<p>Integrins are receptors presented on most cells. By binding ligand they can generate signalling pathways inside the cell. Those pathways are a linkage to proteins in the cytosol. It is known that tumor cells can survive and proliferate in the absence of a solid support while normal cells need to be bound to ligand. To understand why tumour cells act that way, we first have to know how ligand-binding to integrins affect the cell. This research field includes studies on activation of proteins by integrins and the following protein-protein interactions.</p><p>The part of the research that I did, focused on the activation of PI3K by integrins and the question whether Ras is included in that pathway. I also studied the conformation changes of the integrins and tried to identify factors which regulate these changes.</p><p>Known is that Ras can activate PI3K. But we wanted to know if this is a step in the activation of PI3K by integrins. So if this would be a fact then Ras must be activated by integrins.</p><p>To see if integrins could activate Ras I did a pull down assay. GTP loaded Ras was isolated through its affinity for Raf. Only when Ras is in its activated state then it is GTP loaded, otherwise it is GDP loaded. In the experiment we also compared the β1A and the β1B splice variants. As result we could see that both splice variants probably can activate Ras. By blotting with anti-PI3K antibody we looked if PI3K had bound to Ras but no clear result could be obtained.</p><p>Integrins presented on blood cells are mostly in the inactive state while adherent cells have integrins which are mostly in the active state. PI3K has been shown, for blood cells, to be involved in the conformation regulation of integrins. Possibly, there is a positive circle that for blood cells just has to be switched on. It could be that the integrins in adherent cells are active because the cells are adhesive. By being adhesive, PI3K is activated. PI3K may then activate the integrins, through which the integrins stay in the active state. This circle could be broken at two points: we could inhibit PI3K or we could make the cells un-adhesive. I analysed this in cell attachment assay and by binding of conformation-specific integrin antibodies in FACScan. From the results we could not find any evidence that the whole idea around the positive circle is correct. Surprisingly we saw that the integrin value at the surface decrease if you add PI3K inhibitor. This could be due to distribute recirculation of integrins from the cytoplasm to the cell surface.</p><p>β1- and β3-integrins are both widely spread, but no functional difference could be shown already. Previous results suggest that there is a difference between migrations of those two types. To ensure this suggestion I did a wound assay. Hereby I compared the migration of different cell types, with different integrins on their surface and on different ligands.</p>
| Identifer | oai:union.ndltd.org:UPSALLA/oai:DiVA.org:uu-6265 |
| Date | January 2005 |
| Creators | Schelfaut, Roselien |
| Publisher | Uppsala University, Department of Medical Biochemistry and Microbiology, Uppsala : Institutionen för medicinsk biokemi och mikrobiologi |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, text |
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