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.

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 G protein coupled receptors (GPCR) represent the target class for nearly half of the current therapeutic drugs and remain to be the focus of drug discovery efforts. The complexity of receptor signaling continues to evolve. It is now known that many GPCRs are coupled to multiple G-proteins, which lead to regulation of respective signaling pathways downstream. Deciphering this receptor coupling will aid our understanding of the GPCR function and ultimately developing drug candidates. Here, we report the development of four homogenous bioluminescent reporter assays using improved destabilized luciferases and various response elements: CRE, NFAT-RE, SRE, and SRF-RE. These assays allowed measurement of major GPCR pathways including cAMP production, intracellular Ca2+ mobilizations, ERK/MAPK activ-ity, and small G protein RhoA activity, respectively using the same reporter assay format. We showed that we can decipher G protein activation profiles for exogenous m3 muscarinic receptor and endogenous β2-adrenergic receptors in HEK293 cells by using these four reporter assays. Furthermore, we demonstrated that these assays can be readily used for potency rankings of agonists and antagonists, and for high throughput screening.

Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.