Abstract
We have reported previously that a chimeric platelet-derived growth factor receptor (PDGFR) possessing the ligand binding domain of the alpha PDGFR and the intracellular domain of the beta PDGFR (alpha 340 beta 342 R) was markedly more efficient than the wild type alpha PDGFR (alpha RWT) in its ability to enhance PDGF-A transforming activity in NIH/3T3 fibroblasts. To determine the region within the cytoplasmic domain of beta PDGFR that confers this higher transforming activity, we generated several additional alpha/beta PDGFR chimerae. When a chimeric PDGFR possessing the first 933 amino-terminal amino acids from the alpha PDGFR and the final 165 amino acids from the carboxyl-terminal of the beta PDGFR (alpha 933 beta 942 R) was cotransfected with the PDGF-A gene into NIH/3T3 cells, it showed a similar high efficiency to enhance PDGF-A chain transforming activity as alpha 340 beta 342 R. However, when chimeric PDGFRs in which either the kinase insert domain (alpha beta RKI) or the last 79 amino acids from the carboxyl-terminal end of the beta PDGFR (alpha 1024 beta 1028 R) were substituted into alpha PDGFR sequences were cotransfected with PDGF-A, they showed similar low efficiencies in enhancing transforming activity as the alpha RWT. These results predicted that the 86 amino acids following the tyrosine kinase 2 domain of beta PDGFR (amino acid residues 942-1027) were responsible for the higher transforming activity of beta PDGFR. To confirm this finding, we next constructed a chimera in which amino acid residues 942-1028 of the beta PDGFR (alpha beta 942-1028R) were substituted for those in the alpha PDGFR. Cotransfection experiments indicated that alpha beta 942-1028R increased transforming activity of PDGF-A to similar extent as the alpha 933 beta 942R, or alpha 340 beta 342R. Therefore, our findings define a critical domain within the noncatalytic region of beta PDGFR intracellular domain that confers the higher focus forming activity mediated by the beta PDGFR.
Highlights
We have reported previously that a chimeric plateletderived growth factor receptor (PDGFR) possessing the ligand binding domain of the ␣PDGFR and the intracellular domain of the PDGFR (␣340342R) was markedly more efficient than the wild type ␣PDGFR (␣RWT) in its ability to enhance Platelet-derived growth factor (PDGF)-A transforming activity in NIH/ 3T3 fibroblasts
Generation of the Chimeric Receptors between the Cytoplasmic Domain of ␣- and PDGFR—We have reported that cotransfection of an expression vector containing the PDGF-A gene cDNA with another vector encoding a chimeric receptor possessing the first 340 amino acids of the ␣PDGFR fused to the remaining extracellular transmembrane and intracellular domain of the PDGFR (␣340342R) enhanced the transforming activity of PDGF-A by 17-fold
Since the expression levels of ␣RWT and ␣340342R were shown to be comparable, these findings strongly suggested that the greater transforming activity of PDGF-B compare with that of PDGF-A is due to distinct substrate specificities of the ␣- and PDGFR
Summary
Aykut Uren‡§, Jin-Chen Yu§¶, Weiqun Li‡, Il-Yup Chungʈ, Daruka Mahadevan, Jacalyn H. When chimeric PDGFRs in which either the kinase insert domain (␣RKI) or the last 79 amino acids from the carboxyl-terminal end of the PDGFR (␣10241028R) were substituted into ␣PDGFR sequences were cotransfected with PDGF-A, they showed similar low efficiencies in enhancing transforming activity as the ␣RWT These results predicted that the 86 amino acids following the tyrosine kinase 2 domain of PDGFR (amino acid residues 942-1027) were responsible for the higher transforming activity of PDGFR. Identification of a Domain within the Carboxyl-terminal Region findings indicated that the increased transforming activity of PDGF-B in comparison with PDGF-A could be due to distinct substrate specificities of the two PDGFRs. In the present manuscript, we have identified a specific region within the carboxyl-terminal of the PDGFR that is responsible for mediating the enhanced focus formation of PDGF-B
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