Lysosomal traffic of liganded endothelin B receptor.
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.
Foster
Natasha
N
Centre for Immunology, St. Vincent's Hospital, University of NSW, Cnr West and Boundary Street, Darlinghurst NSW 2010, Sydney, Australia.
Loi
To Ha
TH
Owe-Young
Robert
R
Stanley
Keith K
KK
D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002738:Chloroquine / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D019332:Endothelin-1 / Q000378:metabolism* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins
D004220:Disulfides; D019332:Endothelin-1; D008024:Ligands; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D011993:Recombinant Fusion Proteins; D002738:Chloroquine; D002784:Cholesterol; D010447:Peptide Hydrolases