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PrP^c capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction

Department of Biology, University of Konstanz, Konstanz, Germany; and
^* Institute of Biochemistry and Molecular Biology I, Cellular Signal Transduction, Center of Experimental Medicine, University Hospital Hamburg-Eppendorf, Hamburg, Germany

¹Correspondence: Department of Biology; University of Konstanz, 78457 Konstanz, Germany. E-mail: Claudia.Stuermer{at}uni-konstanz.de

SPECIFIC AIMS

The cellular prion protein (PrP^c) is associated with lipid rafts, yet the type of raft PrP^c resides in and the physiological function of PrP^c are unclear. Based on the finding that the noncaveolar lipid raft proteins reggie-1 and reggie-2 provide platforms for T cell signaling, we determined whether PrP^c, when activated by antibody (AB) cross-linking, undergoes a polarized concentration known as capping and if this involves reggies and molecular components of signal transduction cascades.

PRINCIPAL FINDINGS

1. PrP^c cross-linking leads to PrP^c capping and cocapping with reggies
We show here by electron (EM) and light microscopic analysis together with biochemical assays that cross-linking of PrP^c with specific antibodies (ABs) leads to PrP^c capping in Jurkat and human peripheral blood T cells. T lymphocytes are devoid of caveolin and caveolae. The lateral movement of PrP^c during capping results in cocluster formation of PrP^c with the intracellular and noncaveolar lipid raft proteins reggie-1 and reggie-2, which are organized in preformed caps.

2. The association of PrP^c with reggie caps leads to the recruitment of T cell signaling compounds
This association of PrP^c with reggie rafts leads to recruitment of the tyrosine kinases fyn and lck to the cap. The close spatial association of PrP^c, reggies, and lck in Jurkat T cell microdomains was revealed by our double labeling and immunogold EM analysis and coimmunoprecipitation assays. PrP^c aggregation in reggie rafts also guides Thy-1, CD3/TCR, and LAT to the cap and elicits a focal increase of protein phosphorylation and F-actin polymerization (Fig. 1 ). This focal concentration and association imply transduction of signals across the plasma membrane by PrP^c after its cross-linking.

View larger version (25K): [in this window] [in a new window]   Figure 1. Jurkat T cell capping induced by PrPc cross-linking provokes coclustering with reggie-1 and -2 and recruits Thy-1, CD3, lck, and F-actin. The middle vertical row of images shows PrPc after cross-linking by specific primary and multivalent secondary ABs (PrPcX) in living cells (green); arrowheads mark labeled and double-labeled sites in the cap. a–f) AB-mediated cross-linking of PrPc leads to condensation of PrPc in globular aggregates in caps (b, e), also labeled by reggie-1 (a, red) and reggie-2 (d, red), so that PrPc and reggie-1 (c) and PrPc and reggie-2 (f) are colocalized significantly. g–i) After AB cross-linking of PrPc and PrPc accumulation in the cap (h), Thy-1 is detected in the same restricted cap region (g, red) and colocalized with PrPc to a substantial extent (i). k–m) PrPc condensed into globular aggregates after cross-linking (l, green) exhibits a significant degree of colocalization with CD3 (k, red), which shows a similar contraction into globular aggregates as PrPc; both are colocalized to a significant degree (m). n–p) Anti-lck staining (n) coclusters with PrPc (o) in the cap upon PrPc cross-linking. (o, p). q–s) F-actin (Ac) detected by phalloidin (q) increases in the cap after PrPc cross-linking (r, s).

3. PrP^c cross-linking leads to signal transduction involving a distinct Ca²⁺ signal and MAP kinase activation
Indeed, PrP^c cross-linking provoked MAP kinase phosphorylation and a distinct Ca²⁺ signal in the form of a brief elevation of the intracellular Ca²⁺ concentration (Fig. 2 a–d,e), thus showing specific PrP^c-mediated signal transduction events. Blockade of Ca²⁺ signaling by BAPTA/AM prevented PrP^c capping. PrP^c aggregates formed but remained widely distributed over the cell surface and failed to coalesce in the reggie raft platform (Fig. 2f) . Accordingly, none of the other molecules recruited to the cap along with PrP^c under control conditions, accumulated in the cap in BAPTA/AM pretreated cells. Thus, PrP^c association with reggie rafts triggers distinct transmembrane signal transduction events in T cells that enhance the focal concentration of PrP^c itself and other important signaling molecules in the preformed reggie caps.

View larger version (26K): [in this window] [in a new window]   Figure 2. Ca2+ signaling and activation of the MAP kinases ERK 1/2 by PrPc cross-linking. a–d) Ligation of the TCR/CD3 complex by OKT3 induces a biphasic Ca2+ signal with a transient peak (=450 nM±140 nM, n=6) and a long-lasting plateau (a). Addition of the PrPc mAb 6H4 (2 µg/mL) alone induces no Ca2+ signal, but cross-linking by a secondary goat-anti-mouse antibody induces a transient peak (=143 nM±56 nM, n=9) (b). No change in [Ca2+]i is seen after addition of the cross-linking antibody alone (c) (representative tracings of 5–9 independent experiments). A direct comparison of mean [Ca2+]i during the peak (t=80 s after stimulation) and 250 s after stimulation reveals that the Ca2+ signal after PrPc cross-linking was transient; [Ca2+]i returned to baseline levels within 250 s (d, data are mean ±SD). e) PrPc cross-linking induced a slow but sustained phosphorylation of ERK1/2, which was modest compared with cross-linking of the TCR/CD3 complex serving as a positive control. The blot was stripped and reprobed with anti-ERK1/2 as loading control. f) Inhibition of Ca2+ signaling by preincubation with BAPTA/AM blocked PrPc redistribution to the cap region after AB-mediated cross-linking in cells with and without a preformed reggie cap. The % of cells showing a preformed reggie cap was unaffected by BAPTA/AM treatment. Likewise, Thy-1 failed to concentrate in the cap after BAPTA/AM pretreatment and was widely distributed. PrPc and Thy-1 rarely formed coclusters under these conditions.

4. Internalization of PrP^c and reggie-1 and -2 and transfer to lysosomes
Upon longer exposure of Jurkat T cells to the PrP^c cross-linking procedure, a significant proportion of the cells had internalized almost all the protein into globular intracellular organelles, i.e., endosomes and limp-2 positive lysosomes. This suggests that the PrP^c-mediated events were down-regulated by the internalization of PrP^c and its transfer into reggie-positive lysosomes for degradation or recycling.

CONCLUSIONS AND SIGNIFICANCE

Our results demonstrate for the first time that PrP^c cross-linking leads to the lateral movement of PrP^c toward the T cell cap region and results in PrP^c capping. We have identified reggie-1 and -2 as a scaffold/target platform for activated PrP^c (Fig. 3 ). Therefore, we propose that reggie raft microdomains rather than caveolae are target and signaling domains for PrP^c in lymphocytes. This spatial association of PrP^c with reggies recruits relevant T cell signaling components to the cap in response to the transduction of signals by PrP^c across the plasma membrane. In fact, the distinct intracellular Ca²⁺ signal upon PrP^c cross-linking most likely triggers the actin cytoskeletal rearrangement and concentration in the cap region and the focal increase of protein phosphorylation (Fig. 3) . Blockade of the Ca²⁺ signal prevents the lateral movement of PrP^c aggregates formed upon cross-linking by ABs (Fig. 3) , demonstrating that distinct signaling steps regulate the concentration of PrP^c in the cap, which allows further molecular interactions to occur. The MAP kinase activation by PrP^c cross-linking suggests that long-lasting signaling events are also induced. Joint internalization of PrP^c and reggies and transfer into endosomes/lysosomes appears to regulate this pathway by degradation or recycling.

View larger version (32K): [in this window] [in a new window]   Figure 3. Schematic representation of events induced by PrPc capping in relation to reggie caps. In unstimulated Jurkat T cells, reggie-1 and -2 are clustered in preformed caps on the cytoplasmic side of the plasma membrane whereas PrPc and the TCR complex are more or less evenly distributed. AB-mediated cross-linking of PrPc leads to PrPc capping and cocapping with reggie, accumulation of fyn and lck in the cap region, and recruitment of the TCR and its associated signaling molecules. A marked increase in F-actin polymerization occurs in the cap region most likely in response to the Ca2+ signal elicited by PrPc capping. PrPc cross-linking performed in the presence of the Ca2+ chelator BAPTA-AM leads to the appearance of PrPc clusters that are widely distributed over the cell surface and fail to concentrate in the cap. The preformed cap detected by reggie-1 is, however, maintained. The data show that the Ca2+ signal elicited by PrPc leads to the dynamic rearrangement of PrPc clusters into the cap, which recruits other compounds relevant to T cell signaling.

Intensive research is devoted to the physiological function of PrP^c. Our results provide the first evidence for capping of PrP^c in T cells and for the spatial association of PrP^c with preformed reggie caps and internal signaling pathways. In contrast to other views, which propose that PrP^c interacts with caveolin, our results unraveled reggies as PrP^c targets in T lymphocytes that are devoid of caveolae and caveolin. Identification of a distinct signaling pathway evoked by PrP^c which regulates the lateral movement of PrP^c aggregates on the cell surface is consistent with reports showing the contribution of PrP^c to signal transduction. We highlight the events in T cells that gain access to most organs, tissues, and epithelia of an organism. The recruitment of important signaling molecules to the cap by PrP^c activation might also facilitate signaling from the TCR, as suggested by the observation that proliferation of PrP^c–/– lymphocytes is impaired.

The events upon PrP^c capping could imply that during cell contact formation, PrP^sc on the membrane of "infected" cells might act as receptor or ligand for PrP^c on the uninfected cell, or even that shed microparticles exert such functions, so that cell activation through PrP^c clustering in reggie rafts and subsequent reggie-associated internalization might contribute to PrP^sc propagation.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2150fje;

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