Protein Tyrosine Phosphatase Receptor type Gamma

Target ID:

PTPRGA

Aliases:

HPTPGPTPGR-PTP-GAMMARPTPG

Deposition Date:

Thursday 25th May 2006

Families:

Phosphatases

Related Diseases:

CancerSignallingNeurobiology

Structure type:

apo

Download Coordinates:

2H4V

Authors:

E.UGOCHUKWU,A.BARR,S.DAS,J.ESWARAN,P.SAVITSKY,M.SUNDSTROM,A.EDWARDS,C.ARROWSMITH,J.WEIGELT,J.DEBRECZENI,F.VON DELFT,S.KNAPP,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2H4V

tabs

Structure Details

PTPRG (Protein Receptor Tyrosine Phosphatase gamma) belongs together with PTPRÃ¦ to the R5 subfamily which is characterized by an extracellular region containing an N-terminal carbonic anhydrase-like domain, a single transmembrane domain, and a cytoplasmic region with 2 tandem catalytic tyrosine phosphatase domains. Four splice forms have been reported ( PTPRG-A, -B, -C, and -S). The longest isoform (A) is truncated by 29 residues in isoform B, isoform C has only a single phosphatase domain and isoform S is an extracellular variant of PTPRG. All four isoforms are expressed in brain, kidney, lung, and heart.

PTPRG has been discussed as a tumour suppressor gene. The gene was found to be hypermethylated in primary cutaneous T-cell lymphoma (CTCL) which results in downregulation of PTPRG expression. Lower expression levels of PTPRG have been reported for a number of cancerous tissues and several mutations have been isolated from tumour cells. Furthermore expression levels are influenced by estradiol-17beta which suppresses PTPRG expression as well as by linoleic acid, IL-1 and TNFalpha which results in upregulation of PTPRG expression.

Materials and Methods

PTPRG

PDB:2H4V

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|18860898
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:mhhhhhhssgvdlgtenlyfqsmKQFVKHI GELYSNNQHGFSEDFEEVQRCTADMNITAE HSNHPENKHKNRYINILAYDHSRVKLRPLP GKDSKHSDYINANYVDGYNKAKAYIATQGP LKSTFEDFWRMIWEQNTGIIVMITNLVEKG RRKCDQYWPTENSEEYGNIIVTLKSTKIHA CYTVRRFSIRNTKVKKGQKGNPKGRQNERV VIQYHYTQWPDMGVPEYALPVLTFVRRSSA ARMPETGPVLVHCSAGVGRTGTYIVIDSML QQIKDKSTVNVLGFLKHIRTQRNYLVQTEE QYIFIHDALLEAILGKETEV
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:

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 PTPRG 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 PTPRG, 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 11.7 mg/ml using an Amicon 10 kDa cut-off concentrator
Ligand
MassSpec:LC- ESI -MS TOF confirmed the correct mass expected for this construct.
Crystallization:Crystals were grown at 20°C in 300nl sitting drops mixing 200 nl of protein with 100 nl of a solution containing 0.20M NH4(acetate); tris-sodium citrate dihydrate, pH 5.6; 30% PEG-4000.
NMR Spectroscopy:
Data Collection:Resolution: 2.4 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 using a copper Ka radiation generated by a Rigaku FRE X-ray generator and a HTC detector.
Data Processing:

References

Forrest, A. R., Taylor, D. F., Crowe, M. L., Chalk, A. M., Waddell, N. J., Kolle, G., Faulkner, G. J., Kodzius, R., Katayama, S., Wells, C. , et al. (2006). Genome-wide review of transcriptional complexity in mouse protein kinases and phosphatases. Genome Biol 7 , R5.
Pitterle, D. M., Jolicoeur, E. M., and Bepler, G. (1998). Hot spots for molecular genetic alterations in lung cancer. In Vivo 12 , 643-658.
Sorio, C., Mendrola, J., Lou, Z., LaForgia, S., Croce, C. M., and Huebner, K. (1995). Characterization of the receptor protein tyrosine phosphatase gene product PTP gamma: binding and activation by triphosphorylated nucleosides. Cancer Res 55 , 4855-4864.
van Doorn, R., Zoutman, W. H., Dijkman, R., de Menezes, R. X., Commandeur, S., Mulder, A. A., van der Velden, P. A., Vermeer, M. H., Willemze, R., Yan, P. S. , et al. (2005). Epigenetic profiling of cutaneous T-cell lymphoma: promoter hypermethylation of multiple tumor suppressor genes including BCL7a, PTPRG, and p73. J Clin Oncol 23 , 3886-3896.
Wang, Z., Shen, D., Parsons, D. W., Bardelli, A., Sager, J., Szabo, S., Ptak, J., Silliman, N., Peters, B. A., van der Heijden, M. S. , et al. (2004). Mutational analysis of the tyrosine phosphatome in colorectal cancers. Science 304 , 1164-1166.
Wu, C. W., Kao, H. L., Li, A. F., Chi, C. W., and Lin, W. C. (2005). Protein tyrosine-phosphatase expression profiling in gastric cancer tissues. Cancer Lett.