Figure 2.

Expression of a chimera of the exofacial and transmembrane domains of the β₂AR with the cytoplasmic domains of Rfz-2 gene products enables ISO-stimulated differentiation of F9 cells to PE. (A) Schematic representation of the Rfz-2/β₂AR construct. (B) Reverse transcription–PCR and immunoblotting of Rfz-2/β₂AR stably expressed in F9 cells. The RNA of F9 clones harboring either the empty expression vector (EV) or the vector expressing the Rfz-2/β₂AR chimera was reverse-transcribed and amplified. The molecular markers (MK) indicate the relative size in bp of the amplified products. Note the identification of the 55-kDa chimeric receptor species in the immunoblots of clones 1 and 2 when stained with anti-β₂AR antibodies. (C) The β-adrenergic agonist ISO (10 μM) stimulates whereas the β-adrenergic antagonist propranolol (10 μM) blocks differentiation to PE in F9 clones expressing the Rfz-2/β₂AR chimera. Clones were treated with ligand for 4 days and then fixed and stained with TROMA-1 antibody to reveal PE formation. Indirect immunofluorescence (IIF) and phase-contrast (PC) images are displayed. (D) Dose response for ISO-stimulated differentiation to PE in F9 clones expressing the Rfz-2/β₂AR chimera. Formation of PE was revealed by positive staining with the TROMA-1 antibody, shown by IIF. Based on multiple dose responses, the K_d for ISO-induced PE formation appears to be ≈2 μM in the F9 clones stably expressing the chimeric receptor.