Human Protein Tyrosine Phosphatase Receptor G (PTPRG) D1-D2 domain

Target ID:

PTPRGA

Aliases:

HPTPGPTPGR-PTP-GAMMARPTPG

Deposition Date:

Friday 20th October 2006

Families:

Phosphatases

Related Diseases:

CancerSignallingNeurobiology

Structure type:

apo

Download Coordinates:

2NLK

Authors:

P.FILIPPAKOPOULOS,O.GILEADI,C.JOHANSSON,E.UGOCHUKWU,A.EDWARDS,C.ARROWSMITH,M.SUNDSTROM,F.VON DELFT,S.KNAPP,STRUCTURAL GENOMICS CONSORTIUM (SGC)

Site:

Oxford

ICM Datapack:

ICM Datapack

2NLK

tabs

Structure Details

PTPRG (Protein Receptor Tyrosine Phosphatase gamma) belongs together with PTPR&ceta; 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 â€“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 hyper-methylated in primary cutaneous T-cell lymphoma (CTCL),
which results in down-regulation 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. Here, we present the structure of the entire cytoplasmic domain of PTPRG comprising both catalytic domains (D1 and D2).

Materials and Methods

PTPRG D1-D2: Human Protein Tyrosine Phosphatase Receptor type Gamma D1-D2 domain

PDB:2NLK

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|18860898; NM_002841
Entry Clone Source:FivePrime
SGC Clone Accession:
Tag:C-terminal his6 tag
Host:BL21 (DE3) Rosetta R3 (Phage resistant strain)

Construct

Prelude:
Sequence:MAIPVKQFVKHIGELYSNNQHGFSEDFEE VQRCTADMNITAEHSNHPENKHKNRYINI LAYDHSRVKLRPLPGKDSKHSDYINANYV DGYNKAKAYIATQGPLKSTFEDFWRMIWE QNTGIIVMITNLVEKGRRKCDQYWPTENS EEYGNIIVTLKSTKIHACYTVRRFSIRNT KVKKGQKGNPKGRQNERVVIQYHYTQWPD MGVPEYALPVLTFVRRSSAARMPETGPVL VHCSAGVGRTGTYIVIDSMLQQIKDKSTV NVLGFLKHIRTQRNYLVQTEEQYIFIHDA LLEAILGKETEVSSNQLHSYVNSILIPGV GGKTRLEKQFKLVTQCNAKYVECFSAQKE CNKEKNRNSSVVPSERARVGLAPLPGMKG TDYINASYIMGYYRSNEFIITQHPLPHTT KDFWRMIWDHNAQIIVMLPDNQSLAEDEF VYWPSREESMNCEAFTVTLISKDRLCLSN EEQIIIHDFILEATQDDYVLEVRHFQCPK WPNPDAPISSTFELINVIKEEALTRDGPT IVHDEYGAVSAGMLCALTTLSQQLENENA VDVFQVAKMINLMRPGVFTDIEQYQFIYK MLSLVSTKENGNGPMTVDKNGAVLIADES DPAESMESLVahhhhhh
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:1ml from a 10 ml overnight was used to inoculate 1 l of TB medium containing 50 microgram/ml Kanamycine and 34 µg/ml Chloramphenicole. E .coli cells were grown in 2.5-L baffled flasks at 37 degrees until OD reached 2.0. The cells were cooled to 18°C and expression of PTPRG was induced adding 0.5 mM IPTG at an OD600 of 2.2

Purification

Procedure

Extraction

Procedure
50 mM potassium phosphate buffer, pH 7.5, 500 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, Complete® protease inhibitors (1 tablet/50 ml) and 15 units/ml Benzonase.
Frozen cell pellets were thawed at 37 degC and re-suspended in a total volume of 100 ml lysis buffer. The cells were disrupted by a high pressure cell disrupter (20 kpsi) followed by sonication. Nucleic acids and cell debris were removed by adding 0.15% PEI , followed by centrifugation for 30 minutes at 40 000xg. The supernatant was further clarified by filtration (0.45 µm).
Concentration:
Ligand
MassSpec:One peak of 71542 Da was detected for PTPTGAp011, which are 134 Da lower than the expected mass of 71674 . The discrepancy in mass was interpreted as a loss of the N-terminal methionine during expression.
Crystallization:Crystals were grown by vapor diffusion at 4degC from a sitting drop consisting of 100 nl protein (12.8 mg/ml) and 50 nl well solution. The drop was equilibrated against well solution containing 10 % PEG 10K and 100 mM imidazole pH 8.0. The crystal was transferred to a cryoprotectant composed of 20% ethylene glycol before flash-cooling in liquid nitrogen .
NMR Spectroscopy:
Data Collection:Resolution: 2.4 Å diffraction data were collected using a Rigaku FRE X-ray generator equipped with a HTC image plate 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.