Abstract:
:Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (α(v)β(3) and α(5)β(1) integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for α(v)β(3) and α(5)β(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both α(v)β(3) and α(5)β(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, α(v)β(3) sequesters strongly into raft-like domains and α(5)β(1) loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.
journal_name
Biophys Jjournal_title
Biophysical journalauthors
Siegel AP,Kimble-Hill A,Garg S,Jordan R,Naumann CAdoi
10.1016/j.bpj.2011.08.040subject
Has Abstractpub_date
2011-10-05 00:00:00pages
1642-50issue
7eissn
0006-3495issn
1542-0086pii
S0006-3495(11)01016-2journal_volume
101pub_type
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