Guanine nucleotide exchange factor-like factor (Rlf) induces gene expression and potentiates α₁-adrenergic receptor-induced transcriptional responses in neonatal rat ventricular myocytes

Expression of constitutively active Ras (V12Ras) in cultured neonatal rat ventricular myocytes or targeted cardiac expression of V12Ras in transgenic mice induces myocardial cell growth and expression of genes that are markers of cardiac hypertrophy including atrial natriuretic factor (ANF) and myosin light chain-2. However, the signaling pathways that modulate the effects of Ras on acquisition of the various features of cardiac hypertrophy are not known. We identified the Ral guanine nucleotide exchange factor-like factor (Rlf) in a yeast two-hybrid screen of human heart cDNA library using Ras as bait, suggesting that Ras signaling in the heart may involve Rlf. We demonstrate here that Rlf is expressed in human heart. Expression of wild type Rlf or Rlf-CAAX, a membrane-targeted mutant of Rlf, transactivated ANF and myosin light chain-2 promoters but did not activate canonical cAMP responsive elements or phorbol ester responsive elements, suggesting that Rlf expression does not lead to a generalized increase in transcription. Transfection of mutant ANF promoterreporter gene constructs demonstrated that the proximal serum response element is both necessary and sufficient for Rlf-inducible ANF expression. Rlf-induced ANF promoter activation required Ral and Cdc42 but not RhoA, Rac1, ERK, or p38 kinase activation. In addition, Rlf potentiated α₁-adrenergic receptor (α₁-AR)-induced ANF expression. Prolonged activation of the α₁-AR increases RalGTP levels in neonatal rat ventricular myocytes, further emphasizing a role for Ral guanine nucleotide exchange factors in α₁-AR signaling. Overall, this study supports the concept that Rlf and Ral are important previously unrecognized signaling components that regulate transcriptional responses in myocardial cells.