissue- and developmental stage-specific manners.
Fig. 1. Structure of the representative XRPTPβ clones and two cDNAs encoding the largest receptor (XRPTPβ.11) and phosphacan (sXRPTPβ.2). The gray boxes represent coding sequences and the open boxes, 5â²- and 3â²-noncoding sequences. The dotted lines indicate deletion of the DNA segments in between. The clones T8, T11 and T26, and R1 and R20 were isolated using D1/D2 probes (see Section 3.1) from the oligo (dT)-primed and random-primed cDNA libraries, respectively. The clones T3 and T19 were isolated using a fragment of R1 encoding the CAH/FN III sequence. Each clone partially encodes XRPTPβ.1 (R1), XRPTPβ.4 (T26), XRPTPβ.5 (T11), XRPTPβ.10 (R20), XRPTPβ.11 (T8), sXRPTPβ.1 (T3) and sXRPTPβ.2 (T19), respectively (see Table 1). The thick bars 1â3 indicate the sequences used as probes for Northern blot analysis (see Fig. 3), and the arrowheads aâh, positions of the oligonucleotide primers (see Section 2.2).
Fig. 2. (A) The aa sequences of XRPPβ.11 and sXRPTPβ.2. The hydrophobic signal peptide (aa 1â24) and the transmembrane peptide (aa 1602â1627) are underlined. The CAH domain (aa 33â301), the FN III domain (aa 311â404), and the two phosphatase domains, D1 and D2 (aa 1708â1948 and aa 2004â2237, respectively) are boxed. Arrows represent the boundaries of IP 1âIP 5. In sXRPTPβ.2, a single glycine (G) is substituted for the sequence following to the residue 1575, which results in deletion of the transmembrane and intracytoplasmic sequences. The complete nt sequences of XRPPβ.11 and sXRPTPβ.2 have been deposited in the DDBJ/GeneBank/EMBLE database (accession No. AB045237 and AB045238). (B) Comparison of the aa sequences of XRPTPβ.11 and human RPTPβ. Percentage of identical aa residues is shown for each domain.
Fig. 3. (A) Genomic PCR of the XRPTPβ sequence. Genomic DNA was isolated from blood cells and XRPTPβ sequences were amplified with primer combinations indicated. The products were analyzed by electrophoresis on a 0.8% agarose gel. (B) RT-PCR analysis of the different forms of XRPTPβ. Total RNA fractions were isolated from adult brains (Br) and whole embryos at the indicated developmental stages, and used as templates for reverse transcription. EF1α was amplified as a positive control. As a control for reverse transcription (RT-), the reaction was performed without the reverse transcriptase.
Fig. 4. Northern blot analysis of XRPTPβ expression. (A) The blot of poly (A)+ RNA from adult brains (5 μg) was sequentially hybridized with CAH (a), D1 (b) and sXRPTPβ-specific 3′-non-coding (NC; c) probes. Positions of the RNA size marker (Novagen) are indicated on the left. (B) The blot of total RNA fractions (20 μg) from various tissues was sequentially hybridized with the probes indicated. The ethidium bromide (EtBr)-stained gel is for loading control.
Fig. 5. Western blot analysis of XRPTPβ peptides and proteins. (A) Specificity of mAbs to XRPTPβ peptides. The lysates of bacteria producing recombinant CAH/FN III (lane 1), IP 1/IP 2 (lane 2) and D2 (lane 3) peptides were separated on a 10% acrylamide gel containing SDS, and transferred to a PVDF membrane. The blot was detected using mAbs 5G3, 1B10 or 3E5, which are prepared to recombinant CAH/FN III, IP 1/IP 2 and the D2 peptides, respectively (see Section 2.3). The lower band detected by 5G3 mAb (arrowhead on the left) may be due to degradation of the peptide in the upper band. (B) Detection of XRPTPβ proteins in the brain and the spinal cord. Extracts from adult brains (Br) and spinal cords (Sp) were incubated with (+) or without (â) chondroitinase ABC (CHase), and separated on a 6% acrylamide gel (see Section 2.4). XRPTPβ proteins on the blot were detected by the mAbs 5G3, 1B10 or 3E5. The arrowheads on the left show protein bands resolved in the chondroitinase-treated samples.
