IL-8 (or CXCL8) activates the receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB) to induce chemotaxis in leukocytes, but only CXCR1 mediates cytotoxic and cross-regulatory signals. This may be due to the rapid internalization of CXCR2. To investigate the roles of the intracellular domains in receptor regulation, wild-type, chimeric, phosphorylation-deficient, and cytoplasmic tail (C-tail) deletion mutants of both receptors were expressed in RBL-2H3 cells and studied for cellular activation, receptor phosphorylation, desensitization, and internalization. All but one chimeric receptor bound IL-8 and mediated signal transduction, chemotaxis, and exocytosis. Upon IL-8 activation, the chimeric receptors underwent receptor phosphorylation and desensitization. One was resistant to internalization, yet it mediated normal levels of β-arrestin 2 (βarr-2) translocation. The lack of internalization by this receptor may be due to its reduced association with βarr-2 and the adaptor protein-2β. The C-tail-deleted and phosphorylation-deficient receptors were resistant to receptor phosphorylation, desensitization, arrestin translocation, and internalization. They also mediated greater phosphoinositide hydrolysis and exocytosis and sustained Ca2+ mobilization, but diminished chemotaxis. These data indicate that phosphorylation of the C-tails of CXCR1 and CXCR2 are required for arrestin translocation and internalization, but are not sufficient to explain the rapid internalization of CXCR2 relative to CXCR1. The data also show that receptor internalization is not required for chemotaxis. The lack of receptor phosphorylation was correlated with greater signal transduction but diminished chemotaxis, indicating that second messenger production, not receptor internalization, negatively regulates chemotaxis.

The low molecular weight GTP-binding protein RhoA regulates many cellular events, including cell migration, organization of the cytoskeleton, cell adhesion, progress through the cell cycle and gene expression. Physical forces influence these cellular processes in part by regulating RhoA activity through mechanotransduction of cell adhesion molecules (e.g. integrins, cadherins, Ig superfamily molecules). RhoA activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) that are themselves regulated by many different signaling pathways. Significantly, the engagement of many cell adhesion molecules can affect RhoA activity in both positive and negative ways. In this brief review, we consider how RhoA activity is regulated downstream from cell adhesion molecules and mechanical force. Finally, we highlight the importance of mechanotransduction signaling to RhoA in normal cell biology as well as in certain pathological states.

Chemokines are a diverse gene family of chemotactic cytokines that induce leukocyte accumulation and activation at sites of inflammation (1-3). They also mediate tumor cell trafficking and metastasis and participate in many acute and chronic inflammatory diseases (4, 5). Chemokine functions are mediated via cell surface G-protein-coupled receptors that couple predominantly to G i (1-3, 18, 35). Chemokine receptors, most notably CCR5 and CXCR4, also serve as co-receptors for human immunodeficiency virus type 1 (HIV-1) 1 entry into CD4 ϩ cells (6, 7). To date, the relationship between the activation of these receptors and their role in HIV-1 infection is not well understood.Like many members of the G-protein-coupled receptor family, CCR5 and CXCR4 become desensitized upon agonist exposure, resulting in a loss of cellular responsiveness to agonist followed by a decrease in the number of cell surface receptors (8 -13). Phosphorylations of the carboxyl terminus of the receptors are responsible for the desensitization and down-regulation (8 -13). We have previously shown that chemokine receptors cross-regulate the functions of each other (14, 35). The interleukin-8 (IL-8 or CXCL8) receptor CXCR1 cross-phosphorylated and cross-desensitized CCR1-mediated cellular responses to RANTES (CCL5) (14). The formyl peptide chemoattractant receptor also cross-desensitized CCR5-mediated cellular responses to RANTES in monocytes and diminished the ability of RANTES to mediate HIV-1 entry and infection (15,16).While HIV-1 infection requires the CD4 receptor, the role of a chemokine receptor as the fusion cofactor depends on the target cell (17). Both macrophages and T lymphocytes express CCR5 and CXCR4 (18). Macrophages, however, utilize CCR5 for HIV-1 entry (M-tropism), whereas CD4 ϩ T lymphocytes use CXCR4 (T-tropism) (18). In addition to CCR5 and CXCR4, macrophages and CD4 ϩ T lymphocytes express other chemokine receptors including the IL-8 receptors CXCR1 and CXCR2 (1-8 ϫ 10 6 receptors/cell) (1, 18 -24). In the present study we sought to determine the role of cross-regulation by IL-8 receptors in CCR5-and CXCR4-mediated cellular activation and HIV-1 infection. For this purpose, monocytes and MAGIC5 and RBL-2H3 cells stably expressing different combination of receptors were used to study the mechanisms of cross-regulation among IL-8, CCR5, and CXCR4. The results demonstrate that IL-8 led to the cross-phosphorylation and cross-desensitization of both CCR5 and CXCR4. However, IL-8 down-regulated and

Unlike estrogen receptor-positive (ER( þ )) breast cancers, normal human mammary epithelial cells (HMECs) typically express low nuclear levels of ER (ER poor). We previously demonstrated that 1.0 lM tamoxifen (Tam) promotes apoptosis in acutely damaged ER-poor HMECs through a rapid, 'nonclassic' signaling pathway. Interferon-regulatory factor-1 (IRF-1), a target of signal transducer and activator of transcription-1 transcriptional regulation, has been shown to promote apoptosis following DNA damage. Here we show that 1.0 lM Tam promotes apoptosis in acutely damaged ER-poor HMECs through IRF-1 induction and caspase-1/3 activation. Treatment of acutely damaged HMEC-E6 cells with 1.0 lM Tam resulted in recruitment of CBP to the c-IFN-activated sequence element of the IRF-1 promoter, induction of IRF-1, and sequential activation of caspase-1 and -3. The effects of Tam were blocked by expression of siRNA directed against IRF-1 and caspase-1 inhibitors. These data indicate that Tam induces apoptosis in HMEC-E6 cells through a novel IRF-1-mediated signaling pathway that results in activated caspase-1 and -3.