ABCC8 p.Arg486Ala
| Predicted by SNAP2: | A: D (53%), C: D (63%), D: D (85%), E: D (66%), F: D (66%), G: D (66%), H: N (66%), I: D (59%), K: N (93%), L: D (59%), M: D (53%), N: D (59%), P: D (71%), Q: N (66%), S: N (53%), T: N (53%), V: N (53%), W: D (80%), Y: D (63%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: N, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: N, P: D, Q: N, S: N, T: D, V: D, W: D, Y: D, |
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[hide] Evidence of a functional role for interaction betw... J Immunol. 2006 Sep 15;177(6):4113-21. Celli L, Ryckewaert JJ, Delachanal E, Duperray A
Evidence of a functional role for interaction between ICAM-1 and nonmuscle alpha-actinins in leukocyte diapedesis.
J Immunol. 2006 Sep 15;177(6):4113-21., [PMID:16951376]
Abstract [show]
ICAM-1 is involved in both adhesion and extravasation of leukocytes to endothelium during inflammation. It has been shown that the ICAM-1 cytoplasmic domain is important for transendothelial migration of leukocytes but the precise molecular mechanisms involving the intracytoplasmic portion of ICAM-1 is not known. To characterize precisely the molecular scaffolding associated with ICAM-1, we have used the yeast two-hybrid system, and we have identified six different proteins interacting with the ICAM-1 cytoplasmic domain. In this study, we report that the two forms of nonmuscle alpha-actinin (i.e., alpha-actinin 1 and alpha-actinin 4) associate with ICAM-1, and that these interactions are essential for leukocyte extravasation. These interactions were further confirmed by coimmunoprecipitation and immunofluorescence in endothelial cells and in ICAM-1-transfected Chinese hamster ovary cells. The function of these interactions was analyzed by point mutation of charged amino acids located on ICAM-1 cytoplasmic domain. We have identified three charged amino acids (arginine 480, lysine 481, and arginine 486) which are essential in the binding of alpha-actinins to the ICAM-1 cytoplasmic tail. Mutation of these amino acids completely inhibited ICAM-1-mediated diapedesis. Experiments with siRNA inhibiting specifically alpha-actinin 1 or alpha-actinin 4 on endothelial cells indicated that alpha-actinin 4 had a major role in this phenomenon. Thus, our data demonstrate that ICAM-1 directly interacts with cytoplasmic alpha-actinin 1 and 4 and that this interaction is required for leukocyte extravasation.
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No. Sentence Comment
73 ICAM-1 cytoplasmic mutants: CHO ICAM-1 R480A, K481A, K483A, K484A, and R486A were generated using the QuickChange Site-Directed Mutagenesis kit (Stratagene).
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ABCC8 p.Arg486Ala 16951376:73:71
status: NEW197 In this study, we compared the interaction level between ICAM-1, considered as positive control, and each mutant (ICAM-1 R480A, K481A, K483A, K484A, and R486A) with both ␣-actinin 1 and 4.
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ABCC8 p.Arg486Ala 16951376:197:153
status: NEW247 All the rCHO cell lines expressing ICAM-1, ICAM-1⌬Cyt, and mutants (R480A, K481A, K483A, K484A, and R486A) formed monolayers that were nearly impermeable to HRP, with 12.0 Ϯ 3.9%, 9.1 Ϯ 0.8%, 9.8 Ϯ 1.9%, 10.1 Ϯ 5.6%, 16.4 Ϯ 1.9%, 5.7 Ϯ 0.6%, and 12.2 Ϯ 3.3% of controls, respectively; these values are similar to those obtained with WT CHO monolayers (11.9 Ϯ 1.1%) (Fig. 6).
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ABCC8 p.Arg486Ala 16951376:247:107
status: NEW251 As shown in Fig. 7, when ICAM-1/␣-actinin interactions were altered, the number of neutrophils transmigrating across CHO monolayers was reduced to a level similar to CHO WT or CHO ICAM-1⌬Cyt (14.22 Ϯ 3.32% for ICAM-1 R 480A, 14.92 Ϯ 5.25% for ICAM-1 K481A, and 16.2 Ϯ 3.54% for ICAM-1 R486A vs 13.33 Ϯ 2.17% for CHO WT and 14.94 Ϯ FIGURE 5.
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ABCC8 p.Arg486Ala 16951376:251:317
status: NEW336 Given the similar effect of ICAM-1 mutants R480A, K481A, and R486A on both ␣-actinin 1 and 4 interaction, we were not able to conclude which isoform of ␣-actinins was involved in transmigration.
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ABCC8 p.Arg486Ala 16951376:336:61
status: NEW