Receptor-Mediated Endocytosis of an Extracellular Steroid-Binding Protein (TeBG) in MCF-7 Human Breast Cancer Cells*

Abstract

Previous studies suggested that an extracellular steroid-binding protein, testosterone-estradiol-binding globulin (TeBG), can enter a variety of cells. Experiments were conducted to determine whether uptake of TeBG occurs by nonspecific fluid phase endocytosis or via a specific receptor-mediated process. In human breast carcinoma cells (MCF-7) maintained on serum-free medium, exposure to radiolabeled TeBG resulted in cellular uptake, which reached a plateau by 6 h and could be inhibited 80% by competition with unlabeled TeBG. Uptake was temperature dependent with cell-associated radioactivity at 37 C being 1.6-fold higher than at 4 C. Subsequent exposure of cells to pronase resulted in release of the cell-associated TeBG by 88% and 44% at 4 C and 37 C, respectively. After transfer to media devoid of TeBG, approximately 35% of cell-associated radioactivity was release into the medium at 37 C; it was not possible to distinguish whether this was released from the cell surface or from inside the cell. Investigation of the localization of TeBG-gold complexes by electron microscopy revealed that TeBG first binds to the plasmalemma. Within 15 min label appears in receptosomes, which fuse to form multivesicular endosomes. By 1 h all label is observed in multivesicular endosomes and lysosomes, most of which are in the Golgi zone. Localization of the internalized radioactivity using classical cell fractionation techniques showed it appears in a symmetrical band exhibiting the same buoyant density as the lysosomal marker acid phosphatase. The observations reported here show that: 1) TeBG binds to MCF-7 cells; 2) some of the bound TeBG is taken up via a mechanism with all the characteristics of receptor-mediated endocytosis; and 3) within these cells TeBG is localized in endosomes and lysosomes.

Cellular uptake of steroid carrier proteins—Mechanisms and implications

“…Last but not least, surface binding sites have been proposed to mediate the cellular uptake of carrier-bound steroid hormones. For example, uptake of SHBG has been documented in epithelial cells of the epididymis and endometrium (Feldman et al, 1981;Gerard et al, 1988;Noe et al, 1992), in MCF-7 cells (Porto et al, 1991), and in neurons in the brain (Caldwell et al, 2007). A biological process, where uptake of SHBG is suggested to play a physiological role is the delivery of testosterone to principal cells in the epididymis (Fig.…”

Identification of sex hormone binding globulin-interacting proteins in the brain using phage display screening

“…Pope and Lee (27) identified another protein called SNARE associated protein which is also a component of the SNARE complex (53). SHBG internalization was observed in a number of tissues (7,8,10,11,13,54) including the brain (15), so it is possible that some of the proteins we identified here, in particular SNAP-25, are involved in that internalization process.…”

“…SHBG internalization has been demonstrated in multiple tissues (7)(8)(9)(11)(12)(13)54) including the brain (15) suggesting that SHBG along with associated steroids are selectively brought into cells, where cytosolic proteins may bind the SHBG. This work may have identified proteins that are candidates for an intracellular SHBGbinding complex.…”

“…Other studies (5,6) also suggested that there are membrane-associated receptors for SHBG in the uterus. These membrane-associated receptors may be important for internalization of SHBG, which was observed in many tissues (7)(8)(9)(10)(11)(12)(13)(14) including the brain (15). The Nykjaer and Willnow laboratory demonstrated that a membrane-associated protein called megalin, a 600-kDa member of the LDL receptor gene family, is capable of binding to and facilitating the internalization of both SHBG and its bound steroid (16).…”

Steroid hormone enhancement of gene delivery to a human airway epithelial cell line in vitro and mouse airways in vivo

“…The lipophilic nature of steroid hormones means that they can enter cells by diffusion through the plasma membrane although some steroid hormones including estrogen are also internalized by receptormediated endocytosis. 21,22 This could account for the large reduction in the transfection efficiency when the cells are pre-treated with methyl-prednisolone if there was binding and down-regulation of these sites. Interestingly, 17 ␣-estradiol, which does not bind to intracellular estrogen receptors, had no significant effect on luciferase reporter gene expression to confluent 16HBE cells when incorporated into a lipoplex at 200 m. However, when plasmid was visualized by con-focal microscopy ␣-estradiol in the lipoplex was found to increase plasmid entry into cells, much like ␤-estradiol.…”