Binding and uptake of albumin by opossum kidney cells

Assays of both fluid phase endocytosis and receptor mediated endocytosis have been developed in opossum kidney cells. The regulation of the endocytic pathway has been examined using a number of potential inhibitors. In particular, bacterial toxins have been employed to identify potential points of regulation of the pathway by GTP-binding proteins. The apical endocytic pathway in these cells has been examined morphologically using electron microscopy of gold-albumin.;Based on the results of the above experiments a rat cDNA encoding the G-protein subunit G13 has been subcloned into pcDNA3. This vector has been stably transfected into opossum kidney cells by the calcium phosphate method. Over-expressing transfects have been selected and screened by western blotting and immunocytochemistry. These stable transfects have been used to measure albumin endocytosis and the results compared to control transfects and wild type cells.;Two binding sites for albumin have been identified, each with a different affinity. Based on the lectin competition studies the receptors appear to be glycoproteins carrying O-linked sugars. Specificity experiments indicate that the receptors share many similar characteristics to the family of scavenger receptors. [125I]-albumin ligand blotting has revealed the presence of three specific albumin binding proteins.;Endocytosis has been visualised using electron microscopy, with gold- albumin being seen in multiple intracellular vesicular structures. These endocytic pathways can be regulated by GTP-binding protein modulating agents. Opossum kidney cells have been successfully transfected with the Gi3 protein subunit. These cells show enhanced uptake of albumin compared to controls.;Therefore the experiments described in the thesis document the characteristics of albumin binding to opossum kidney cells, identify the potential receptors involved, and explore the mechanism of regulation of the subsequent endocytic uptake of albumin by the cells.