Differential coupling of human endothelin type A receptor to G(q/11) and G(12) proteins: the functional significance of receptor expression level in generating multiple receptor signaling.
This study examines the influence of receptor expression level on signaling pathways activated via endothelin type A receptor (ET(A)R) expressed in Chinese hamster ovary cells at 32,100 (ET(A)R-high-CHO) and 893 (ET(A)R-low-CHO) fmolmg protein(-1). Endothelin-1 (ET-1) elicited a sustained increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), which was dependent on G(q/11) protein, phospholipase C (PLC), Na(+)/H(+) exchanger (NHE), and p38 mitogen-activated protein kinase (p38MAPK) in ET(A)R-high-CHO, whereas the sustained [Ca(2+)](i) increase was negligible in ET(A)R-low-CHO. Functional study with Cytosensor(TM) microphysiometer showed that ET-1 evoked an NHE1-mediated increase in extracellular acidification rate (ECAR) in ET(A)R-high-CHO and ET(A)R-low-CHO. In ET(A)R-high-CHO, the ECAR response at 30 min after ET-1 stimulation was insensitive to G(q/11) and PLC inhibitors, but sensitive to the p38MAPK inhibitor. In ET(A)R-low-CHO, the ECAR response at 30 min was sensitive to these inhibitors. Western blot analysis demonstrated that ET-1-induced p38MAPK phosphorylation in ET(A)R-low-CHO but not in ET(A)R-high-CHO was mediated via G(q/11) and PLC. The G(q/11)/PLC-independent p38MAPK phosphorylation in ET(A)R-high-CHO was suppressed by expression of the C terminus of G(alpha12) protein to disrupt receptor-G(12) protein coupling. These results provide evidence for multiple signaling pathways of ET(A)R that were activated via at least the G(q/11)/PLC/NHE, G(12)/p38MAPK/NHE, and G(q/11)/PLC/p38MAPK/NHE cascades in an expression level-dependent manner.
Horinouchi
Takahiro
T
Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Japan.
Asano
Hiroshi
H
Higa
Tunaki
T
Nishimoto
Arata
A
Nishiya
Tadashi
T
Muramatsu
Ikunobu
I
Miwa
Soichi
S
D000818:Animals; D016466:CHO Cells; D020013:Calcium Signaling / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D019332:Endothelin-1 / Q000494:pharmacology; D005260:Female; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000378:metabolism*; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects* / Q000235:genetics; D011869:Radioligand Assay; D044022:Receptor, Endothelin A / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
D019332:Endothelin-1; D044022:Receptor, Endothelin A; D017923:Sodium-Hydrogen Exchangers; D048051:p38 Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D043802:GTP-Binding Protein alpha Subunits, Gq-G11