Professor Elizabeth Smythe: Research
Regulation of the early endocytic pathway
Endocytosis allows the cellular uptake of a variety of biologically important macromolecules including nutrients and signaling molecules. The pathway is also hijacked by a number of pathogens to gain entry into the cell. A further emerging paradigm is that interactions between the endocytic pathway and intracellular signaling are key to fine-tuning signaling output in developmental and organismal contexts. We are interested in the regulation of the early endocytic pathway and how endocytosis and intracellular signaling are integrated.
Rab5 and its GEFs regulate trafficking and signaling
Members of the rab family are small GTPases of the ras superfamily and they act as molecular switches, being active in their GTP conformation and inactivated following GTP hydrolysis.
Rab5 is a major regulator of the early endocytic pathway, modulating clathrin coated vesicle formation, endosomal fusion and motility and signaling. It carries out this myriad of functions by interacting with a large number (>20) effector molecules.
A key question is how the spatial regulation of rab5 on the endocytic pathway is established. Emerging evidence from our lab and others supports the hypothesis that rab guanine nucleotide exchange factors (GEFs) specify recruitment of functional pools of rab5 to particular endocytic intermediates.
We are particularly interested in hRME-6, a rab5GEF, that functions at clathrin coated pits. Our recent studies have demonstrated that hRME-6 recruits an active pool of rab5 to clathrin coated vesicles to participate in uncoating. hRME-6 is a multi-domain protein which contains a classical VPS9 domain that has GEF activity towards rab5 and an N-terminal RasGAP domain, making it an ideal candidate to integrate trafficking and signaling.
Regulation by phosphorylation
Peripheral membrane proteins are recruited to endocytic membranes to regulate transport vesicle formation, cargo sorting and fusion. Following membrane dissociation, these proteins need to be recycled through the cytosol for further rounds of uptake. Reversible phosphorylation is thus an attractive and feasible mechanism to regulate the endocytic pathway.
We, and others, have shown how reversible phosphorylation effects specific regulation of individual events on the endocytic pathway and current studies in the lab are focusing on the regulation of clathrin and rab5 function via phosphorylation.
Endocytosis and signaling in biological contexts
To further understand the relationship between endocytosis and signaling we have extended our studies from cultured cell lines to more physiological systems. One avenue that we are pursuing is the regulation of Tie2, a receptor tyrosine kinase essential for vasculogenesis, by its endocytic trafficking in human umbilical vein endothelial cells (HUVECs). We are also using Drosophila as a model organism to understand the role of different rab5GEFs in signaling.