G_q/G₁₃ signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ET_A and CPI-17 dephosphorylation via ET_B

Abstract

We analyzed the signaling pathways initiated by endothelin receptors ETA and ETB in intestinal circular and longitudinal smooth muscle cells. The response to endothelin-1 (ET-1) consisted of two phases in both cell types. The initial, transient phase of contraction and phosphorylation of 20-kDa myosin light chain (MLC20) was mediated additively by ETA and ETB receptors and initiated by Galphaq-, Ca2+/calmodulin-dependent activation of MLC kinase. In contrast, the sustained phase was mediated selectively by ETA receptors via a pathway involving sequential activation of Galpha13, RhoA, and Rho kinase, resulting in phosphorylation of MYPT1 at Thr696 and phosphorylation of MLC20. Although PKC was activated, CPI-17 was not phosphorylated and hence did not contribute to inhibition of MLC phosphatase. The absence of CPI-17 phosphorylation by PKC reflected active dephosphorylation of CPI-17 by protein phosphatase 2A (PP2A). PP2A was activated via a pathway involving ETB-dependent stimulation of p38 MAPK activity. CPI-17 phosphorylation was unmasked in the presence of the ETB antagonist BQ-788, but not the ETA antagonist BQ-123, and in the presence of a low concentration of okadaic acid, which selectively inactivates PP2A. The resultant phosphorylation of CPI-17 was blocked by bisindolylmaleimide, providing direct confirmation that it was PKC dependent. We conclude that the two phases of the intestinal smooth muscle response to ET-1 involve distinct receptors, G proteins, and signaling pathways. The sustained response is mediated via selective ETA-dependent phosphorylation of MYPT1. In contrast, ETB initiates an inhibitory pathway involving p38 MAPK-dependent activation of PP2A that causes dephosphorylation of CPI-17.

Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase

“…Previous studies indicate that endothelin A (Gohla et al, 1999;Hersch et al, 2004), endothelin B (Kitamura et al, 1999;Liu and Wu, 2003), and bombesin receptors activate heterotrimeric G-protein subunits, which includes Ga 12 and Ga 13 (Yuan et al, 2001). Activated Ga 12/13 in turn binds to and activate rhoGEFs Suzuki et al, 2003), which then activate RhoA (Dhanasekaran and Dermott, 1996;Kurose, 2003).…”

“…Bombesin and ET-1 receptors activate RhoA through the heterotrimeric G-protein a-subunits Ga 13 (Hersch et al, 2004) and Ga 13 /Gaq (Mao et al, 1998;Yuan et al, 2001), respectively. There is a small subfamily of Dbl RhoGEFs whose members bind and serve as effectors for heterotrimeric G-protein a-subunits.…”

Cross-regulation of VPAC2 receptor desensitization by M3 receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

“…PKC activity was measured in the particulate fraction as described previously (10). Muscle cells (2 ϫ 10 6 cells/ml) were incubated with ACh in the presence of methoctramine, and the reaction was terminated by rapid freezing.…”

Increased expression of G.ALPHA.q protein in bronchial smooth muscle of mice with allergic bronchial asthma

“…It is therefore suggested that the enhanced contractile response via muscarinic M3 receptors in AHR may reflect the over-expression of Gq proteins in airway smooth muscles. As well as muscarinic M3 receptors, many other receptors participate in airway smooth muscle contraction, such as histamine H1 (Daykin et al, 1993), serotonin 5-HT2 (Yang et al, 1994c), leukotrienes (Dumitriu et al, 1997), tachykinin NK2 (Grandordy et al, 1988), bradykinin B2 (Yang et al, 1994a) and endothelin ET (Hersch et al, 2004), are known to couple to phospholipase C via the Gq proteins. In addition, recent studies also revealed that the Gαq directly activates a monomeric GTP-binding protein, RhoA, via a formation of Gαq-p63RhoGEF-RhoA complex (Lutz et al, 2005;2007).…”