Gq/G13 signaling by ET-1 in smooth muscle: MYPT1 phosphorylation via ETA and CPI-17 dephosphorylation via ETB.
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.
Department of Physiology, Virginia Commonwealth University Medical School, Richmond, Virginia 23298, USA.
Eric
E
Hersch
Jiean
J
Huang
John R
JR
Grider
Karnam S
KS
Murthy
D000818:Animals; D019332:Endothelin-1 / Q000502:physiology*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D007422:Intestines; D009119:Muscle Contraction / Q000502:physiology; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D054648:Protein Phosphatase 2; D011817:Rabbits; D044022:Receptor, Endothelin A / Q000378:metabolism*; D044023:Receptor, Endothelin B / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology
D019332:Endothelin-1; D009124:Muscle Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D011493:Protein Kinase C; D054648:Protein Phosphatase 2; D043263:Myosin-Light-Chain Phosphatase; D043802:GTP-Binding Protein alpha Subunits, Gq-G11