Abstract
Recent literature suggests that tetraspanin proteins (transmembrane 4 superfamily; TM4SF proteins) may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with alpha(3)beta(1) (an integrin) and complexes of CD63 (a TM4SF protein) with phosphatidylinositol 4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like microdomains, as seen by three different criteria. First, these complexes localize to low density light membrane fractions in sucrose gradients. Second, CD9 and alpha(3) integrin colocalized with ganglioside GM1 as seen by double staining of fixed cells. Third, CD9-alpha3beta1 and CD81-alpha3beta1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K complex formation itself was not dependent on localization into raftlike lipid microdomains. These complexes did not require cholesterol for stabilization, were maintained within well solubilized dense fractions from sucrose gradients, were stable at 37 degrees C, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.
Highlights
Transmembrane 4 superfamily (TM4SF)1 proteins comprise a large group of ubiquitously expressed proteins that function in diverse contexts such as Tand B-cell activation, platelet aggregation, and cell fusion, motility, and proliferation [1,2,3]
Recent literature suggests that tetraspanin proteins may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts
To examine the detergent resistance of TM4SF-integrin protein complexes, A431 human carcinoma cells were surface labeled with biotin and lysed using four different detergent conditions, and immunoprecipitations were carried out
Summary
The possible location of these complexes in raft-type microdomains, coupled with the appearance of level 2 and level 3 complexes only under mild detergent conditions, suggests that possibly these complexes may exist only in the context of large and poorly solubilized membrane vesicles In this regard, the integrin ␣L2-CD63 complex was excluded from a Sepharose CL4B column, suggesting a size equal to or greater than 20 million daltons [49]. We asked whether these complexes only exist in the context of large raftlike vesicles or whether they may exist in a truly soluble form of a reasonable size These studies have been carried out using A431 and HT1080 cell lines because they express significant levels of ␣31, CD9, and/or CD81. A431 cells were selected for study because they were used previously to establish that PtdIns 4-K may localize into lipid rafts [42]
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