Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 109 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

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Cellular pH homeostasis plays crucial roles in cellular functions, and it is now widely recognized that Na⁺/H⁺ exchangers are among the most prominent players in this process. Although recently described mammalian Na⁺/H⁺ exchanger NHE7 has attracted much attention, its biological functions remain largely unknown. Most proteins exist as protein complexes in the cell and elicit their unique functions in collaboration with their binding partners. Therefore, identification and characterization of binding proteins will often unveil unexpected functions of the protein of interest. To begin to elucidate biological roles of the novel class of Na⁺/H⁺ exchanger NHE7, yeast two-hybrid screening was conducted and several binding candidates were identified. Among these candidates, I show that Secretory Carrier Membrane Proteins (SCAMPs) are novel NHE7 binding proteins and that SCAMPs regulate endocytic trafficking of NHE7 from the recycling endosomes to the trans-Golgi network (TGN). In agreement with this finding, I found that NHE7 can also be targeted to the plasma membrane and then internalized. Caveolins, structural proteins for caveolae, were identified as NHE7-binding proteins and it was initially hypothesized that caveolins might regulate NHE7-internalization. Interestingly, caveolins bound to NHE7 through a novel binding domain and facilitated its association to caveolae/lipid rafts, but did not affect NHE7-internalization. I also show that SCAMP2 associates with the heterotrimeric G protein β subunit (Gβ) and regulates the ERK1/2 signaling. Moreover, NHE7 was found to associate with both SCAMP2 and Gβ in the cell, suggesting that ERK1/2 signaling mediated by the SCAMP2-Gβ complex might regulate NHE7.

The role of NUMB, a protein important for cellular differentiation and endocytosis in non-placental cells, was investigated in syncytiotrophoblast development and function in the human placenta. The BeWo choriocarcinoma cell line was used as a model for villous cytotrophoblast cells and syncytiotrophoblast to investigate NUMB’s involvement in differentiation and epidermal growth factor receptor (EGFR) endocytosis. NUMB isoforms 1 and 3 were found to be the predominant isoforms and were upregulated following forskolin-induced differentiation. Overexpression of NUMB isoforms 1 and 3 did not mediate differentiation or EGFR signaling. Immunofluorescence analysis revealed that NUMB colocalized with EGFR at perinuclear late endosomes and lysosomes following EGF stimulation. We have demonstrated for the first time that NUMB isoforms 1 and 3 are expressed in BeWo cells, are upregulated in forskolin-differentiated BeWo cells and are involved in ligand-dependent EGFR endocytosis in BeWo cells.

The p85 subunit of phosphatidylinositol 3-kinase (PI3K) has long been thought of as a regulatory subunit that has no other function than the regulation of the p110 catalytic subunit. Our laboratory is studying other roles of the p85 subunit, in particular determining the role of the p85 BH domain. The BH domain has homology to GTPase activating protein (GAP) domains that are involved in the stimulation of monomeric G proteins to hydrolyze their bound GTP to GDP. This converts the G protein from its active conformation to its inactive conformation. We have determined that p85 interacts with Rab proteins, monomeric G proteins that regulate vesicle fusion during the endocytosis of receptors. We have shown that p85 binds to Rab5 regardless of nucleotide-bound state of Rab5. The p85 subunit of PI3K has in vitro GAP activity towards Rab5. It was determined that p85 also has in vitro GAP activity towards Rab4, Rab7, Rab6 as well as the Rho-family G proteins, Rac1 and Cdc42. This GAP activity was localized to the BH domain of p85 and mutation of Arg 274 to Ala abolishes the GAP activity of p85. When this p85R274A mutant was expressed in cells, PDGFR degradation was severely inhibited and there was a corresponding increase in the duration of MAPK and Akt signalling. This increase in cell signalling caused a transformed phenotype in cells expressing the p85 protein with the Arg 274 mutation. These cells have lost contact inhibition for growth, are able to grow independent of attachment as well as in the presence of limited growth factors. They also form tumours in nude mice. These cellular effects seem to be due to an increase in receptor recycling because of the loss of the GAP activity of p85. This increase in receptor recycling may interfere with receptor targeting to the late endosome, which would cause the decrease in receptor degradation that is seen in the p85R274A cells.

The p85 subunit of phosphatidylinositol 3-kinase (PI3K) has long been thought of as a regulatory subunit that has no other function than the regulation of the p110 catalytic subunit. Our laboratory is studying other roles of the p85 subunit, in particular determining the role of the p85 BH domain. The BH domain has homology to GTPase activating protein (GAP) domains that are involved in the stimulation of monomeric G proteins to hydrolyze their bound GTP to GDP. This converts the G protein from its active conformation to its inactive conformation. We have determined that p85 interacts with Rab proteins, monomeric G proteins that regulate vesicle fusion during the endocytosis of receptors. We have shown that p85 binds to Rab5 regardless of nucleotide-bound state of Rab5. The p85 subunit of PI3K has in vitro GAP activity towards Rab5. It was determined that p85 also has in vitro GAP activity towards Rab4, Rab7, Rab6 as well as the Rho-family G proteins, Rac1 and Cdc42. This GAP activity was localized to the BH domain of p85 and mutation of Arg 274 to Ala abolishes the GAP activity of p85. When this p85R274A mutant was expressed in cells, PDGFR degradation was severely inhibited and there was a corresponding increase in the duration of MAPK and Akt signalling. This increase in cell signalling caused a transformed phenotype in cells expressing the p85 protein with the Arg 274 mutation. These cells have lost contact inhibition for growth, are able to grow independent of attachment as well as in the presence of limited growth factors. They also form tumours in nude mice. These cellular effects seem to be due to an increase in receptor recycling because of the loss of the GAP activity of p85. This increase in receptor recycling may interfere with receptor targeting to the late endosome, which would cause the decrease in receptor degradation that is seen in the p85R274A cells.

Clathrin-coated pits on the plasma membrane invaginate into coated vesicles to internalize receptors and membrane. The clathrin adaptor epsin contains an aminoterminal ENTH domain that binds PI(4,5)P₂ and a carboxy-terminal domain that binds clathrin, and accessory proteins such as AP2. Here, we assessed how inter- and intramolecular factors affect the contribution of epsin to coated-pit function in living cells. We found Dictyostelium epsin was not required for global clathrin function, but plays an essential role in spore development. We demonstrated that clathrin, but not AP2, was critical for epsin to associate with clathrin-coated pits. We found that the carboxy-terminal region of epsin was essential, but not sufficient, for targeting epsin within clathrin-coated pits on the plasma membrane. In addition to targeting epsin to the membrane, the amino-terminal ENTH domain regulates the interaction between epsin and clathrin, an essential property that cannot be replaced by an alternate PI(4,5)P₂ binding domain. Moreover, the ENTH domain facilitates the functional interaction between clathrin and actin during late stages of endocytosis, possibly by regulating the activity of the adaptor Hip1r. Both the ability to bind PI(4,5)P₂ and another function mediated by residue T107 are critical for the activity of the ENTH domain. Our results support a model where the ENTH domain coordinates with the clathrin-binding C-terminal domain to allow a dynamic interaction of epsin with coated pits. Furthermore, we propose that the ENTH domain of epsin facilitates the membrane recruitment and phosphorylation of Hip1r, which in turn mediates the productive interaction of clathrin with the actin cytoskeleton at the plasma membrane. / text

Cellular pH homeostasis plays crucial roles in cellular functions, and it is now widely recognized that Na⁺/H⁺ exchangers are among the most prominent players in this process. Although recently described mammalian Na⁺/H⁺ exchanger NHE7 has attracted much attention, its biological functions remain largely unknown. Most proteins exist as protein complexes in the cell and elicit their unique functions in collaboration with their binding partners. Therefore, identification and characterization of binding proteins will often unveil unexpected functions of the protein of interest. To begin to elucidate biological roles of the novel class of Na⁺/H⁺ exchanger NHE7, yeast two-hybrid screening was conducted and several binding candidates were identified. Among these candidates, I show that Secretory Carrier Membrane Proteins (SCAMPs) are novel NHE7 binding proteins and that SCAMPs regulate endocytic trafficking of NHE7 from the recycling endosomes to the trans-Golgi network (TGN). In agreement with this finding, I found that NHE7 can also be targeted to the plasma membrane and then internalized. Caveolins, structural proteins for caveolae, were identified as NHE7-binding proteins and it was initially hypothesized that caveolins might regulate NHE7-internalization. Interestingly, caveolins bound to NHE7 through a novel binding domain and facilitated its association to caveolae/lipid rafts, but did not affect NHE7-internalization. I also show that SCAMP2 associates with the heterotrimeric G protein β subunit (Gβ) and regulates the ERK1/2 signaling. Moreover, NHE7 was found to associate with both SCAMP2 and Gβ in the cell, suggesting that ERK1/2 signaling mediated by the SCAMP2-Gβ complex might regulate NHE7.