The effect of C truncPDGFRb was examined by wes tern blotting with general phospho tyrosine or site speci fic phospho antibodies as indicated. To prevent the effect from being masked by signal amplification, the cells were stimulated with submaximal concentrations of either PDGF BB or dopamine. Immunoprecipitation with anti FLAG antibody was performed prior to blotting with general phospho tyrosine antibodies. Relative to the lacZ control plasmid, transfection of C truncPDGFRb reduced basal PDGFRb general tyrosine phosphorylation, as well as receptor phosphorylation in response to both PDGF BB and dopamine. A corresponding reduc tion was also observed at two of the SH2 domain binding sites.

The phosphorylation of the Grb2 site and the major PLC g binding site in unstimulated CHO/DRD4 PR and those stimulated by either PDGF BB or DRD4 activation was reduced by the C truncPDGFRb to near basal levels. Similar to its effect on PDGF stimulated PDGFRb tyrosine phosphorylation, the C truncPDGFRb also blocked ERK1/2 phosphorylation in response to PDGF BB. In contrast, the DRD4 mediated ERK1/2 phosphorylation was unaffected by expression of C truncPDGFRb. These results suggest that unlike PDGF mediated signaling, DRD4 induced ERK1/2 phosphorylation is not contin gent on PDGFRb cross phosphorylation. We further confirmed that the differential effect of the C truncPDGFRb on PDGF and DRD4 mediated signal ing was not due to signal amplification caused by our overexpression of recombinant PDGFRb by examining ERK1/2 phosphorylation in CHO/DRD4 cells, which express the PDGFRb endogenously.

Consistent with our observations in CHO/DRD4 PR cells, the C truncPDGFRb blocked PDGF BB mediated ERK1/2 phosphorylation in CHO/DRD4 cells. In contrast, C truncPDGFRb did not inhibit the ERK1/2 phosphorylation that was stimulated by submaximal AV-951 concentrations of dopamine. To ascertain that the lack of effect of C truncPDGFRb on DRD4 mediated ERK1/2 phosphorylation is not due to overexpression of PDGFRb, the experiment was also performed in CHO/ DRD4. The p values for the 0. 3 ng/mL and 1 ng/mL PDGF BB treated groups are 0. 02 and 0. 01, respectively, according to the paired t test. Moreover, in the presence of C truncPDGFRb, the DRD4 mediated ERK1/2 phos phorylation remained sensitive to PDGFR kinase inhibi tion, showing that basal kinase activity of PDGFRb is still required for DRD4 mediated PDGFRb transactiva tion.

The lack of the need for PDGFRb cross phosphoryla tion in DRD4 mediated ERK1/2 activation suggests that PDGFRb dimerization is also not required. We investi gated the need for receptor dimerization, by utilizing a glutathione S transferase fusion protein to inhibit the formation of PDGFRb dimers. Previous reports have shown that a glutathione S transferase PDGFRa Ig4 domain fusion protein perturbs PDGFRa dimerization. The extracellular Ig4 domain of PDGFRb is known to provide the interface for subunit subunit interaction without playing a role in ligand binding.