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 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. Therefore, to specifically block PDGFRb dimerization, a glutathione S transferase PDGFRb Ig4 fusion protein was con structed. Pre incubation of CHO/DRD4 PR cells with GST alone had little effect on the ability of dopamine or PDGF BB to elicit an ERK1/2 response.

How ever, incubation with GST Ig4b prevented PDGF BB sti mulated ERK1/2 phosphorylation. Conversely, blocking Batimastat PDGFRb dimerization with GST Ig4b did not affect DRD4 mediated ERK1/2 phosphorylation. These results suggest that DRD4 can activate ERK1/2 by utilizing the PDGFRb in a way that does not require receptor dimerization. Previous data from our laboratory suggested the involvement of PI3 kinase in the DRD4 mediated activa tion of PDGFRb.