Human Protein Tyrosine Phosphatase Receptor type O

Target ID:

PTPROA

Aliases:

GLEPP1PTP-U2PTPU2

Deposition Date:

Friday 31st March 2006

Families:

Phosphatases

Related Diseases:

SignallingCancerNeurobiology

Structure type:

apo

Download Coordinates:

2GJT

Authors:

A.BARR,E.UGOCHUKWU,J.ESWARAN,S.DAS,F.NIESEN,P.SAVITSKY,A.TURNBULL,M.SUNDSTROM,C.ARROWSMITH,A.EDWARDS,J.WEIGELT,F.VON DELFT,E.PAPAGRIGORIOU,S.KNAPP,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2GJT

tabs

Structure Details

PTPRO (also known as Glomerular epithelial protein 1, GLEPP1; PTPU2) is a receptor type p rotein tyrosine phosphatase related to PTPRB-like PTPs. This phosphatase has an extended extracellular domain containing eight repeats of fibronectin type III-like motifs, a transmembrane region and a single intracellular catalytic domain and thus belongs to the R3 class of PTPs. It is primarily expressed in kidney and brain with lower expression also found in heart, placenta, spleen, colon, lymph nodes, lung and bone marrow. In mouse PTPRO mRNA is detected in the cerebral cortex, olfactory bulb and nucleus, hippocampus, motor neurons, and the spinal cord midline, dorsal root, cranial, and sympathetic ganglia and within kidney glomeruli. Multiple alternatively spliced PTPRO transcripts have been reported such as truncated PTPRO (PTPROt) which lacks the extracellular domain and is found predominantly in B lymphocytes and macrophages. Expression is developmentally regulated and it has been implicated in G0/G1 arrest.

Several functional roles have been reported for PTPRO. Studies with PTPRO -/- mice revealed that in the kidney PTPRO regulates the glomerular pressure/filtration rate through an effect on podocyte structure and function, and as a consequence the mice show a tendency to hypertension. P TPRO has also been implicated in axon growth and guidance in the developing chick motor neurons, consistent with studies in Drosophila. In monoblastoid leukaemia U937 cells PTPRO expression is induced during PMA-induced differentiation and a functional role of PTPRO in apoptosis has been suggested. In rat hepatomas, primary human lung tumors and in several human lung cancer cell lines reduced PTPRO expression levels have been detected associated with gene promoter methylation. Overexpression of PTPRO in a lung cancer cell line (A549) suppressed cell growth, delayed reentry of the cells into the cell cycle and increased susceptibility of the cells to apoptosis, consistent with a role for PTPRO as a tumour suppressor.

The protein, neuronal pentraxin with chromo domain (NPCD), has been identifed as a PTPRO-interacting protein in a yeast-two hybrid screen. Co-immunoprecipitation and pull-down approaches have confirmed the interaction and shown that at least one isoform of NPCD can serve as a substrate in vitro. Normal localization of NPCD at the plasma membrane requires PTPRO expression suggesting a possible functional role for the interaction.

Materials and Methods

PTPRO

PDB:2GJT

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|13677213|ref|NM_030667.1|
Entry Clone Source:Purely Proteins Ltd.
SGC Clone Accession:
Tag:mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.
Host:BL21 (DE3)R3 (Phage resistant strain)

Construct

Prelude:
Sequence: mhhhhhhssgvdlgtenlyfqsmNPVQLD DFDAYIKDMAKDSDYKFSLQFEELKLIGL DIPHFAADLPLNRCKNRYTNILPYDFSRV RLVSMNEEEGADYINANYIPGYNSPQEYI ATQGPLPETRNDFWKMVLQQKSQIIVMLT QCNEKRRVKCDHYWPFTEEPIAYGDITVE MISEEEQDDWACRHFRINYADEMQDVMHF NYTAWPDHGVPTANAAESILQFVHMVRQQ ATKSKGPMIIHCSAGVGRTGTFIALDRLL QHIRDHEFVDILGLVSEMRSYRMSMVQTE EQYIFIHQCVQLMWMKKKQQFCISDV
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight culture containing 50µg/ml kanamycin was used to inoculate 1 litre of LB containing 50µg/ml kanamycin. Cultures were grown at 37°C until the OD 600 reached ~0.3 then the temperature was adjusted to 18°C. Expression was induced for 4 hours using 1 mM IPTG at an OD 600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer : 50mM HEPES pH 7.5; 500 mM NaCl; 5 mM imidazole, 5% glycerol; 0.5 mM TCEP.

Purification

Procedure

Column 1 : Ion exchange - Nucleic acid removal. DEAE cellulose (DE52, Whatman), 10 g of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, then washed with 20 ml binding buffer prior to loading the sample.
Supernatant was applied by gravity flow, followed by a wash with 100 ml binding buffer. The column flow-through was collected.

Column 2: Ni-affinity. Ni-NTA (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.
The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 100 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 and 250 mM); fractions were collected until essentially all protein was eluted.

Enzymatic treatment: (His tag cleavage using TEV) Samples containing PTPRO were pooled and TEV protease added for overnight incubation at 4°C. Cleaved products and TEV protease were removed by binding to Ni-NTA agarose after buffer exchange to 50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 0.5 mM TCEP using a 10 kDa cut-off concentrator .

Column 3: Size Exclusion Chromatography. Superdex S200 16/60 HiLoad
TEV-cleaved PTPRO, after re-binding to NiNTA, was directly applied to a S200 16/60 HiLoad gel filtration column equilibrated in 50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM DTT using either an ÄKTAprime or ÄKTAxpress system

Extraction

Procedure
Frozen pellets were thawed and cells lysed using a high pressure cell disrupter. The lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification.
Concentration:Protein was concentrated to 10 mg/ml using an Amicon 10 kDa cut-off concentrator
Ligand
MassSpec:LC- ESI -MS TOF confirmed the correct mass expected for this construct. Predicted mass of TEV-cleaved protein = 34596; Measured = 34596
Crystallization:Crystals were grown at 4°C in 600 nl sitting drops mixing 450 nl of protein with 150 nl of a solution containing 0.1M HEPES pH 8.0; 10% isopropanol; 20% PEG 4K
NMR Spectroscopy:
Data Collection:Resolution: 2.19 Å; Crystals were cryo-protected using the well solution and 20% ethylene glycol and flash frozen in liquid nitrogen. X-ray source: Diffraction data were collected at the SLS beamline X10 at a single wavelength (0.979 Å).
Data Processing:

References

Stepanek L, Stoker AW, Stoeckli E, Bixby JL. (2005). Receptor tyrosine phosphatases guide vertebrate motor axons during development. J Neurosci. 25:3813-23.

Chen B, Bixby JL. (2005). A novel substrate of receptor tyrosine phosphatase PTPRO is required for nerve growth factor-induced process outgrowth. J Neurosci. 25 :880-888.

Chen B, Bixby JL. (2005).Neuronal pentraxin with chromo domain (NPCD) is a novel class of protein expressed in multiple neuronal domains. J Comp Neurol. 481:391-402.

Motiwala T, Kutay H, Ghoshal K, Bai S, Seimiya H, Tsuruo T, Suster S, Morrison C, Jacob ST. (2004). Protein tyrosine phosphatase receptor-type O (PTPRO) exhibits characteristics of a candidate tumor suppressor in human lung cancer. Proc Natl Acad Sci U S A. 101:13844-9.

Wharram BL, Goyal M, Gillespie PJ, Wiggins JE, Kershaw DB, Holzman LB, Dysko RC, Saunders TL, Samuelson LC, Wiggins RC. (2000). Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low glomerular filtration rate. J Clin Invest. 106:1281-1290.

Aguiar RC, Yakushijin Y, Kharbanda S, Tiwari S, Freeman GJ, Shipp MA. (1999). PTPROt: an alternatively spliced and developmentally regulated B-lymphoid phosphatase that promotes G0/G1 arrest. Blood 94: 2403-2413.