title: Subtype-specific trafficking of endothelin receptors.
abstract: We investigated the subcellular localization of two endothelin receptors (ET(A)R and ET(B)R). To visualize these receptors directly, the C terminus of each receptor was fused to the N terminus of enhanced green fluorescent protein (designated as ETR-EGFP). When transiently expressed in various mammalian cell lines, ET(A)R-EGFP was predominantly localized on the plasma membrane. By contrast, ET(B)R-EGFP was, independent of ligand stimulation, predominantly localized on the intracellular vesicular structures containing Lamp-1. Immunoblot analyses revealed that at steady state ET(B)R-EGFP was highly degraded, and its degradation was inhibited by bafilomycin A(1). Antibody uptake experiments suggested that the ET(B)R-EGFP molecules were internalized from the plasma membrane. It is therefore likely that ET(B)R is first transported to the plasma membrane and then internalized, irrespective of ligand stimulation, to lysosomes where it undergoes proteolytic degradation. Exchanging the C-terminal cytoplasmic tails of the two ETRs revealed that the cytoplasmic tail is responsible for both the intracellular localization and the degradation of the receptors. Deletion of the extreme C-terminal 35 amino acids from both receptors allowed the receptor proteins to localize predominantly in the intracellular vesicles and to degrade. These observations indicate that the cytoplasmic tail of ET(A)R determines its plasma membrane localization. Stimulation with endothelin-1 increased the amount of intact ETR-EGFP fusion proteins without increasing their de novo synthesis, suggesting that binding of endothelin-1 stabilizes the ETRs.
authors.affiliation: Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Abe
authors.forename: K
authors.initials: K
authors.lastname: Nakayama
authors.forename: A
authors.initials: A
authors.lastname: Yamanaka
authors.forename: T
authors.initials: T
authors.lastname: Sakurai
authors.forename: K
authors.initials: K
authors.lastname: Goto
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000302:isolation & purification; D001692:Biological Transport; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D019332:Endothelin-1 / Q000378:metabolism; D019334:Endothelin-3 / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000302:isolation & purification; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D051381:Rats; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: D015703:Antigens, CD; D019332:Endothelin-1; D019334:Endothelin-3; D008024:Ligands; D008164:Luminescent Proteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins