6533b7dafe1ef96bd126e39c

RESEARCH PRODUCT

Subtype-specific endothelin-A and endothelin-B receptor desensitization correlates with differential receptor phosphorylation.

subject

description

In the rat cardiovascular system endothelin-1 (ET-1) elicits prolonged physiologic responses mediated by the ET A receptor, whereas the effects mediated by the ET B receptor are transient. The molecular mechanisms for the subtype-specific responses are not yet clear. However, post-translational modifications such as phosphorylation and palmitoylation may play an important role. In Sf9 cells overexpressing the human ET A and ET B receptors, both subtypes are palmitoylated. However, only the ET B but not the ET A receptor is phosphorylated in a ligand-dependent manner. Because phosphorylation is believed to play an important role in ligand-dependent receptor inactivation, we analyzed whether the differential phosphorylation of the ET A and ET B receptors reflects a differential mechanism of receptor inactivation. Using a modified inositol phosphate accumulation assay, we analyzed CHO cells that expressed the ET A or ET B receptor. The ET B receptor was deactivated almost completely within 5 min after agonist stimulation, whereas stimulation of the ET A receptor resulted in sustained activation, i.e., >90% of the initial activity was maintained after 5 min of ligand stimulation and >30% after 20 min. A strong correlation was observed between the time course of ET A receptor inactivation and ET A receptor internalization. The endogenous ET A receptor in Rat-1 cells produced a prolonged stimulation of phospholipase C similar to that seen in CHO cells. Therefore, the sustained signaling activity of the ET A receptor is not a property only of recombinant cell lines. Together, our data suggest rapid ET B receptor inactivation due to phosphorylation and delayed ET A receptor inactivation by internalization. These mechanisms adequately reflect the differential response patterns of the ET receptors under physiologic conditions.