Figure 3

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.