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The ubiquitin-dependent endocytosis motif is required for efficient incorporation of growth hormone receptor in clathrin-coated pits, but not clathrin-coated lattices

¹ Department of Cell Biology, AZU G02.525, Heidelberglaan 100, Utrecht University Medical Center and Institute of Biomembranes, 3584 CX Utrecht, The Netherlands
² Center for Biomedical Genetics, PO Box 80042, 3508 TA Utrecht, The Netherlands

View larger version (134K): [in a new window]   Fig. 1. Ultrathin cryosections of wild-type GHR cells showing the endocytic compartments involved in GH endocytosis. GH was added to cells for 30 minutes, after which cells were fixed and GH was visualized by immunogold labeling. (A) GH (10 nm gold) is internalized from the plasma membrane (P) via clathrin-coated pits. The electron dense layer (arrowhead) at the cytoplasmic site of the coated pit is characteristic for the presence of clathrin. (B) GH (10 nm gold) is present at the plasma membrane in close vicinity to caveolae (small arrowheads), which are always devoid of GH label. (C) Early after uptake, GH (10 nm gold) is transported to vacuolar early endosomes (EE). (D) At a later stage of endocytosis, GH (10 nm gold) is found on the intra-endosomal vesicles of early (EE) and late (LE) endosomes, indicative of targeting to lysosomes. The 5 nm gold represents internalized BSA-gold that was added for 1 hour. (E) Double-labeling of GH (10 nm gold) and internalized Tf (15 nm gold) shows co-localization in vacuolar early endosomes (EE), but not in the Tf-rich recycling endosomes (arrows). N, nucleus. Scale bars: 200 nm.

View larger version (57K): [in a new window]   Fig. 2. Wild-type GHR cells incubated for 30 minutes with GH were double immunolabeled for GH (15 nm gold) and clathrin (5 nm gold) to unequivocally identify coated areas. (A) GH was found at non-coated areas of the plasma membrane as well as in flat clathrin-coated lattices (arrowheads). (B) In addition, GH was regularly seen in clathrin-coated pits (arrow). P, plasma membrane. Scale bars: 200 nm.

View larger version (154K): [in a new window]   Fig. 3. Cryosections of wild-type GHR cells co-incubated with BSA-5 nm gold and GH for 1 hour. (A-C) BSA-gold (5 nm) and GH (10 nm gold) co-localize extensively in late endosomes (LE). (B,D) Lysosomes (L) are reached by BSA gold but lack GH staining, which is probably due to rapid degradation. EE, early endosome; N, nucleus; P, plasma membrane. Scale bars: 200 nm.

View larger version (132K): [in a new window]   Fig. 4. Cryosections of GHR-F327A cells incubated for 30 minutes with GH, showing the exclusion of GH from clathrin-coated pits and vesicles. (A) GH (15 nm gold) accumulates at the plasma membrane (P). (B) GH (15 nm gold) can also be found associated with clathrin-coated lattices (arrowheads) at the plasma membrane, which positively label for clathrin (5 nm gold). The arrows point to clathrin-coated vesicles. (C) GH (15 nm gold) is found in a clathrin (5 nm gold) -coated lattice (arrowhead), but not in the clathrin-coated pit (arrow). (D) GH (10 nm gold) and (E) GH (15 nm gold) are two more examples, showing that GH is excluded from clathrin-coated pits (arrow). Scale bars: 200 nm.