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 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.