Organization of signaling networks during receptor-mediated membrane protein trafficking

Abstract

The mechanisms by which receptor signals are organized in a synergic/hierarchical manner in time and space during regulation of integral membrane protein traffic remain a present challenge. The aim of this study was to address some critical aspects of these processes. Our model was the Na+,K+-ATPase (NKA), because it represents an integral membrane protein that is physiologically regulated within the cell by altering the amount of active molecules in the plasma membrane in response to G-protein-coupled receptor (GPCR) signals such as dopamine, and angiotensin II, among others.

The following conclusions were drawn from the results obtained in this thesis:

・Binding of phosphatidylinositol 3-kinase (PI-3K) to the NKA and its activation in response to dopamine requires that the NKA becomes phosphorylated by protein kinase C (PKC)-zeta. Phosphorylation of Ser-18 promotes the binding of 14-3-3-epsilon/beta to the alpha-subunit which then functions as an anchor for the PI-3K.

・Binding of adaptor proteins to the NKA is a necessity for its internalization by clathrin-coated vesicle formation. Phosphorylation of adaptor protein-2 (AP2) μ2 subunit is under the control of PKC-zeta, which represents an essential signal that triggers the association of AP2 to the NKA alpha-subunit.

・Phosphorylation of AP2 μ2 subunit has a wide biological significance during NKA endocytosis. Studies in lung epithelial cells demonstrated that in the presence of AP2 μ2 subunit negative mutant (lacking the phosphorylation site), hypoxia-generated reactive oxygen species failed to promote the removal of NKA from the plasma membrane.

・INAD [a multiple PSD-95/Discs-large/ZO-1 (PDZ) domain-containing protein] provides a higher level of GPCR signaling organization during dopamine-, angiotensin II-, and insulin-dependent regulation of NKA activity in renal epithelial cells.

・It appears that selective PDZ domains within inactivation-no-after potential (INAD) provide selectivity to the GPCR response, depending on whether the signals are inhibitory (dopamine) or stimulatory (angiotensin II and insulin). The latter also reflects that while inhibitory signals need to be assembled at the plasma membrane (endocytosis), stimulatory signals (recruitment) may do so at the endosomal membrane.

Taken together these results have provided additional new information, have clarified and improved our understanding on relevant mechanisms by which signals originated at the plasma membrane could be translated and integrated in a hierarchical manner to avoid deleterious cross-talk during regulation of membrane protein traffic.