Human Protein Tyrosine Phosphatase Receptor T

Target ID:

PTPRTA

Aliases:

KIAA0283PTPRTRPTPrhRPTPrho

Deposition Date:

Friday 26th January 2007

Families:

Phosphatases

Related Diseases:

CancerSignallingNeurobiology

Structure type:

apo

Download Coordinates:

2OOQ

Authors:

E.UGOCHUKWU,I.ALFANO,A.BARR,T.KEATES,J.ESWARAN,E.SALAH,P.SAVITSKY,G.BUNKOCZI,A.EDWARDS,C.H.ARROWSMITH,J.WEIGELT,M.SUNDSTROM,F.VON DELFT,S.KNAPP,STRUCTURAL GENOMICSCONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2OOQ

tabs

Structure Details

PTPRT belongs together with PTPμ, PTPκ and PCP-2 to the receptor type IIb (2B) subfamily of PTPases. The full-length protein contains a large extracellular region containing a meprin-A5 antigen-PTP (MAM) domain and Ig-like and fibronectin type III-like repeats, a single transmembrane region and two intracellular catalytic domains.

PTPRT has been suggested to play roles in both signal transduction and cellular adhesion regulating cadherin-mediated cell adhesion. This phosphatase is mainly expressed in the central nervous system with highest expression levels reported in the hippocampus and dentate gyrus, olfactory bulb, subiculum, cerebral cortex, pyriform cortex, medullary motor nuclei, and anterior cerebellum. Lower expression levels have also been detected in colon, heart, lung and pancreas. Expression of PTPRT is developmentally regulated and is high in the cortex and olfactory bulbs during perinatal development, but down-regulated during postnatal week two.

PTPRT is mutated in several cancer types in particular in colon cancer. Five missense mutations have been detected in cancer tissues reducing phosphatase activity suggesting a tumor suppressor function for this phosphatase. PTPRT has also been found to be upregulated in ERalpha positive breast cancer.

PTPRT interacts with the catalytically inactive phosphatase PTPRN, Islet antigen-2 (IA-2), a major autoantigen in type 1 diabetes as well as with several adherens junctional proteins including E-cadherin, p120ctn, N-cadherin, VE-cadherin, desmoglein, α, β and γ catenin as well as α-actinin.

Materials and Methods

PTPRTA: Human Protein Tyrosine Phosphatase Receptor T

PDB:2OOQ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi 5031765
Entry Clone Source:synthetic
SGC Clone Accession:
Tag:N terminus: mgsshhhhhhssgrenlyfqgh. C terminus: gs
Host:BL21(DE3)

Construct

Prelude:
Sequence:
Vector:p11

Growth

Medium:
Antibiotics:
Procedure:The 11β-HSD1 construct was expressed in the E. coli expression strain BL21(DE3), in TB medium with a plasmid encoding the chaperone GroEL/ES, under control of the arabinose promoter ( Takara , Japan ). High-level soluble protein production was achieved by IPTG induction (3 mM) at 18oC for 12 hrs in the presence of 1% sucrose, 1% glucose, 0.05% arabinose and 100 µM carbenoxolone (CBX, 3ß-hydroxy-11-oxoolean-12-en-30-oic acid, 3-hemisuccinate, Sigma).

Purification

Procedure
Column 1: HiTrap chelating 5ml_ Ni_Column
Procedure: Add supernatant to column at 3 ml/min, filtrate if necessary prior to application. Wash with buffer A to baseline. Wash with 9 % B. Wash with 16 % B. Go back to buffer A to baseline. Incubate column in buffer C at room temp over night. Wash with 9 % E. Wash with 16 % E to baseline. Elute with 60 % E

Column 2: Hi Load 16/60 Superdex 200

Extraction

Procedure
Collected/resuspended cells (50 mM Tris/Cl, 300 mM NaCl, 5 mM Imidazole, 2 mM TCEP, 0.5 uM CBX, 5% (w/v) glycerol, pH8.0) were disrupted in a high-pressure homogenizer, the supernatant after centrifugation at 15000 g for 40 min of the cell lysate was subjected to immobilized metal affinity chromatography, followed by incubation overnight in 0.05% Triton X-100 (Anapoe X-100; Anatrace, Maumee, OH), 20 µM CBX, 50 mM Tris/Cl, pH 8.0, 300 mM NaCl, 2 mM TCEP, 0.5 mM MgCl2, 2 mM ATP and a final gel filtration chromatographic step on a Superdex 200 HiLoad 16/60 column (GE Healthcare, Uppsala, Sweden). The protein was concentrated in 20 mM Tris-HCl, pH 8.0, containing 2mM TCEP, 5% (w/v) glycerol, 0.05 % (w/v) Triton X-100, and 2 µM of the inhibitor to around 12 mg/ml using a 30000 kDa MW cutoff Amicon Ultra concentration device (Millipore, Bedford, MA).
Concentration:12 mg/ml
Ligand
MassSpec:observed mass 31064 Da, theoretical mass 31063.9 Da
Crystallization:Crystals were grown using the hanging-drop vapor diffusion technique. Two microliter drops containing equal volumes of protein solution and a buffer solution composed of 200 mM MgCl2, 17% PEG 3350, 100 mM Bis-Tris, pH 5.5 were equilibrated against a well containing 300 µl of the buffer solution. Crystals reached a size of ~200 microns over a period of 3-7 days.
NMR Spectroscopy:
Data Collection:Resolution: 2.1 Å, X-ray source: Synchrotron SLS-X06, single wavelength.
Data Processing:

References

Besco, J., Popesco, M. C., Davuluri, R. V., Frostholm, A., and Rotter, A. (2004). Genomic structure and alternative splicing of murine R2B receptor protein tyrosine phosphatases (PTPkappa, mu, rho and PCP-2). BMC Genomics 5 , 14.
Besco, J. A., Frostholm, A., Popesco, M. C., Burghes, A. H., and Rotter, A. (2001). Genomic organization and alternative splicing of the human and mouse RPTPrho genes. BMC Genomics 2 , 1.
Besco, J. A., Hooft van Huijsduijnen, R., Frostholm, A., and Rotter, A. (2006). Intracellular substrates of brain-enriched receptor protein tyrosine phosphatase rho (RPTPrho/PTPRT). Brain Res 1116 , 50-57.
Tozlu, S., Girault, I., Vacher, S., Vendrell, J., Andrieu, C., Spyratos, F., Cohen, P., Lidereau, R., and Bieche, I. (2006). Identification of novel genes that co-cluster with estrogen receptor alpha in breast tumor biopsy specimens, using a large-scale real-time reverse transcription-PCR approach. Endocr Relat Cancer 13 , 1109-1120.
Wang, Z., Shen, D., Parsons, D. W., Bardelli