β adrenergic receptor-mediated atrial specific up-regulation of RGS5
Introduction
Regulators of G-protein Signalling (RGS) are identified by the presence of a conserved 120–125 amino acid motif, which is referred to as the RGS box (Chidiac and Roy, 2003, Hollinger and Hepler, 2002, Wieland and Mittmann, 2003). RGS containing proteins have been classified into six subfamilies. Most RGS containing proteins are relatively large proteins (>300 residues) and they possess a number of previously identified sequence motifs that suggest specific functions. For example, GRK2 has a kinase domain that serves to phosphorylate GPCRs while its RGS box is capable of inhibiting Gαq (Carman et al., 1999). In contrast, the R4 subfamily of RGS containing proteins are small (ca. 200 residues). These RGSs negatively regulate G-Protein Coupled Receptor (GPCR) signalling mainly by decreasing the levels of the receptor activated Gα-proteins (Hollinger and Hepler, 2002, Wieland and Mittmann, 2003). Since this inhibition occurs without affecting the receptor per se, RGSs are strong candidates to be involved in mediating decreases in GPCR responsiveness that are not due to the internalisation of cell surface receptors. This role is strongly supported by the fact that a number of RGSs are induced in response to a wide variety of stimuli and that increased levels of RGSs lead to significant decreases in GPCR responsiveness. Evidence now exists that the basal cellular level of RGS proteins will also serve to regulate the responsiveness of GPCRs to agonist stimulation. For example, Heximer et al. (Heximer et al., 2003) have demonstrated an increased responsiveness of the P2Y receptor in smooth muscle cells of mice lacking the RGS2 gene. These animals develop a number of abnormalities, including hypertension, indicating that basal control of cardiovascular GPCR responses by RGS2 is of critical importance for regulating signaling.
The β adrenergic receptors (β1- and β2AR) are the most powerful receptors for stimulating an increase in cardiac output in the failing heart (Post et al., 1999). Therefore the loss of βAR responsiveness that occurs in chronic heart failure is a prominent feature in the progression of the disease. Although decreased βAR responsiveness in some forms of cardiac failure is due to a loss of β1AR on the cell surface, there is no decrease in cell surface receptors associated with reduced βAR responsiveness in sepsis and other forms of heart failure (Dorn et al., 2000, Patten et al., 2002). Up-regulation of RGSs may account for the hyporesponsiveness to vasoactive GPCR agonists that occur in cardiovascular tissues during sepsis and in the heart during heart failure. For example, RGS1, RGS4, RGS7 and RGS16 mRNA levels have been shown to increase in animal models of sepsis (Benzing et al., 1999, Panetta et al., 1999, Patten et al., 2002) while RGS3 and RGS4 levels are elevated in failing human hearts (Mittmann et al., 2002, Owen et al., 2001).
A number of transgenic animal models that overexpress adrenergic receptors in the heart have been developed (Koch et al., 2000). In addition to serving as models of cardio pathologies, the study of these animals has also generated insight into the basic physiology of cardiac AR signaling. TG4 mice, which have a 195-fold increase in cardiac β2AR levels, are a widely used model (Heubach et al., 1999, Heubach et al., 2003, Milano et al., 1994). The overexpressed β2AR are partly constitutively active and, consequently, the heart rate of these animals remains maximal even at rest. In spite of these observations, there is experimental evidence to suggest that most of the overexpressed β2AR do not properly couple to Gα-proteins (Heubach et al., 1999). Since RGSs are capable of functionally uncoupling GPCRs and a number of RGSs are induced in response to GPCR stimulation, we hypothesized that some RGS(s) may be up regulated in the heart of TG4 animals. Using a degenerate RT-PCR based screen, we have identified RGS5 as being up regulated in the atria but not in the ventricles of TG4 mice. We further demonstrate that the chronic administration of the βAR agonist isoproterenol to rats is sufficient to cause an increase in the levels of RGS5 in the atria.
Section snippets
Animals and tissues
TG4 and their non-transgenic littermates were generated and identified as previously described (Heubach et al., 1999). Sprague Dawley (male, 280–320 g) rats were used to study the effects of isoproterenol on RGS5 gene expression. The animals were given a daily intra-peritoneal (i.p.) injection of isoproterenol (2.4 mg/kg) for 14 days and the animals were sacrificed 4 hours after the last treatment (Szabo et al., 1975). Control animals were injected with the same volume of physiological saline.
RGS5 is up-regulated in the atria of TG4 mice
A degenerate RT-PCR based strategy was used in order to identify RGSs that may be up regulated in the hearts of transgenic TG4 mice that overexpress the β2AR. We have previously used the same strategy to identify RGS1 and RGS16 as being up regulated in the heart of septic animals (Panetta et al., 1999). Total RNA from the atria and ventricles of TG4 mice as well as their non-transgenic littermates was isolated, reverse transcribed into DNA, amplified by PCR using degenerate RGS-specific oligos
Discussion
RGS5 is a member of the R4 subfamily of RGS containing proteins. These RGSs are small (ca. 200 residues) and are characterized by the presence of an N-terminal region consisting of an amphipathic α-helix (De Vries et al., 2000, Heximer et al., 2001). Overexpression of these RGSs in cultured mammalian cells has revealed that they are capable of inhibiting signalling by acting as a GTPase Accelerating Protein (GAP) for GPCR activated Gqα as well as Giα proteins (Cho et al., 2003b, Zhou et al.,
Acknowledgments
We thank Dr. W. Claycomb for the gift of HL-1 cells and A. Rahman and W. Song for technical assistance. This work was supported by the Canadian Institutes of Health Research (CIHR). The Natural Sciences and Engineering Research Council of Canada (NSERC) and the Heart and Stroke Foundation (HSF) of Québec were also instrumental in the completion of this work. MTG is a FRSQ scholar.
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