How CD82 regulates cancer cell migration: A lipid-dependent process

Membrane protein CD82 is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Tetraspanins are integral transmembrane proteins, but lack common transmembrane proteins functions, such as membrane receptor, membrane transporter, membrane enzyme or adhesion molecule functions, which could hardly be observed in tetraspanins. Tetraspanins are generally believed to act as membrane protein scaffolds that interact with multiple membrane protein complexes and domains, and regulate membrane micro-domains organization and function. Tetraspanin CD82 plays a pivotal role in suppressing cancer progression especially cancer metastasis, down-regulation of CD82 or loss of CD82 expression was frequently observed in aggressive and late stage solid tumor cancers. In contrast, normal or high CD82 expression in solid tumor cancers generally leads to favorable prognosis and patient survival. However, the fundamental mechanisms of solid tumor cancer metastasis suppression by CD82 remain unclear.;Studies have shown that CD82 is associated with lipid raft which was determined in multiple ways, such as density gradient floatation technique and detergent resistant membrane centrifugation method. CD82 association with outer leaflet lipid raft components such as gangliosides GM2/GM3 was proven to be crucial for CD82 inhibitory function on growth factor receptor activation and downstream signaling. Though lipid raft association plays an important role in CD82-mediated inhibition of cancer cell migration, how CD82 is localized to lipid raft, how CD82 is associated with membrane lipids, and why lipid raft association is important for CD82 function remain unclear. In order to explain how CD82 is localized to lipid raft and how CD82 interacts with lipid raft components, cholesterol recognition/interaction amino acid consensus (CRAC) sequences, were identified by analyzing the sequences of CD82 and other tetraspanins transmembrane domains. Cholesterol binding motifs were found in most tetraspanin family proteins, which is likely to be a common feature of tetraspanins, but ganglioside binding motifs were not found in any of the tetraspanins transmembrane domains. CD82 contains four cholesterol binding motifs in total, localized to three different transmembrane domains. We studied the cholesterol binding motif spans through the outer leaflet segment of the 3rd transmembrane domain of CD82. By conducting site-directed mutagenesis on this cholesterol binding motif, we observed in the mutant transfectant loss of inhibition on cell migration determined by cell migration assays and loss of inhibition on cell adhesion determined by cell-matrix adhesion assay, as well as reduced lipid raft localization determined by sucrose density gradient floatation procedures, and the mutant linked fewer CD82-associated proteins to outer leaflet lipid rafts. CD82-binding to gangliosides GM2/GM3 was also decreased in the mutant. However, CD82 association with Tetraspanin enriched micro-domain (TEM) was not disrupted upon mutagenesis of the cholesterol binding motif. These observations suggest that CD82-cholesterol binding property 1) promotes the interaction between outer leaflet lipid rafts and CD82-containing TEMs, which is needed for CD82 function as a suppressor of cancer cell migration, and 2) determines CD82 association with outer leaflet lipid raft components such as gangliosides GM2/GM3. Endocytosis of CD82 and CD82-associated proteins such as integrin alpha3, integrin beta1, and membrane lipid cholesterol were reduced, compared to the CD82 WT transfected cells. These results suggested that CD82-cholesterol interaction also plays a role in CD82-mediated membrane endocytic vesicle assembly. Moreover, we found inhibition of galectins with lactose promotes CD82 localization to the lipid raft but reduces CD82-associated integrin beta1. Our study reveals that the tetraspanin CD82 links CD82-associated proteins such as integrins and growth factor receptors, to the lipid raft through cholesterol binding, and CD82 association with CD82-associated integrins depends on galectins binding. Galectins connect CD82 and other TEM proteins by interacting with the glycosylated moieties of TEM proteins. With impaired lipid raft localization, CD82 mutant failed to efficiently translocate, internalize and recycle itself as well as CD82-associated TEM proteins through lipid raft-mediated endocytosis pathway. Therefore, CD82 induced down-regulation of membrane integrins and growth factor receptor levels was impaired, which results in loss of inhibition on cancer cell migration upon cholesterol binding motif mutagenesis.