The Transmembrane Region Is Responsible for Targeting of Adaptor Protein LAX into “Heavy Rafts”

BACKGROUND: The importance of membrane compartmentalization into specific membrane microdomains has been shown in many biological processes such as immunoreceptor signaling, membrane trafficking, pathogen infection, and tumor progression. Microdomains like lipid rafts, caveolae and tetraspanin enriched microdomains are relatively resistant to solubilization by some detergents. Large detergent-resistant membrane fragments (DRMs) resulting from such membrane solubilization can be conveniently isolated by density gradient ultracentrifugation or gel filtration. Recently, we described a novel type of raft-like membrane microdomains producing, upon detergent Brij98 solubilization, "heavy DRMs" and containing a number of functionally relevant proteins. Transmembrane adaptor protein LAX is a typical "heavy raft" protein. The present study was designed to identify the molecular determinants targeting LAX-derived constructs to heavy rafts. METHODOLOGY/PRINCIPAL FINDINGS: We prepared several constructs encoding chimeric proteins containing various informative segments of the LAX sequence and evaluated their effects on targeting to heavy rafts. Replacement of the polybasic membrane-proximal part of LAX by CD3ε-derived membrane-proximal part had no effect on LAX solubilization. Similarly, the membrane-proximal part of LAX, when introduced into non-raft protein CD25 did not change CD25 detergent solubility. These results indicated that membrane-proximal part of LAX is not important for LAX targeting to heavy rafts. On the other hand, the replacement of transmembrane part of CD25 by the transmembrane part of LAX resulted in targeting into heavy rafts. We also show that LAX is not S-acylated, thus palmitoylation is not involved in LAX targeting to heavy rafts. Also, covalent dimerization was excluded as a cause of targeting into heavy rafts. CONCLUSIONS/SIGNIFICANCE: We identified the transmembrane domain of LAX as a first motif targeting transmembrane protein constructs to detergent-resistant heavy rafts, a novel type of membrane microdomains containing a number of physiologically important proteins

Association of CD99 short and long forms with MHC class I, MHC class II and tetraspanin CD81 and recruitment into immunological synapses

<p>Abstract</p> <p>Background</p> <p>CD99, a leukocyte surface glycoprotein, is broadly expressed in many cell types. On the cell surface, CD99 is expressed as two distinct isoforms, a long form and a short form. CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T cell activation. However, the molecular mechanisms by which CD99 participates in such processes are unclear. As CD99 contains a short cytoplasmic tail, it is unlikely that CD99 itself takes part in its multi-functions. Association of CD99 with other membrane proteins has been suggested to be necessary for exerting its functions.</p> <p>Results</p> <p>In this study, we analyzed the association of CD99 with other cell surface molecules involved in T cell activation. We demonstrate the association of MHC class I, MHC class II and tetraspanin CD81 with CD99 molecules on the cell surface. Association of CD99 with its partners was observed for both isoforms. In addition, we determined that CD99 is a lipid raft-associated membrane protein and is recruited into the immunologic synapse during T cell activation. The implication of CD99 on T cell activation was investigated. Inhibition of anti-CD3 induced T cell proliferation by an anti-CD99 monoclonal antibody was observed.</p> <p>Conclusions</p> <p>We provide evidence that CD99 directly interact and form the complex with the MHC class I and II, and tetraspanin CD81, and is functionally linked to the formation of the immunologic synapse. Upon T cell activation, CD99 engagement can inhibit T cell proliferation. We speculate that the CD99-MHC-CD81 complex is a tetraspanin web that plays an important role in T cell activation.</p

Palmitoylation of LAX-LAX-LAX protein.

<p>Protein acylation of the LAX-LAX-LAX protein construct was examined using the acyl-biotinyl exchange chemistry-based method as described in Experimental procedures. Lysate that was not treated with NH₂OH (hydroxylamine) served as a negative control. A palmitoylated heavy raft protein, transferrin receptor (TfR, CD71), was used as a positive control. IP: SA, material isolated on streptavidin beads.</p

Schematic representation of the chimeric proteins used in this study.

<p>EC – extracellular part, TM – transmembrane part, SM – membrane-proximal part, IC – intracellular part, OFP – orange fluorescent protein, N – N-terminus of the protein.</p

The effect of membrane-proximal sequence of LAX on protein targeting to heavy rafts.

<p>Membranes isolated from cells expressing the indicated constructs were solubilized in Brij98, subjected to gel filtration on Sepharose 4B, and analyzed by immunoblotting. The first collected fraction was No. 3, the last one was No. 9. The blots were probed with antibodies to LAT and Lck, a standard marker of lipid rafts. One representative blot is shown for each construct. The results of densitometric analysis were plotted as relative densities of LAT in each fraction. The amino acid sequence of LAX sub-membrane part is provided.</p

The effect of the transmembrane segment of LAX on targeting to heavy rafts.

<p>Membranes isolated from cells expressing the indicated constructs were solubilized in Brij98, subjected to gel filtration on Sepharose 4B, and analyzed by immunoblotting as described in legend to Fig. 2.</p

Lack of covalent dimerization of the chimeric protein constructs.

<p>Transfectant cell lines expressing the indicated constructs were analyzed by SDS-PAGE under reducing or non-reducing conditions, followed by immunoblotting. Jurkat cells were used as a control expressing endogenous LAT. Positions of m.w. standards (in kDa) are indicated.</p

Inefficient Cross-Presentation Limits the Cd8+ T Cell Response to a Subdominant Tumor Antigen Epitope

CD8+ T lymphocytes (TCD8) responding to subdominant epitopes provide alternate targets for the immunotherapy of cancer, particularly when self-tolerance limits the response to immunodominant epitopes. However, the mechanisms that promote TCD8 subdominance to tumor Ags remain obscure. We investigated the basis for the lack of priming against a subdominant tumor epitope following immunization of C57BL/6 (B6) mice with SV40 large tumor Ag (T Ag)-transformed cells. Immunization of B6 mice with wild-type T Ag-transformed cells primes TCD8 specific for three immunodominant T Ag epitopes (epitopes I, II/III, and IV) but fails to induce TCD8 specific for the subdominant T Ag epitope V. Using adoptively transferred T CD8 from epitope V-specific TCR transgenic mice and immunization with T Ag-transformed cells, we demonstrate that the subdominant epitope V is weakly cross-presented relative to immunodominant epitopes derived from the same protein Ag. Priming of naive epitope V-specific TCR transgenic TCD8 in B6 mice required cross-presentation by host APC. However, robust expansion of these TCD8 required additional direct presentation of the subdominant epitope by T Ag-transformed cells and was only significant following immunization with T Ag-expressing cells lacking the immunodominant epitopes. These results indicate that limited cross-presentation coupled with competition by immunodominant epitope-specific TCD8 contributes to the subdominant nature of a tumor-specific epitope. This finding has implications for vaccination strategies targeting TCD8 responses to cancer. © 2005 The American Association of Immunologists, Inc