Characterization and chromosomal localization of PTP-NP-2, a new isoform of protein tyrosine phosphatase-like receptor, expressed on synaptic boutons.
Recently, there have been several reports describing the cloning and characterization of the novel family of protein tyrosine phosphatase-like receptor molecules (known as IA-2 and PTP-NP/PTP-IAR/IA-2beta/phogrin), which may act as autoantigens in diabetes. Here, we report the molecular characterization and chromosomal localization of a new isoform of this family in brain termed PTP-NP-2 (for PTP-NP tyrosine phosphatase isoform), and its function in rat primary hippocampal neurons. PTP-NP-2 has 48% identity to IA-2. The principal difference between PTP-NP-2 and PTP-NP is a 17-amino-acid insert near the N-terminus of PTP-NP that is absent in PTP-NP-2. Genomic DNA analysis indicates that the 17-amino-acid insert is coded by a separate exon, suggesting that both IA-2beta and PTP-NP-2 are isoforms arising by alternate splicing of the same gene. Reverse transcriptase-PCR revealed that both isoforms are present in human SH-SY5Y neuroblastoma cells. PTP-NP-2 mRNA expression is highly restricted, with a 5.5-kb specific transcript in human fetal and adult brain and 5.5 and 3. 8 kb in human adult pancreas. SH-SY5Y neuroblastoma and U87-MG glioblastoma cells showed specific transcripts of 5.5 and 3.8<HSP SP = "0.25">kb, respectively, indicating the existence of several isoforms of this molecule in the nervous system. The human gene encoding PTP-NP-2 was assigned to human chromosome 7q22-qter using Southern blot analysis of genomic DNAs from rodent/human somatic hybrid cell lines. Confocal microscopy analyses of rat primary hippocampal neurons revealed that PTP-NP-2 is abundantly expressed on synaptic boutons in primary neurons. Wild-type PTP-NP-2 showed no measurable tyrosine phosphatase activity using an in-vitro pNPP assay. Examination of the PTP-NP-2 catalytic consensus sequence revealed that this sequence differed from the typical tyrosine phosphatase-domain consensus sequence by an alanine to aspartate change (amino acid 930). Mutation of aspartate 930 to alanine produced a catalytically active enzyme, suggesting that native PTP-NP and its isoform PTP-NP-2 are catalytically inactive receptor protein tyrosine phosphatase homologues. Taken together, these results indicate that the tyrosine phosphatase PTP-NP-2 is a new isoform of PTP-NP tyrosine phosphatase, is expressed on synaptic boutons and may participate in the regulation of synaptic bouton endocytosis.
Division of Experimental Medicine, Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115, USA.
S
S
Jiang
A G
AG
Tulloch
T A
TA
Kim
Y
Y
Fu
R
R
Rogers
A
A
Gaskell
R A
RA
White
H
H
Avraham
S
S
Avraham
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000196:embryology / Q000378:metabolism*; D002874:Chromosome Mapping; D002897:Chromosomes, Human, Pair 7; D005260:Female; D005333:Fetus; D018507:Gene Expression Regulation, Developmental; D005909:Glioblastoma; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D008565:Membrane Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009447:Neuroblastoma; D009474:Neurons / Q000378:metabolism*; D011487:Protein Conformation; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D051381:Rats; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8; D013569:Synapses / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C102889:PTPRN2 protein, human; D017027:Protein Tyrosine Phosphatases; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8