Localization of autocrine motility factor receptor to caveolae and clathrin-independent internalization of its ligand to smooth endoplasmic reticulum.
Autocrine motility factor receptor (AMF-R) is a cell surface receptor that is also localized to a smooth subdomain of the endoplasmic reticulum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF-R concentrates within smooth plasmalemmal vesicles or caveolae in both NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell surface AMF-R labeled by the addition of anti-AMF-R antibody to viable cells at 4 degreesC exhibits partial colocalization with caveolin, confirming the localization of cell surface AMF-R to caveolae. Labeling of cell surface AMF-R by either anti-AMF-R antibody or biotinylated AMF (bAMF) exhibits extensive colocalization and after a pulse of 1-2 h at 37 degreesC, bAMF accumulates in densely labeled perinuclear structures as well as fainter tubular structures that colocalize with AMF-R tubules. After a subsequent 2- to 4-h chase, bAMF is localized predominantly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-mediated endocytosis, results in the essentially exclusive distribution of internalized bAMF to AMF-R tubules. By confocal microscopy, the tubular structures labeled by internalized bAMF show complete colocalization with AMF-R tubules. bAMF internalized in the presence of a 10-fold excess of unlabeled AMF labels perinuclear punctate structures, which are therefore the product of fluid phase endocytosis, but does not label AMF-R tubules, demonstrating that bAMF targeting to AMF-R tubules occurs via a receptor-mediated pathway. By electron microscopy, bAMF internalized for 10 min is located to cell surface caveolae and after 30 min is present within smooth and rough endoplasmic reticulum tubules. AMF-R is therefore internalized via a receptor-mediated clathrin-independent pathway to smooth ER. The steady state localization of AMF-R to caveolae implicates these cell surface invaginations in AMF-R endocytosis.
Benlimame
N
N
Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Québec, Canada H3C 3J7.
Le
P U
PU
Nabi
I R
IR
D016475:3T3 Cells; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D002966:Clathrin / Q000502:physiology*; D004705:Endocytosis / Q000502:physiology*; D018871:Endoplasmic Reticulum, Smooth / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D051379:Mice; D016253:Microscopy, Immunoelectron; D060285:Receptors, Autocrine Motility Factor; D018121:Receptors, Cytokine / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D044767:Ubiquitin-Protein Ligases
D002966:Clathrin; D008024:Ligands; D018121:Receptors, Cytokine; C555000:AMFR protein, human; C555002:Amfr protein, mouse; D060285:Receptors, Autocrine Motility Factor; D044767:Ubiquitin-Protein Ligases