Differential Distribution of Both IL-12Rβ Chains in the Plasma Membrane of Human T Cells

Abstract

IL-12 is a cytokine that stimulates the expression of CD26, a T cell- and raft-associated ectopeptidase. IL-12 also enhances the interaction between CD26 and CD45RO, which removes the phosphatase CD45RO from raft microdomains. Since Janus kinases are known CD45 substrates, our hypothesis was that this relocation of CD45RO in nonraft areas of the membrane could be important to switch off the signaling via cytokine receptors, e.g., the IL-12 receptor (IL-12R). Accordingly, both IL-12R and CD45RO should be equally positioned in the cell membrane upon IL-12R ligation. However, there were no data available on the membrane distribution of IL-12R on human T cells. Working with phytohemagglutinin (PHA) lymphoblasts, we tried to fill that gap. The high-affinity IL-12R is made of two chains: IL-12Rbeta1 and IL-12Rbeta2. Using flow cytometry, Western blot and confocal microscopy, we obtained data suggesting that IL-12Rbeta1 is mainly associated to phospholipid-rich membrane areas, a location even enhanced upon IL-12 incubation of PHA blasts. Instead, IL-12Rbeta2 is found more segregated into membrane rafts, which could explain why two IL-12-triggered events, T-cell proliferation and ERK1/2 activation, are both methyl-beta-cyclodextrin-sensitive events. Ligation of IL-12R with IL-12 seems to induce a partial enrichment of IL-12Rbeta2 in phospholipid-rich areas, where according to our data IL-12Rbeta1 is already present. Therefore, although new data will be required, the present results support the initial hypothesis.

Inflammatory Signals Direct Expression of Human IL12RB1 into Multiple Distinct Isoforms

“…This first IL-12R component was cloned by Chua et al (10) from COS cells transfected with a cDNA library from PHA-activated PBMCs; a COS clone exhibiting both surface reactivity with mAb 2.4E6 and an affinity for extracellular IL-12 was chosen for sequencing (10). Rigorous studies since that time have shown that isoform 1 is integral to the plasma membrane (60,61), binds the p40 domain of IL-12/IL-23 (11,62), associates with IL-12Rb2/IL-23R to form high-affinity complexes for IL-12/IL-23 (12,32), and, because it lacks intrinsic kinase activity, relies on its association with cytoplasmic Tyk2 for relaying IL-12 signals (31, 63). However, the original cloning scheme used by Chua et al (10) , and CD56 + lineages from the same PBMC pool were magnetically sorted, and the levels of IL12RB1 isoform 1 and 2 expression were determined using real-time PCR.…”

IL-12 family cytokines: immunological playmakers

“…Why STAT1:STAT4 heterodimers are only generated following IL-35 ligation remains to be determined, but could be due to the proximity of STAT1 and STAT4 phosphorylation favoring heterodimer formation. Indeed, the distribution of the IFNGR and the IL12R may not be uniform and is altered following T cell activation, suggesting that these receptors might even be actively segregated as a mechanism to actively prevent STAT1:STAT4 heterodimer formation 62–64 . Alternatively, IL-35 quantitatively promotes the generation of almost equal levels of pSTAT1 and pSTAT4 promoting heterodimerization, thereby leaving little free pSTATs for homodimerization.…”

Molecular characterization, tissue distribution and expression analysis of Interleukin-12 receptor β2 chain in sheep

“…IL-12Rβ2 is not present on resting T cells and a T-cell receptor (TCR)-mediated stimulus is required in order to express this protein (Rogge et al, 1999;Watford et al, 2004;Wu et al, 2000). IL-12Rβ2 is found more segregated into membrane rafts, ligation of IL-12R with IL-12 seems to induce a partial nrichment of IL-12Rβ2 in phospholipid-rich areas, where IL-12Rβ1 is already present (Canda-Sánchez et al, 2009). Based on the report of Canda-Sánchez et al and the analysis of putative protein structure of ovine IL-12Rβ2, we speculated that IL-12Rβ2 is initially expressed in membrane rafts of T lymphoblasts at very low level, and it would increase and reinforce signaling and T cell proliferation generated by the TCR/CD3 complex (Canda-Sánchez et al, 2009).…”

“…IL-12Rβ2 is found more segregated into membrane rafts, ligation of IL-12R with IL-12 seems to induce a partial nrichment of IL-12Rβ2 in phospholipid-rich areas, where IL-12Rβ1 is already present (Canda-Sánchez et al, 2009). Based on the report of Canda-Sánchez et al and the analysis of putative protein structure of ovine IL-12Rβ2, we speculated that IL-12Rβ2 is initially expressed in membrane rafts of T lymphoblasts at very low level, and it would increase and reinforce signaling and T cell proliferation generated by the TCR/CD3 complex (Canda-Sánchez et al, 2009). However, the report about the membrane distribution of the IL-12R indicated that both FcRIIIa and IL-12R proteins stained diffusely around the cell membrane in resting human NK cells (Kondadasula et al, 2008).…”

“…The expression of IL-12R on different kinds of cells was investigated (Airoldi et al, 2000;Airoldi et al, 2008;Canda-Sánchez et al, 2009;Kondadasula et al, 2008;Waldvogel et al, 2002). Airoldi et al first investigated expression and function of IL-12Rβ2 in normal polyclonal plasmablastic cells (PPCs) generated in vitro from normal peripheral blood or isolated from tonsil, as well as in normal plasma cells (PCs) purified from bone marrow (BM) or tonsil (Airoldi et al, 2008).…”