N. Benlimame et al., LOCALIZATION OF AUTOCRINE MOTILITY FACTOR-RECEPTOR TO CAVEOLAE AND CLATHRIN-INDEPENDENT INTERNALIZATION OF ITS LIGAND TO SMOOTH ENDOPLASMIC-RETICULUM, Molecular biology of the cell, 9(7), 1998, pp. 1773-1786
Autocrine motility factor receptor (AMF-R) is a cell surface receptor
that is also localized to a smooth subdomain of the endoplasmic reticu
lum, the AMF-R tubule. By postembedding immunoelectron microscopy, AMF
-R concentrates within smooth plasmalemmal vesicles or caveolae in bot
h NIH-3T3 fibroblasts and HeLa cells. By confocal microscopy, cell sur
face AMF-R labeled by the addition of anti-AMF-R antibody to viable ce
lls at 4 degrees C exhibits partial colocalization with caveolin, conf
irming 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 degrees C, bAMF accumulates in densely labeled perinuclear structu
res as well as fainter tubular structures that colocalize with AMF-R t
ubules. After a subsequent 2- to 4-h chase, bAMF is localized predomin
antly to AMF-R tubules. Cytoplasmic acidification, blocking clathrin-m
ediated endocytosis, results in the essentially exclusive distribution
of internalized bAMF to AMF-R tubules. By confocal microscopy, the tu
bular structures labeled by internalized bAMF show complete colocaliza
tion with AMF-R tubules. bAMF internalized in the presence of a 10-fol
d excess of unlabeled AMF labels perinuclear punctate structures, whic
h are therefore the product of fluid phase endocytosis, but does not l
abel 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 tubul
es. AMF-R is therefore internalized via a receptor-mediated clathrin-i
ndependent pathway to smooth ER. The steady state localization of AMF-
R to caveolae implicates these cell surface invaginations in AMF-R end
ocytosis.