Angel Y.F. Kam

Rac and Cdc42‐dependent regulation of c‐Jun N‐terminal kinases by the δ‐opioid receptor

Heptahelical opioid receptors utilize Gi proteins to regulate a multitude of effectors including the classical adenylyl cyclases and the more recently discovered mitogen‐activated protein kinases (MAPKs). The c‐Jun NH2‐terminal kinases (JNKs) belong to one of three subgroups of MAPKs. In NG108‐15 neuroblastoma × glioma hybrid cells that endogenously express δ‐opioid receptors, δ‐agonist dose‐dependently stimulated JNK activity in a pertussis toxin‐sensitive manner. By using COS‐7 cells transiently transfected with the cDNAs of δ‐opioid receptor and hemagglutinin (HA)‐tagged JNK, we delineated the signaling components involved in this pathway. Sequestration of Gβγ subunits by transducin suppressed the opioid‐induced JNK activity. The possible involvement of the small GTPases was also examined. Expression of dominant negative mutants of Rac and Cdc42 blocked the opioid‐induced JNK activation, and a partial inhibition was observed in the presence of the dominant negative mutant of Ras. In contrast, the dominant negative mutant of Rho did not affect the opioid‐induced JNK activation. In addition, the receptor‐mediated JNK activation was dependent on Src family tyrosine kinases, but independent of phosphatidylinositol‐3 kinase and EGF receptor tyrosine kinases. Collectively, these results demonstrate functional regulation of JNK by the δ‐opioid receptor, and this pathway requires Gβγ, Src kinases and the small GTPases Rac and Cdc42.

Phosphatidylinositol-3 kinase is distinctively required for mu-, but not kappa-opioid receptor-induced activation of c-Jun N-terminal kinase.

Opioid receptors are the therapeutic targets of narcotic analgesics. All three types of opioid receptors (µ, δ and κ) are prototypical Gi‐coupled receptors with common signaling characteristics in their regulation of intracellular events. Nevertheless, numerous signaling processes are differentially regulated by the three receptors. We have recently demonstrated that stimulation of δ‐opioid receptor can up‐regulate the activity of the c‐Jun N‐terminal kinase (JNK) in a pertussis toxin‐sensitive manner ( Kam et al. 2003 ; J. Neurochem. 84, 503–513). The present study revealed that the µ‐opioid receptor could stimulate JNK in both SH‐SY5Y cells and transfected COS‐7 cells. The mechanism by which the µ‐opioid receptor stimulated JNK was delineated with the use of specific inhibitors and dominant‐negative mutants of signaling intermediates. Activation of JNK by the µ‐opioid receptor was mediated through Gβγ, Src kinase, son‐of‐sevenless (Sos), Rac and Cdc42. Interestingly, unlike the δ‐opioid receptors, the µ‐opioid receptor required phosphatidylinositol‐3 kinase (PI3K) to activate JNK. The µ‐opioid receptor‐induced JNK activation was effectively inhibited by wortmannin or the coexpression of a dominant negative mutant of PI3Kγ. Like the δ‐opioid receptor, activation of JNK by the κ‐opioid receptor occurred in a PI3K‐independent manner. These studies revealed that the µ‐opioid receptor utilize a distinct mechanism to regulate JNK.

Formyl peptide‐receptor like‐1 requires lipid raft and extracellular signal‐regulated protein kinase to activate inhibitor‐κB kinase in human U87 astrocytoma cells

Formyl peptide‐receptor like‐1 (FPRL‐1) may possess critical roles in Alzheimer’s diseases, chemotaxis and release of neurotoxins, possibly through its regulation of nuclear factor‐κB (NFκB). Here we illustrate that activation of FPRL‐1 in human U87 astrocytoma or Chinese hamster ovary cells stably expressing the receptor resulted in the phosphorylations of inhibitor‐κB kinase (IKK), an onset kinase for NFκB signaling cascade. FPRL‐1 selective hexapeptide Trp‐Lys‐Tyr‐Met‐Val‐Met (WKYMVM) promoted IKK phosphorylations in time‐ and dose‐dependent manners while pre‐treatment of pertussis toxin abrogated the Gαi/o‐dependent stimulations. The FPRL‐1‐mediated IKK phosphorylation required extracellular signal‐regulated protein kinase (ERK), phosphatidylinositol 3‐kinase and cellular Src (c‐Src), but not c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase. Despite its ability to mobilize Ca2+, WKYMVM did not require Ca2+ for the modulation of IKK phosphorylation. Activation of FPRL‐1 also induced NFκB‐driven luciferase expression. Interestingly, cholesterol depletion from plasma membrane by methyl‐β‐cyclodextrin abolished the FPRL‐1‐stimulated IKK phosphorylation, denoting the important role of lipid raft integrity in the FPRL‐1 to IKK signaling. Furthermore, we demonstrated that in U87 cells, several signaling intermediates in the FPRL‐1‐IKK pathway including Gαi2, c‐Src and ERK were constitutively localized at the raft microdomains. WKYMVM administration not only resulted in higher amount of ERK recruitment to the raft region, but also specifically stimulated raft‐associated c‐Src and ERK phosphorylations. Taken together, these results demonstrate that FPRL‐1 is capable of activating NFκB signaling through IKK phosphorylation and this may serve as a useful therapeutical target for FPRL‐1‐related diseases.

Activation of the Human FPRL-1 Receptor Promotes Ca2+ Mobilization in U87 Astrocytoma Cells

The human formyl peptide receptor like 1 (FPRL-1) is a variant of the Gi-coupled formyl-peptide receptor. Functional FPRL-1 is endogenously expressed in the U87 astrocytoma cell line and there is accumulating evidence to suggest that FPRL-1 may be involved in neuroinflammation associated with the pathogenesis of Alzheimer’s disease. In this study, we examined the ability of FPRL-1 to mobilize intracellular Ca2+ in U87 astrocytoma cells, as well as in Chinese hamster ovary (CHO) cells stably expressing FPRL-1. We showed that Trp–Lys–Tyr–Met–Val–Met–NH2 (WKYMVM), a specific agonist for FPRL-1, stimulated Ca2+ influx in both U87 and FPRL-1/CHO cells. These effects can be inhibited by the FPRL-1 selective antagonist, WRW4. Involvement of Gi proteins was demonstrated with the use of pertussis toxin, while inhibitors of store-operated channels (SOC) including 1-[2-(4-methoxyphenyl)]-2-[3-(4-methpxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF96365) and 2-aminoethoxydiphenyl borate (2-APB) were found to abolish the WKYMVM-induced Ca2+ increase. However, intracellular Ca2+ mobilization in both cell lines were unaffected by the phospholipase Cβ inhibitor U73122 or selective ryanodine receptor inhibitors. Our data demonstrated that activation of Gi-coupled FPRL-1 can lead to Ca2+ influx possibly via SOCs in U87 and FPRL-1/CHO cells.