Lysosomal traffic of liganded endothelin B receptor

Abstract

The endothelin B receptor (ETB) is an endothelial cell receptor found in caveolae. Studies with GFP-tagged ETB have suggested that the protein is constitutively endocytosed and targeted to lysosomes where it is rapidly degraded. We report that iodinated endothelin-1 ligand (ET-1) is taken up by cells transfected with ETB and remains undegraded for at least 17 h. Analysis of the intracellular traffic of endocytosed ET-1 on isotonic Ficoll gradients shows that it is rapidly internalised to lysosomes by a chloroquine sensitive and cholesterol dependent pathway. Low-temperature nonreducing SDS gels show that the ET-1 initially binds to full-length GFP-tagged ETB, which is rapidly clipped at the amino-terminus and is then stable for at least 6 h. Analysis of GFP tagged ETB on reducing SDS gels shows that it is proteolytically cleaved with a half time of approximately 3 h. However, nonreducing gels show that the receptor is virtually intact, suffering only a similar cleavage to the liganded receptor. We conclude that the ETB receptor shows remarkable stability in lysosomes, held together by disulfide bonds, and maintaining ligand binding for long periods of time.

Vascular Matrix Metalloproteinase-9 Mediates the Inhibition of Myogenic Reactivity in Small Arteries Isolated from Rats after Short-Term Administration of Relaxin

“…Because immunohistochemistry is not quantitative unless the signal is rendered virtually on or off by the experimental intervention, we did not attempt to assess differences using this technique and instead we relied upon the zymography for quantitation. This exclusive localization to vascular smooth muscle with increased density of MMP-9 in the immediate subendothelial region beneath the internal elastic lamina, raises the possibility that MMP-9 may be secreted toward the endothelium where it processes big ET to ET 1-32 at a gly-leu bond, thereby activating the endothelial ET B receptor and endothelial nitric oxide synthase localized in caveolae (21,22). Indeed, we demonstrated in the present study that the inhibition of myogenic reactivity after shortterm rhRLX administration is mediated by the endothelial ET B receptor and nitric oxide by using specific inhibitors.…”

“…One possible explanation for this apparent paradox relates to anatomical location. That is, after chronic rhRLX administration or during midterm pregnancy, vascular MMP-2 may be up-regulated specifically in caveolae where it would be ideally situated to interact with the endothelial ET B receptor (21) and endothelial nitric oxide synthase (22). Indeed, MMP-2 as well as membrane type 1-MMP FIG.…”

Intracellular Endothelin Type B Receptor-driven Ca2+ Signal Elicits Nitric Oxide Production in Endothelial Cells

“…Our results indicate that degradation is not the only fate of endolysosomal ET B ; they are also functional and involved in ET-1 signaling. This may correlate with the remarkable stability of ET B receptors in these organelles (44).…”

Mechanisms of Renal Vasodilation and Hyperfiltration During Pregnancy: Current Perspectives and Potential Implications for Preeclampsia

“…Big ET is released from Weibel-Palade bodies in response to pulsatile pressure in vivo or increases of intraluminal pressure in vitro (Russell et al 1998;Macarthur et al 1994;Hishikawa et al 1995). MMP-2 (Puyraimond et al 2001), eNOS (Shaul 2002), and possibly the ET B receptor (Foster et al 2003) are localized in caveolae of endothelial cells. Whether the relaxin receptor also resides in caveolae requires further investigation.…”