Binding of cytohesin 2 to the plasma membrane and its relationship with the EGFR

Abstract

Eukaryotic cells are compartmentalized by lipid membranes to achieve the spatial separation of biological processes and signaling pathways. Controlled trafficking of proteins between these compartments as well as the recruitment of proteins to the membranes themselves are crucial for trouble-free function of the cell. <br /> Previous research has revealed several possibilities for interaction between proteins and membranes. Phosphoinositides can specifically interact with certain protein domains, anionic lipids attract positively charged protein domains by electrostatic force and lipids can even be attached to proteins post-translationally to enable integration into the membrane. <br /> This work focuses on the guanine nucleotide exchange factor ARNO, a multidomain protein that activates small GTPases like Arf6 and therefore is directly involved in the vesicle trafficking machinery of the cell.<br /> In its autoinhibited form, ARNO is localized in the cytoplasm, whereas recruitment to the plasma membrane is a prerequisite for its activation of Arf. So far, research has been centered on the interaction of the PH-domain and the PBR-domain of ARNO with artificial membrane systems. To expand these findings and account for the enormous complexity of the inner leaflet of the cellular plasma membrane, in this study, membrane sheets are employed. The ability of a variety of protein constructs consisting of different ARNO domains to bind to these sheets is analyzed.<br /> It seems that the different domains of ARNO aid to the interaction with the membrane in a cooperative manner. While the PH-domain is absolutely required for association with the membrane, it is not sufficient for sequestration of ARNO in the membrane. Moreover, its interaction with the phosphoinositides could be altered by the concentration of calcium in the binding buffer. Hypothetically, this might be due to the formation of PIP-bridges permitted by the crosslinking of individual PIP molecules by the Ca²⁺ ions. This results in a loss of accessibility of the PIPs for binding by the ARNO PH-domain.<br /> In this work, binding studies of the other ARNO domains conclusively show that the PBR-domain, the Sec7-domain as well as the coiled-coil domain participate in plasma membrane binding. Moreover, dimerization of ARNO also improves its binding ability, most probably by an increase of the local avidity. Once bound to the plasma membrane, ARNO proteins form clusters. Brightfield as well as STED microscopy reveals that these overlap with clusters of the endogenous EGFR to a non-random degree.<br /> Having observed this colocalization, a possible biological relationship between ARNO and the EGFR is assessed. Overexpression of ARNO in HeLa cells results in a tendency towards increased activation of the EGFR after stimulation with EGF.<br /> Upon activation, the EGFR can be translocated to the nucleus by retrograde endosomal trafficking. Its JM-domain of the EGFR has been proposed as a possible binding partner for ARNO in previous studies and contains the nuclear localization signal of the EGFR. This work explores in how far overexpression or inhibition of ARNO influences the nuclear translocation of the EGFR. However, this does not seem to be the case.