Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), members of the 7 transmembrane receptor family, have classically been shown to result from G s -dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby β-arrestins mediate β 1 AR signaling to the EGFR. This β-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via β-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.Introduction β-Adrenergic receptors (βARs) belong to the family of 7 transmembrane receptors (7TMRs) (1) and mediate the powerful regulatory effects on cardiac function of the catecholamine neurotransmitters epinephrine and norepinephrine. β 1 ARs constitute more than 70% of the cardiac βARs. Catecholamine stimulation of β 1 ARs results in activation of heterotrimeric G proteins followed by rapid phosphorylation of the receptor, resulting in desensitization (2). Homologous desensitization of β 1 ARs is brought about by phosphorylation of the receptor by G protein-coupled receptor kinases (GRKs), leading to the recruitment of β-arrestin, which then sterically interdicts further coupling to G proteins (3) and targets the receptor for internalization (3). In addition to β-arrestin's role in terminating G protein signaling, recent studies demonstrate that β-arrestins also function as adapter molecules, allowing for the assembly of multiprotein signaling complexes such as ERKs and tyrosine kinases (4, 5). For the angiotensin II type 1A receptor (AT 1A R), this second wave of β-arrestin-mediated signaling has recently been demonstrated to be independent of G protein signaling (6) and to require the activity of GRKs 5 and 6 (7).The signaling mechanisms that underlie the activation of the mitogenic ERK growth response by 7TMRs are complex and likely result from both classical G protein-regulated effectors such as PKA and PKC and non-G protein-mediated crosstalk, such as EGFR transactivation (8). The current paradigm of transactivation involves agonist stimulation of a 7TMR, which through a number of undefined steps leads to MMP-mediated cleavage and

Background-Although the renin-angiotensin and the ␤-adrenergic systems are interrelated, a direct interaction between ␤-adrenergic receptors (␤ARs) and angiotensin II type 1 receptors (AT 1 Rs) has not been identified. Methods and Results-Here, we provide evidence for a functional and physiological interaction between 2 G protein-coupled receptors: the ␤AR and the AT 1 R. Selective blockade of ␤ARs in mouse cardiomyocytes inhibits angiotensin-induced contractility with an IC 50 that is similar to its inhibition of isoproterenol-mediated contractility. Furthermore, administration of the angiotensin receptor blocker valsartan to intact mice results in a significant reduction in the maximal response to catecholamine-induced elevation of heart rate. The mechanism for this transinhibitory effect of ␤-blockers and angiotensin receptor blockers is through receptor-G protein uncoupling; ie, ␤-blockers interfere with AT 1 R-G q coupling, and valsartan interferes with ␤AR-G s coupling.

Agonist-induced phosphorylation of ␤-adrenergic receptors (␤ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization؊ /GRK ؊ ␤ 1 AR was independent of ␤-arrestin recruitment. Importantly, clathrin inhibitors abolished agonistdependent internalization for both the WT␤ 1 AR and PKA ؊ ␤ 1 AR, whereas caveolae inhibitors prevented internalization only of the GRK ؊ ␤ 1 AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the ␤ 1 AR and 2) the pathway selected for ␤ 1 AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits. ␤-Adrenergic receptors (␤ARs)1 belong to the large family of G protein-coupled receptors (GPCRs) characterized by a typical structure of seven transmembrane domains (1, 2). Three types of ␤ARs, designated ␤ 1 , ␤ 2 , and ␤ 3 ARs, have been cloned from mammalian tissues (1, 3). Both ␤ 1 and ␤ 2 ARs contain phosphorylation sites located in the third intracellular loop and the C-terminal tail of the receptor, which serve as targets for cAMPdependent protein kinase A (PKA), protein kinase C (PKC), and G protein-coupled receptor kinases (GRKs) (2). Furthermore, site-specific mutagenesis studies of the human ␤ 2 AR suggest that low concentrations of agonist preferentially induce phosphorylation at PKA sites, whereas higher concentrations of agonist induce phosphorylation at both PKA and GRK sites (4).Continuous exposure of cells to a stimulus causes ␤ARs to undergo rapid phosphorylation in a process that dampens receptor signaling known as desensitization (4 -8). ␤ARs demonstrate two different mechanisms of desensitization. Agonistspecific or homologous desensitization of ␤ARs consists of a two-step process in which phosphorylation at the C terminus of the ␤AR is mediated by GRKs followed by binding to an arrestin protein, which sterically interrupts signaling to the G protein (5,8). Heterologous or non-agonist-specific desensitization is mediated by the second messenger-stimulated protein kinases A and C, which phosphorylate the receptor and effect a change in receptor conformation such that interaction with the G protein is impaired (5). An important consequence of agonistmediated receptor phosphorylation and desensitization by GRKs is the subsequent internalization of phosphorylated receptors into the cell (9). This process is mediated by ␤-arrestin, which binds to components of the clathrin-mediated endocytic machinery and targets the ligand-bound receptor to clathrincoated pits for endocytosis (10, 11). Interestingly, PKA phosphorylation, although an important mechanism for desensitization (4 -8), appears to play only a small role in ␤ 2 AR

Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.