Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts.
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.
Claas
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Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Stipp
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Hemler
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D015703:Antigens, CD / Q000737:chemistry* / Q000502:physiology; D002784:Cholesterol / Q000737:chemistry; D006801:Humans; D039201:Integrin alpha3beta1; D016023:Integrins / Q000737:chemistry* / Q000502:physiology; D008562:Membrane Glycoproteins; D021962:Membrane Microdomains / Q000737:chemistry* / Q000502:physiology; D008565:Membrane Proteins; D017830:Octoxynol / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry; D060225:Tetraspanin 28; D060245:Tetraspanin 29; D060149:Tetraspanin 30; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; C554586:CD63 protein, human; C554798:CD81 protein, human; C554869:CD9 protein, human; D039201:Integrin alpha3beta1; D016023:Integrins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D060225:Tetraspanin 28; D060245:Tetraspanin 29; D060149:Tetraspanin 30; D017830:Octoxynol; D002784:Cholesterol