Jab1 regulates levels of endothelin type A and B receptors by promoting ubiquitination and degradation.

Endothelin type A receptor (ET(A)R) plays an important role in some cardiovascular disorders where ET(A)R levels are increased. However, regulatory mechanisms for ET(A)R levels are unknown. Here, we identified Jun activation domain-binding protein 1 (Jab1) as an ET(A)R-interacting protein by yeast two-hybrid screening of human heart cDNA library using carboxyl terminal tail (C-tail) of ET(A)R as a bait. The interaction was confirmed by glutathione S-transferase pull-down assay, co-immunoprecipitation in HEK293T cells expressing ET(A)R-myc and FLAG-Jab1, and confocal microscopy. Jab1 knockdown increased whole cell and cell surface levels of ET(A)R and ET-1-induced ERK1/2 phosphorylation in HEK293T cells expressing ET(A)R, whereas Jab1 overexpression decreased them. Jab1 overexpression accelerated disappearance rate of ET(A)R after protein synthesis inhibition as an index of a degradation rate. ET(A)R was constitutively ubiquitinated, and the level of ubiquitination was enhanced by Jab1 overexpression. Long-term ET-1 stimulation markedly accelerated the rate of ET(A)R degradation and increased the amount of Jab1 bound to ET(A)R with a maximal level of 500% at 3h. In the absence of ET-1 stimulation, the level of ET(B)R was lower than that of ET(A)R and the degradation rate of ET(B)R was markedly faster than that of ET(A)R. Notably, the amount of Jab1 bound to ET(B)R and ubiquitination level of ET(B)R were markedly higher than those for ET(A)R. Taken together, these results suggest that the amount of Jab1 bound to ETR regulates the degradation rate of ET(A)R and ET(B)R by modulating ubiquitination of these receptors, leading to changes in ET(A)R and ET(B)R levels. Copyright 2009 Elsevier Inc. All rights reserved.