Role of CD31 Binding Partners in Viable Leukocyte Detachment from Macrophages
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CD31 mediates homophilic interactions between leukocytes and macrophages during inflammation, apoptotic cells remain attached and are engulfed whereas viable cells actively detach. We hypothesised that differential recruitment of signalling and adapter molecules to the cytoplasmic domain were responsible for the disengagement of the viable leukocyte from macrophages. Investigation with a static attachment assay using THP-1 as a macrophage model showed the ITIM of CD31 on the leukocyte was important for viable cell detachment. Our data also implicated a role for the recruitment of SHP-2 which we attempted to knock-down by siRNA delivered by lentivirus. SHP-2, in addition to its phosphatase activity, also acts as a docking protein. To examine for potential interacting partners we fused the cytoplasmic domain of CD31 to GST which we used in pulldown assays from lysates of viable and apoptotic leukocytes. We demonstrated that the recruitment of SHP-1 and SHP-2 were dependent on an intact ITIM (immunoreceptor tyrosine-based inhibitory motif). Interestingly, apoptotic cell lysates promoted dephosphorylation of the in vitro phosphorylated GST-CD31, suggesting an increase in phosphatase activity in aged neutrophils. We were unable to demonstrate an interaction between the cytoplasmic domain of CD31 with putative binding partners β-catenin, src, RasGAPp120, RhoGAPp190, talin or calmodulin. A proteomic approach by MALDI-TOF and MS/MS identified Hsp90 as a novel binding partner of CD31 irrespective of the phosphorylation state. In contrast, 14-3-3ε bound to phosphorylated CD31, whereas eIF3 specifically bound to an ITIM double tyrosine mutant. The binding of Hsp90 to CD31 was proposed to occur via a TPR motif within the cytoplasmic domain of CD31 which comprises a surface fold of basic amino acids complexing a highly acidic carboxy tail of Hsp90. Truncation and site directed mutagenesis of the cytoplasmic domain revealed multiple binding sites for Hsp90; specifically two regions containing the sequences (KAFYLRKAKAK), previously shown to be the calmodulin binding region, and a novel area (SNNEKMSDMEANSHY) which has significant homology with other TPR-containing proteins. Systematic mutagenesis of the putative basic charged amino acids within the cytoplasmic domain of CD31 which may mediate the interaction with Hsp90 also supports the presence of a TPR motif. The importance of Hsp90, 14-3-3ε and eIF3 is currently unknown, although it is interesting to note that CD31 was recently found within this laboratory to associate with the voltage-gated potassium channel HERG which also binds 14-3-3ε and Hsp90. eIF3 is an RNA helicase that may link CD31 and leukocyte motility to spreading initiation centres where motility can be viewed as a rapid turnover of focal adhesion complexes. Together, these studies have identified novel binding partners of CD31 may form a macromolecular complex to promote CD31-dependent leukocyte motility and detachment from macrophages.