Abstract 13719: Direct Cross-Talk and Reciprocal Regulation Between β-1-Adrenergic Receptor and Sphingosine1-Phosphate Receptor
Sphingosine-1-phosphate receptor (S1P1) and ß-1-adrenergic receptor (ß1AR) are both regulated by the G-protein coupled receptor kinase-2 (GRK2) and are expressed at high levels in the heart. While ß1AR acts via stimulatory G-alpha protein subunit (Gs) with a consequent increase in contractility, S1P1 couples to the inhibitory G-alpha protein subunit (Gi), antagonizing the effects mediated by isoproterenol (ISO, βARs agonist). The molecular interaction between different receptors has become a very interesting topic in understanding cardiac pathophysiology. While chronic stimulation of ß1AR can be detrimental, S1P1 has shown a cardioprotective effect in experimental settings. Therefore, we decided to test the hypothesis of a cross-talk between these two receptors. We used HEK293 cells overepressing the mouse wild type ß1AR carrying a Flag epitope (WT ß1AR) or 2 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-ß1AR) or the putative GRK phosphorylation sites (GRK-ß1AR). All cells were transiently transfected with S1P1-GFP. By confocal microscopy we evaluated the receptor-receptor interaction following ISO or sphyngosine (S1P, S1P1 agonist) stimulation. Either ISO or S1P are able to induce internalization of both WT ß1AR and S1P1 and the two receptors co-localize in the cytosol. Metoprolol (β1AR antagonist) abolished the effect of ISO on both ß1AR and S1P1 internalization. The same reciprocal downregulation (with ISO or S1P) was observed for PKA-ß1AR and S1P1. The interaction between S1P1 and either WT ß1AR or PKA-ß1AR induced a ERK 1/2 and GRK2 significant activation. Surprisingly, GRK-ß1AR and S1P1 were co-internalized after agonist stimulation. In fact, ISO stimulation only induced GRK-ß1AR but not S1P1 internalization and S1P stimulation downregulated S1P1 but not GRK-ß1AR. The lack of reciprocal downregulation was associated to a very blunted activation of both GRK2 and ERK 1/2. In conclusion we demonstrate a strictly correlation between S1P1 and ß1AR signaling, which is GRK-dependent. The absence of GRK phosphorylation sites not only abolishes this reciprocal downregulation but also affects the ability of this molecular interaction to activate important downstream signaling or regulating molecules.
- © 2011 by American Heart Association, Inc.