Ligand-induced trafficking of the sphingosine-1-phosphate receptor EDG-1.
The endothelial-derived G-protein-coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-1 interaction. SPP binds to EDG-1-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a tau1/2 of approximately 15 min. The EDG-1-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1-GFP recycles back to the plasma membrane with a tau1/2 of approximately 30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.
Liu
C H
CH
Center for Vascular Biology, Department of Physiology, University of Connecticut School of Medicine, Farmington, Connecticut 06030, USA.
Thangada
S
S
Lee
M J
MJ
Van Brocklyn
J R
JR
Spiegel
S
S
Hla
T
T
D000595:Amino Acid Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D049452:Green Fluorescent Proteins; D006801:Humans; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D007668:Kidney; D007700:Kinetics; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D008246:Lysophospholipids; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D017874:Immediate-Early Proteins; D008024:Ligands; D008164:Luminescent Proteins; D008246:Lysophospholipids; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049312:Receptors, Lysophospholipid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C060506:sphingosine 1-phosphate; D013110:Sphingosine