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Chloride intracellular channel proteins have homology to ion channels and omega class of glutathione-S-transferases but channel activity is not well established, suggesting roles in other signaling pathways. Among the six CLICs, CLIC1 and CLIC4 are expressed in endothelial cells (EC) and act to promote EC proliferation, capillary-like networks, and lumen formation. We and others determined that Sphingosine-1-phosphate (S1P) signaling promotes transient CLIC4 membrane localization. We report that CLIC1 and CLIC4 have distinct roles in endothelial S1P signaling. In knockdown studies, CLIC1 and CLIC4 were independently required for S1PR1-mediated Rac1 activation, enhanced EC barrier integrity, and EC migration. CLIC1 was uniquely required for S1PR2/3-driven RhoA activation and actin stress fiber formation, while CLIC4 was uniquely required for thrombin/PAR-driven RhoA activation and endothelial permeability. CLICs were not required for other GPCR-mediated pathways measured, including S1PR1-mediated cAMP regulation downstream of Gαi, or Ras and ERK activation downstream of Gβγ. Endothelial β-adrenergic signaling, which uses Gαs, was unaltered by loss of CLICs. Further investigation of receptor tyrosine kinase signaling (VEGF, EGF) in endothelial cells reveals their signaling cascades do not depend on CLICs as well. We conclude that CLICs mediate S1PR-driven RhoA and Rac1 regulation, and thrombin/PAR-driven RhoA activation, and a possible mediator for endothelial GPCR by modulating Rac1 and RhoA. CLIC N-termini contain membrane insertion motifs and the putative ion channel domain, while the C-termini contain two predicted SH domains. Chimeric proteins generated by swapping N and C-termini of CLIC1 and CLIC4 were used in rescue experiments. The C-terminal domain was determined to confer S1PR1-CLIC-Rac1 mediated barrier function and migration. We further characterized N-termini of CLIC4 and membrane localization of by generating CLIC4 C-termini truncated protein, along with CLIC4 C-termini fusing with Lck-peptide for myristylation and plasma membrane re-localization. CLIC4 C-termini alone fails to rescue S1PR1-CLIC-Rac1 mediated barrier function, while membrane localization of the CLIC4 C-terminal domain functions in S1P signaling, suggesting the N-terminal domain confers membrane localization but not signaling function. Thus, we conclude S1P promotes cell localization of CLIC4 to the EC plasma membrane through N-termini, which then regulates Rac1 mediated events through C-termini. Through these findings, our work defines a molecular mechanism through which CLICs function in endothelium.

The ITS sequences of Morus were analyzed in this study, which have an average length of 576 bp and a GC content of 59.55%. There are many mutations in the ITS sequences, and a total of 166 variants are identified (100 in ITS1, 60 in ITS2, and 0 in 5.8S). The systematic location of Morus is between Milicia excelsa (MEU93585) and Streblus heterophyllus (DQ499105), and much closer to the former one based on the maximum parsimony (MP) analysis. Under Bayesian analysis, Xinjiang M. nigra and North America M. murrayana (FJ605515) were firstly separated in cladogram as the most primitive species with the time of origin at about 55 mya, while other species were divided into three branches: (1) Lichuan M. wittiorum and Xianfeng M. wittiorum with a time of origin at about 2 mya; (2) M. cathayana related species with a time of origin at about 15 mya; (3) species that morphology related to M. alba with a time of origin at about 28 mya. Morus was originated from the high latitude regions in Laurasia and then migrated from north to south. The mountains between 20-40°N latitudes in East Asia are the secondary origin centers of Morus.