The 3 human RAS genes, KRAS, NRAS, and HRAS, encode 4 different RAS proteins which belong to the protein family of small GTPases that function as binary molecular switches involved in cell signaling. Activating mutations in RAS are among the most common oncogenic drivers in human cancers, with KRAS being the most frequently mutated oncogene. Although KRAS is an excellent drug discovery target for many cancers, and despite decades of research, no therapeutic agent directly targeting RAS has been clinically approved. Using structure-based drug design, we have discovered BI-2852 (1), a KRAS inhibitor that binds with nanomolar affinity to a pocket, thus far perceived to be “undruggable,” between switch I and II on RAS; 1 is mechanistically distinct from covalent KRASG12C inhibitors because it binds to a different pocket present in both the active and inactive forms of KRAS. In doing so, it blocks all GEF, GAP, and effector interactions with KRAS, leading to inhibition of downstream signaling and an antiproliferative effect in the low micromolar range in KRAS mutant cells. These findings clearly demonstrate that this so-called switch I/II pocket is indeed druggable and provide the scientific community with a chemical probe that simultaneously targets the active and inactive forms of KRAS.

An understanding of chemotaxis at the level of cell-molecule interactions is important because of its relevance in cancer, immunology, and microbiology, just to name a few. This study quantifies the effects of flow on cell migration during chemotaxis in a microfluidic device. The chemotaxis gradient within the device was modeled and compared to experimental results. Chemotaxis experiments were performed using the chemokine CXCL8 under different flow rates with human HL60 promyelocytic leukemia cells expressing a transfected CXCR2 chemokine receptor. Cell trajectories were separated into x and y axis components. When the microchannel flow rates were increased, cell trajectories along the x axis were found to be significantly affected (p < 0.05). Total migration distances were not affected. These results should be considered when using similar microfluidic devices for chemotaxis studies so that flow bias can be minimized. It may be possible to use this effect to estimate the total tractile force exerted by a cell during chemotaxis, which would be particularly valuable for cells whose tractile forces are below the level of detection with standard techniques of traction-force microscopy.

The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/ caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors.

Using transgenic Nicotiana plumbaginifolia seedlings in which the calcium reporter aequorin is targeted to the chloroplast stroma, we found that darkness stimulates a considerable flux of Ca 2 ϩ into the stroma. This Ca 2 ϩ flux did not occur immediately after the light-to-dark transition but began ‫ف‬ 5 min after lights off and increased to a peak at ‫ف‬ 20 to 30 min after the onset of darkness. Imaging of aequorin emission confirmed that the dark-stimulated luminescence emanated from chloroplast-containing tissues of the seedling. The magnitude of the Ca 2 ϩ flux was proportional to the duration of light exposure (24 to 120 h) before lights off; the longer the duration of light exposure, the larger the darkstimulated Ca 2 ϩ flux. On the other hand, the magnitude of the dark-stimulated Ca 2 ϩ flux did not appear to vary as a function of circadian time. When seedlings were maintained on a 24-h light/dark cycle, there was a stromal Ca 2 ϩ burst after lights off every day. Moreover, the waveform of the Ca 2 ϩ spike was different during long-day versus short-day light/dark cycles. The dark-stimulated Ca 2 ϩ flux into the chloroplastidic stroma appeared to affect transient changes in cytosolic Ca 2 ϩ levels. DCMU, an inhibitor of photosynthetic electron transport, caused a significant increase in stromal Ca 2 ϩ levels in the light but did not affect the magnitude of the dark-stimulated Ca 2 ϩ flux. This robust Ca 2 ϩ flux likely plays regulatory roles in the sensing of both light/dark transitions and photoperiod.

Agonist-stimulated internalization followed by recycling to the cell membrane play an important role in fine-tuning the activity of chemokine receptors. Because the recycling of chemokine receptors is critical for the reestablishment of the cellular responsiveness to ligand, it is crucial to understand the mechanisms underlying the receptor recycling and resensitization. In the present study, we have demonstrated that the chemokine receptor CXCR2 associated with myosin Vb and Rab11-family interacting protein 2 (FIP2) in a ligand-dependent manner. Truncation of the C-terminal domain of the receptor did not affect the association, suggesting that the interactions occur upstream of the C terminus of CXCR2. After ligand stimulation, the internalized CXCR2 colocalized with myosin Vb and Rab11-FIP2 in Rab11a-positive vesicles. The colocalization lasted for ϳ2 h, and little colocalization was observed after 4 h of ligand stimulation. CXCR2 also colocalized with myosin Vb tail or Rab11-FIP2 (129 -512), the N-terminal-truncated mutants of myosin Vb and Rab11-FIP2, respectively, but in a highly condensed manner. Expression of the enhanced green fluorescent protein-tagged myosin Vb tail significantly retarded the recycling and resensitization of CXCR2. CXCR2 recycling was also reduced by the expression Rab11-FIP2 (129 -512). Moreover, expression of the myosin Vb tail reduced CXCR2-and CXCR4-mediated chemotaxis. These data indicate that Rab11-FIP2 and myosin Vb regulate CXCR2 recycling and receptor-mediated chemotaxis and that passage of internalized CXCR2 through Rab11a-positive recycling system is critical for physiological response to a chemokine. INTRODUCTIONChemokine receptors belong to the large family of seventransmembrane G protein-coupled receptors (GPCRs) that function in immune and inflammatory response by regulating the activation and migration of leukocytes, immune cell development, and angiogenesis (Nagasawa et al., 1996;Luster 1998;Belperio et al., 2000;Murphy et al., 2000;Zlotnik and Yoshie, 2000). Some of them (e.g., CCR5 and CXCR4) participate in HIV infection of CD4 ϩ T lymphocytes as coreceptors (Berger et al., 1999). Ligand binding to the chemokine receptors triggers various signaling cascades, including activation of G proteins, phosphotidylinositol 3-kinase, Janus kinase/signal transducers and activators of transcription proteins, the Rho-p160 ROCK axis, and the MAPK pathway (Wu et al., 1993;Ganju et al., 1998;Mellado et al., 1998;Vicente-Manzanares et al., 1999;Vicente-Manzanares et al., 2002). Chemokine activation of these intracellular signals is often accompanied by chemokine receptor internalization and trafficking back to the cell membrane. The intracellular trafficking of chemokine receptors controls their activities, and the balance between the chemokine receptor recycling and degradation dictates the leukocyte responsiveness to chemokines (Sabroe et al., 1997;Asagoe et al., 1998;Khandaker et al., 1998;Mack et al., 1998).Ligand stimulated chemokine receptor internalization can be accomplished ...