Differential Coupling of Human Endothelin Type A Receptor to Gq/11 and G12 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 (ETAR) expressed in Chinese hamster ovary cells at 32,100 (ETAR-high-CHO) and 893 (ETAR-low-CHO) fmol·mg protein−1. Endothelin-1 (ET-1) elicited a sustained increase in intracellular Ca2+ concentration ([Ca2+]i), which was dependent on Gq/11 protein, phospholipase C (PLC), Na+/H+ exchanger (NHE), and p38 mitogen–activated protein kinase (p38MAPK) in ETAR-high-CHO, whereas the sustained [Ca2+]i increase was negligible in ETAR-low-CHO. Functional study with CytosensorTM microphysiometer showed that ET-1 evoked an NHE1-mediated increase in extracellular acidification rate (ECAR) in ETAR-high-CHO and ETAR-low-CHO. In ETAR-high-CHO, the ECAR response at 30 min after ET-1 stimulation was insensitive to Gq/11 and PLC inhibitors, but sensitive to the p38MAPK inhibitor. In ETAR-low-CHO, the ECAR response at 30 min was sensitive to these inhibitors. Western blot analysis demonstrated that ET-1–induced p38MAPK phosphorylation in ETAR-low-CHO but not in ETAR-high-CHO was mediated via Gq/11 and PLC. The Gq/11/PLC-independent p38MAPK phosphorylation in ETAR-high-CHO was suppressed by expression of the C terminus of Gα12 protein to disrupt receptor-G12 protein coupling. These results provide evidence for multiple signaling pathways of ETAR that were activated via at least the Gq/11/PLC/NHE, G12/p38MAPK/NHE, and Gq/11/PLC/p38MAPK/NHE cascades in an expression level–dependent manner. Keywords:: endothelin type A receptor, receptor expression level, intracellular free Ca2+ concentration, Na+/H+ exchanger, p38 mitogen–activated protein kinase