Chemotaxis mediated by chemokine receptors such as CXCR4 plays a key role in lymphocyte homing and hematopoiesis as well as in breast cancer metastasis. We have demonstrated previously that ␤-arrestin2 functions to attenuate CXCR4-mediated G protein activation and to enhance CXCR4 internalization. Here we show further that the expression of ␤-arrestin2 in both HeLa and human embryonic kidney 293 cells significantly enhances the chemotactic efficacy of stromal cell-derived factor 1␣, the specific agonist of CXCR4, whereas the suppression of ␤-arrestin2 endogenous expression by antisense or RNA-mediated interference technology considerably attenuates stromal cell-derived factor 1␣-induced cell migration. Expression of ␤-arrestin2 also augmented chemokine receptor CCR5-mediated but not epidermal growth factor receptor-mediated chemotaxis, indicating the specific effect of ␤-arrestin2. Further analysis reveals that expression of ␤-arrestin2 strengthened CXCR4-mediated activation of both p38 MAPK and ERK, and the suppression of ␤-arrestin2 expression blocked the activation of two kinases. Interestingly, inhibition of p38 MAPK activation (but not ERK activation) by its inhibitors or by expression of a dominantnegative mutant of p38 MAPK effectively blocked the chemotactic effect of ␤-arrestin2. Expression of a dominant-negative mutant of ASK1 also exerted the similar blocking effect. The results of our study suggest that ␤-arrestin2 can function not only as a regulator of CXCR4 signaling but also as a mediator of stromal cellderived factor 1␣-induced chemotaxis and that this activity probably occurs via the ASK1/p38 MAPK pathway.

Norepinephrine released by the sympathetic nerve terminals regulates the immune system primarily via its stimulation of beta(2)-adrenergic receptor (beta(2)AR), but the underlying molecular mechanisms remain to be elicited. Beta(2)AR, a well-studied G protein-coupled receptor (GPCR), is functionally regulated by beta-arrestin2, which not only causes receptor desensitization and internalization but also serves as a signaling molecule in GPCR signal transduction. Here we show that beta-arrestin2 directly interacts with IkappaBalpha (inhibitor of NF-kappaB, the key molecule in innate and adaptive immunity) and thus prevents the phosphorylation and degradation of IkappaBalpha. Consequently, beta-arrestin2 effectively modulates activation of NF-kappaB and expression of NF-kappaB target genes. Moreover, stimulation of beta(2)AR significantly enhances beta-arrestin2-IkappaBalpha interaction and greatly promotes beta-arrestin2 stabilization of IkappaBalpha, indicating that beta-arrestin2 mediates a crosstalk between beta(2)AR and NF-kappaB signaling pathways. Taken together, the current study may present a novel mechanism for regulation of the immune system by the sympathetic nervous system.

The chemokine receptor CXCR4 has recently been shown to be a co-receptor involved in the entry of human immunodeficiency virus type 1 into target cells. This study shows that coexpression of ␤-arrestin with CXCR4 in human embryonic kidney 293 cells attenuated chemokine-stimulated G protein activation and inhibition of cAMP production. Truncation of the C-terminal 34 amino acids of CXCR4 (CXCR4-T) abolished the effects of ␤-arrestin on CXCR4/G protein signaling, indicating the functional interaction of the receptor C terminus with ␤-arrestin. On the other hand, receptor internalization and the subsequent activation of extracellular signal-regulated kinases were significantly promoted by coexpression of ␤-arrestin with CXCR4, whereas the C-terminal truncation of CXCR4 did not affect this regulation of ␤-arrestin, suggesting that ␤-arrestin can functionally interact with CXCR4 with or without the C terminus. Moreover, ␤ 2 V54D, the dominant inhibitory mutant of ␤-arrestin 2, exerted no effects on CXCR4/G protein signaling, but strongly influenced receptor internalization and extracellular signalregulated kinase activation. Further cross-linking experiments demonstrated that ␤-arrestin as well as ␤ 2 V54D could physically contact both CXCR4 and CXCR4-T. Glutathione S-transferase pull-down assay showed that ␤-arrestin was able to bind efficiently in vitro to both the third intracellular loop and the 34-amino acid C terminus of CXCR4. Taken together, our data clearly establish that ␤-arrestin can effectively regulate different functions of CXCR4 and that this is mediated through its distinct interactions with the C terminus and other regions including the third loop of CXCR4.

The Epidermal Growth Factor Receptor (EGFR) is upregulated in numerous human cancers. Inhibition of EGFR signaling induces autophagy in tumor cells. Here we report an unanticipated role for the inactive EGFR in autophagy initiation. Inactive EGFR interacts with the oncoprotein LAPTM4B that is required for the endosomal accumulation of EGFR upon serum starvation. Inactive EGFR and LAPTM4B stabilize each other at endosomes and recruit the exocyst subcomplex containing Sec5. We show that inactive EGFR, LAPTM4B, and the Sec5 subcomplex are required for basal and starvation induced autophagy. LAPTM4B and Sec5 promote EGFR association with the autophagy inhibitor Rubicon, which in turn disassociates Beclin 1 from Rubicon to initiate autophagy. Thus, the oncoprotein LAPTM4B facilitates the role of inactive EGFR in autophagy initiation. This pathway is positioned to control tumor metabolism and promote tumor cell survival upon serum deprivation or metabolic stress.

The purpose of this study was to investigate invasion- and metastasis-related genes in gastric cancer. To this end, we used the transwell system to select a highly invasive subcell line from minimally invasive parent cells and compared gene expression in paired cell lines with high- and low-invasive potentials. Lysyl oxidase-like 2 (LOXL2) was overexpressed in the highly invasive subcell line. Immunohistochemical analysis revealed that LOXL2 expression was markedly increased in carcinoma relative to normal epithelia, and this overexpression in primary tumor was significantly associated with depth of tumor invasion, lymph node metastasis and poorer overall survival. Moreover, LOXL2 expression was further increased in lymph node metastases compared with primary cancer tissues. RNA interference-mediated knockdown and ectopic expression of LOXL2 showed that LOXL2 promoted tumor cell invasion in vitro and increased gastric carcinoma metastasis in vivo. Subsequent mechanistic studies showed that LOXL2 could activate both the Snail/E-cadherin and Src kinase/Focal adhesion kinase (Src/FAK) pathways. However, secreted LOXL2 induced gastric tumor cell invasion and metastasis exclusively via the Src/FAK pathway. Expression correlation analysis in gastric carcinoma tissues also revealed that LOXL2 promoted invasion via the Src/FAK pathway but not the Snail/E-cadherin pathway. We then evaluated secreted LOXL2 as a target for gastric carcinoma treatment and found that an antibody against LOXL2 significantly inhibited tumor growth and metastasis. Overall, our data revealed that LOXL2 overexpression, a frequent event in gastric carcinoma progression, contributes to tumor cell invasion and metastasis, and LOXL2 may be a therapeutic target for preventing and treating metastases.

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Summary Polarized delivery of signaling and adhesion molecules to the leading edge is required for directional migration of cells. Here, we describe a role for the PIP2 synthesizing enzyme, PIPKIγi2, in regulation of exocyst complex control of cell polarity and polarized integrin trafficking during migration. Loss of PIPKIγi2 impaired directional migration, formation of cell polarity, and integrin trafficking to the leading edge. Upon initiation of directional migration PIPKIγi2 via PIP2 generation controls the integration of the exocyst complex into an integrin-containing trafficking compartment(s) that requires the talin-binding ability of PIPKIγi2, and talin for integrin recruitment to the leading edge. A PIP2 requirement is further emphasized by inhibition of PIPKIγi2-regulated directional migration by an Exo70 mutant deficient in PIP2 binding. These results reveal how phosphoinositide generation orchestrates polarized trafficking of integrin in coordination with talin that links integrins to the actin cytoskeleton, processes that are required for directional migration.

Summary Endosomal trafficking and degradation of epidermal growth factor receptor (EGFR) play an essential role in control of its signaling. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2) is an established regulator of endocytosis, whereas PtdIns3P modulates endosomal trafficking. However, here we demonstrate that type Igamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes PtdIns4,5P2, controls endosome to lysosome sorting of EGFR. In this pathway, PIPKIγi5 interacts with sorting nexin 5 (SNX5), a protein that binds PtdIns4,5P2 and other phosphoinositides. PIPKIγi5 and SNX5 localize to endosomes, and loss of either protein blocks EGFR sorting into intraluminal vesicles (ILVs) of the multivesicular body (MVB). Loss of ILV sorting greatly enhances and prolongs EGFR signaling. PIPKIγi5 and SNX5 prevent Hrs ubiquitination and this facilitates the Hrs association with EGFR that is required for ILV sorting. These findings reveal that PIPKIγi5 and SNX5 form a unique signaling nexus that controls EGFR endosomal sorting, degradation, and signaling.