Product Pathways - Tyrosine Kinase/ Adaptors
PDGF Receptor β Kinase #7392
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Description
Purified recombinant human PDGFRbetaβ (Gln557-Leu1106) kinase, supplied as a GST fusion protein.
Source / Purification
The GST-Kinase fusion protein was produced using a baculovirus expression system with a construct expressing human PDGFRβ (Gln557-Leu1106) (GenBank Accession No. NM_002609) with an amino-terminal GST tag. The protein was purified by one-step affinity chromatography using glutathione-agarose.
Gel Staining
Figure 1. The purity of the GST-PDGFRβ fusion protein was analyzed using SDS/PAGE followed by Coomassie stain.
Kinase Assay - Radiometric
Figure 2. PDGFRβ kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 10 mM MnCl2, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 40 ng/μL BSA, 50 μM ATP, Substrate: Poly(Glu-Tyr), 400 ng/μL, and recombinant PDGFRbeta: variable.
Quality Control
The theoretical molecular weight of the GST-PDGFRbeta fusion protein is 96 kDa. The purified kinase was quality controlled for purity using SDS-PAGE followed by Coomassie stain [Fig.1]. PDGFRbeta kinase activity was determined using a radiometric assay [Fig.2].
Background
The proteins of the platelet derived growth factor (PDGF) family exist as several disulphide-bonded, dimeric isoforms (PDGF AA, PDGF AB, PDGF BB, PDGF CC and PDGF DD) that bind in a specific pattern to two closely related receptor tyrosine kinases, PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα and PDGFRβ share 75% to 85% sequence homology between their two intracellular kinase domains while the kinase insert and carboxy-terminal tail regions display a lower level (27% to 28%) of homology (1). PDGF Receptor α homodimers bind all PDGF isoforms except those containing PDGF D. PDGF Receptor β homodimers bind PDGF BB and DD isoforms, as well as the PDGF AB heterodimer. The heteromeric PDGFα/β receptor binds PDGF B, C, and D homodimers as well as the PDGF AB heterodimer (2). PDGFRα and PDGFRβ can each form heterodimers with EGFR, which is also activated by PDGF (3). Various cells differ in the total number of receptors present and in the receptor subunit composition, which may account for responsive differences among cell types to PDGF binding (4). Ligand binding induces receptor dimerization and autophosphorylation, followed by binding and activation of cytoplasmic SH2 domain-containing signal transduction molecules such as Grb2, Src, GAP, PI3 kinase, PLCγ and Nck. A number of different signaling pathways are initiated by activated PDGF receptors and lead to control of cell growth, actin reorganization, migration and differentiation (5). Tyr751 in the kinase-insert region of PDGFRβ is the docking site for PI3 kinase (6). Phosphorylated pentapeptides derived from Tyr751 of PDGFRβ (pTyr751-Val-Pro-Met-Leu) inhibit the association of the carboxy-terminal SH2 domain of the p85 subunit of PI3 kinase with PDGFRβ (7). Tyr740 is also required for PDGFRβ mediated PI3 kinase activation (8).
- Deuel, T.F. et al. (1988) Biofactors 1, 213-217.
- Bergsten, E. et al. (2001) Nat. Cell Biol. 3, 512-516.
- Betsholtz, C. et al. (2001) Bioessays 23, 494-507.
- Coughlin, S.R. et al. (1988) Prog. Clin. Biol. Res. 266, 39-45.
- Ostman, A. and Heldin, C.H. (2001) Adv. Cancer Res. 80, 1-38.
- Panayotou, G. et al. (1992) EMBO J. 11, 4261-4272.
- Ramalingam, K. et al. (1995) Bioorg. Med. Chem. 3, 1263-1272.
- Kashishian, A. et al. (1992) EMBO J. 11, 1373-1382.
Application References
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Companion Products
- 7450 Tyrosine Kinase Substrate Screening Kit
- 9804 ATP (10 mM)
- 9805 HTScan® Tyrosine Kinase Buffer (4X)
- 1305 FLT3 (Tyr589) Biotinylated Peptide
- 9411 Phospho-Tyrosine Mouse mAb (P-Tyr-100)
- 7770 HTScan® PDGF Receptor β Kinase Assay Kit
This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.
