The regulation of the cellular distribution and intracellular signaling properties of the alpha(1B)- and alpha(1D)- adrenoceptor (alpha(1)-AR) subtypes was examined in stably transfected Rat 1 fibroblasts. In unstimulated cells, alpha(1B)-AR expression was noted primarily on the cell surface. Treatment with phenylephrine induced internalization of the alpha(1B)-AR and promoted association with arrestin 2. The internalized alpha(1B)-AR colocalized with the transferrin receptor, an endosomal marker. In unstimulated fibroblasts, the alpha(1D)-AR was detected in a perinuclear orientation and was colocalized with arrestin 2 in a compartment also containing the transferrin receptor. After treatment with prazosin, which exhibits inverse agonist properties, the alpha(1D)-AR was redistributed from intracellular sites to the cellular periphery and was no longer associated with the transferrin receptor or arrestin 2. alpha(1D)-AR-expressing cells exhibited a high degree of basal activity for both inositol phosphate formation and extracellular signal regulated kinase (ERK), which was reduced by treatment with prazosin. In these cells, phenylephrine induced a dose-dependent increase in inositol phosphate formation but had no effect on ERK activity. In alpha(1B) -AR-expressing cells, phenylephrine stimulated both inositol phosphate formation and ERK activity. These data show that: 1) there are differences in the cellular localization of the alpha(1)-AR subtypes; 2) the alpha(1B)-AR exhibits expected G protein-coupled receptor activity regarding cellular localization, agonist-mediated internalization, and coupling to second messengers; and 3) the alpha(1D)-AR is constitutively active and, as a result, is localized to intracellular compartments involved in receptor recycling.
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 ␣ 1 -adrenergic receptor (␣ 1 -AR)-induced ANF expression. Prolonged activation of the ␣ 1 -AR increases RalGTP levels in neonatal rat ventricular myocytes, further emphasizing a role for Ral guanine nucleotide exchange factors in ␣ 1 -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.
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