PDGFRβ siRNAs inhibit dopamine-mediated ERK1/2 activation. (A) 100 nM of double-stranded PDGFRβ interference RNA (PDGFRβ silencer 1) or (B) 30 nM of double-stranded PDGFRβ RNA (PDGFRβ silencer 2) was transfected into the CHO/DRD4 cells. Cells were harvested at 72 h post transfection and PDGFRβ expression was determined. For phospho-ERK1/2 expression (Thr202/Tyr204), cells were serum-starved overnight (beginning at 48 h post-transfection) and subsequently treated with dopamine (1 βM) or PDGF-BB (10 ng/ml).

Phosphorylation of PDGFRβ following DRD4 versus PDGF-BB stimulation. (A) Differential pattern of phosphorylation of the PDGFRβ elicited by PDGF-BB and DRD4 in CHO-K1 cells overexpressing DRD4 and FLAG-PDGFRβ (CHO/DRD4-PR). (B) Time course of dopamine-stimulated phosphorylation of endogenous PDGFRβ and ERK1/2 in CHO/DRD4 in the presence or absence of AG1295 (10 μM). AG1295 was pre-incubated with the cells for 1 h before agonist stimulation. (A-B) CHO/DRD4 or CHO/DRD4-PR was stimulated for 5 min, or for the time indicated, with 10 ng/mL PDGF-BB or 1 μM dopamine. IP: immunoprecipitation; IB: immunoblot.

Cross-tyrosine phosphorylation of PDGFRβ is not required for DRD4-mediated ERK1/2 activation. (A) Dose-response of DRD4- and PDGF-BB-mediated ERK1/2 phosphorylation. CHO/DRD4 (black square) or CHO/DRD4-PR (black triangle) cells were treated with different concentrations of dopamine or PDGF-BB for 5 min. (B) Effect of C-truncPDGFRβ on the phosphorylation of full-length PDGFRβ and ERK1/2 in CHO/DRD4-PR cells mediated by PDGF-BB and DRD4. CHO/DRD4-PR cells were transfected with C-truncPDGFRβ, or with lacZ as a control. IP: immunoprecipitation; IB: immunoblot. (C) DRD4-mediated phosphorylation of ERK1/2 in CHO/DRD4 was not blocked by C-truncPDGFRβ. (D) Requirement for PDGFRβ in the DRD4-mediated phosphorylation of ERK1/2 in C-truncPDGFRβ-transfected cells. Upper blot shows the phosphorylation of ERK1/2 in lacZ-transfected and C-truncPDGFRβ-transfected CHO/DRD4 cells that have been pre-treated with DMSO or 1 μM tyrphostin A9 prior to stimulation with dopamine. (B-D) The blots were stripped and re-probed with antibodies for PDGFRβ or ERK1/2 to verify that the total protein did not vary between lanes. In (C) and (D), the bar graphs show densitometric measurements of the phosphorylation signal from the respective experiments. The results are expressed as percentages relative to lacZ control and indicated as mean ± SEM. The number of experiments is indicated in parentheses.

Blocking PDGFRβ dimerization inhibits ERK1/2 activation in PDGF-BB-treated, but not in dopamine-stimulated CHO-K1 cells. (A) CHO/DRD4-PR cells were pretreated with 4 μg/mL of either GST or GST-PDGFRβ immunoglobulin domain 4 (GST-Ig4β) fusion proteins for 20 min at 37°C to prevent PDGFRβ dimerization and were then stimulated with 1 μM dopamine or 10 ng/mL PDGF-BB for 5 min. Lysates were taken for western blotting with phospho-ERK1/2 antibody (upper blot). The same blot was stripped and reprobed for total PDGFRβ to demonstrate equal loading of all lanes (lower blot). (B) Pre-treatment with 100 nM wortmannin for one hour abolished the dopamine-mediated ERK1/2 signal following PDGFRβ dimerization block with GST-Ig4β. The same blot was stripped and reprobed for β-tubulin to demonstrate equal loading of all lanes (lower blot). The bar graphs show the densitometric measurement of the relative signals from phospho-ERK1/2 over two to four experiments, and the quantities were given as mean ± SEM.

Absence of a paracrine mediator in DRD4-mediated PDGFRβ transactivation. (A) mRNA expression of known PDGF ligands and the α and β subtypes of PDGFR. RT-PCR was performed on total RNA extracted from CHO-K1 cells or as controls, on total RNA taken from the cardiac tissues of hamster or C57/black mouse. The primers were designed with the primer3 software to target sequences that are conserved between mouse and human and to yield PCR products of sizes between 200 to 300 bp. Expression of PDGF-A, PDGF-C and PDGFRβ were detected in CHO-K1 cells, with PDGF-D being a non-specific band. (B) CHO/DRD4 or COS-7 cells were treated with the diphtheria toxin mutant CRM197 (10 μg/mL) or the metalloproteinase inhibitor GM6001 (5 μM) for 30 min. The cells were subsequently stimulated with 1 μM dopamine (CHO/DRD4) or 10 μM LPA (COS-7), and lysates were taken for western blotting with phospho-ERK1/2 (CHO/DRD4) or phospho-Shc (COS-7). The inhibitors had no effect on dopamine-stimulated ERK1/2 phosphorylation. (C) CHO/DRD4 and CHO-K1 cells stably expressing FLAG-PDGFRβ (CHO/PR) were cultured separately or together at 90% confluency in a ratio of 1:1. The cells were stimulated with 10 ng/mL PDGF-BB or 1 μM dopamine as indicated. Lysates were collected to probe with phospho-ERK1/2 antibody (left blot) or taken for immunoprecipitation with anti-FLAG antibody and immunoblotted with phosphotyrosine antibody (pTyr) (right blot). IP: immunoprecipitation; IB: immunoblot.