Growth Factor Receptor Pathway Substrate Research Articles

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Parallel Dimers and Anti-parallel Tetramers Formed by Epidermal Growth Factor Receptor Pathway Substrate Clone 15 (EPS15)

The recently discovered localization of epidermal growth factor receptor pathway substrate clone 15 (Eps15) to plasma membrane clathrin-coated pits and its constitutive association with the endocytic clathrin adaptor protein complex, AP-2, strongly suggest that Eps15 has an important role in the pathway of clathrin-dependent endocytic traffic. We report here that Eps15 forms dimers and tetramers of distinct shape. The Eps15 dimer is an elongated molecule, 32 nm in length. There is a globular "head" at one end of the molecule and an extended "stalk" of 25 nm which is kinked at about 17 nm away from the head. In the Eps15 dimer, two subunits are arranged parallel to each other, so that the head corresponds to two side by side copies of the N-terminal region I, which contains the three Eps15 homology domains. The proximal part of the stalk is the coiled-coil central region II containing 20 heptad repeats. The kink is at the boundary between region II and the C-terminal region III, which contains the AP-2 binding site, 15 aspartic-proline-phenylalanine repeats, and proline-rich Src homology domain ligand sites. The Eps15 tetramer has a "dumbbell" shape, approximately 31 nm in length; it is formed by the anti-parallel association of two Eps15 dimers. Formation of these Eps15 tetramers appears to require contacts between regions I of one dimer and regions III of a second apposing dimer. The extended shapes of the Eps15 dimers and tetramers suggest how Eps15 oligomers are located in the clathrin coat. We discuss the implications for accessibility to partners and for proposed functions of Eps15.

Epidermal Growth Factor Induces Ubiquitination of Eps15

Epidermal growth factor (EGF) receptor pathway substrate clone 15 (Eps15) has been described as a 142-kDa EGF receptor substrate. It has been shown to bind to the EGF receptor, adaptor protein-2, and clathrin and is present at clathrin-coated pits and vesicles. Upon stimulation of cells with EGF or transforming growth factor alpha, Eps15 becomes rapidly and transiently phosphorylated on tyrosine residues. This phosphorylation coincides with an increase of 8 kDa in molecular mass. Here we show that this increase in molecular mass is not due to tyrosine phosphorylation. Instead, we found both by Western blotting and protein sequencing that this EGF-induced increase in molecular mass is the result of monoubiquitination. Eps15 ubiquitination but not tyrosine phosphorylation was inhibited under conditions that blocked EGF-induced internalization of the EGF receptor. Our results establish ubiquitination as a second form of EGF-stimulated covalent modification of Eps15.

Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal.

The kinetics of spermatogenesis in mammals are reviewed with special emphasis on the seminiferous epithelium cycle and spermatogonial renewal . 2 features of the spermatogenic process are that for a given species each step of spermatogenesis has a constant duration and that many spermatogonial stem cells enter spermatogenesis simultaneously and almost synchronously. The seminiferous epithelium composed of 5 or 6 generations of germ cells form cellular associations of fixed compositi on. Spermatids at a given step of spermiogenesis are always associated with spermatocytes and spermatogonia at given steps of development. These cellular groupings were taken to represent stages of a cycle of the seminferous epithelium. The number of stages composing the cycle vary with the species and the criteria used to characterize them. The classifications of cell associations are particulary useful in investigations of the mode of renewal of the spermatogonial population. The stem cells that belong to the Type A category may be classified as "reserve" and "renewing" stem cells. The former appear to be actively involved in the production of spermatocytes in normal adult animals but the latter initiate at each cycle of the seminiferous epithelium a series of mitoses during which they renew themselves and simultaneously give rise to "differentiating" spermatogonia. The "nodal" or key mitosis that result appear to be equivalent in type and not bivalent.