title: Unraveling the role of membrane proteins Notch, Pvr, and EGFR in altering integrin diffusion and clustering.
abstract: The role of three membrane proteins in altering the diffusion and clustering of integrin receptors has been measured. Integrins are membrane proteins responsible for integrating intracellular and extracellular signaling events and anchoring cells to the extracellular matrix. The methodology used to elucidate the role of other membrane proteins in altering integrin diffusion and clustering combines fluorescence microscopy with RNA interference (RNAi), which is a technique to reduce the expression of a target protein. The three RNAi-targeted membrane proteins were epidermal growth factor receptor (EGFR), platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr), and Notch. Real-time polymerase chain reaction or quantitative immunocytochemistry was used to measure a reduction in mRNA or protein concentration after RNAi treatment, respectively. Fluorescence recovery after photobleaching showed that reducing the concentration of EGFR or Notch results in less constrained integrin diffusion and, in the case of Notch RNAi, 4 % more mobile integrins. Fluorescence resonance energy transfer measurements performed before and after RNAi treatments indicate that clustering decreases for wild-type integrin, but increases for a high-ligand-affinity integrin mutant after reducing the expression of EGFR, Pvr, or Notch. A model to explain the measured changes after reducing the expression of these three membrane proteins involving cholesterol-enriched nanodomains is proposed.
authors.affiliation: Department of Chemistry, Iowa State University, Ames, IA 50011, USA.
authors.forename: Neha
authors.initials: N
authors.lastname: Arora
authors.forename: Dipak
authors.initials: D
authors.lastname: Mainali
authors.forename: Emily A
authors.initials: EA
authors.lastname: Smith
mesh_terms: D002478:Cells, Cultured; D004058:Diffusion; D066246:ErbB Receptors / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D016023:Integrins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D051880:Receptors, Notch / Q000378:metabolism; D011991:Receptors, Virus / Q000378:metabolism
chemical_list: D016023:Integrins; D008565:Membrane Proteins; D012333:RNA, Messenger; D051880:Receptors, Notch; D011991:Receptors, Virus; C066100:poliovirus receptor; D066246:ErbB Receptors