Chemokine-induced biological events. The binding of a cognate chemokine ligand to its receptor modifies the tertiary structure of the receptor, such that its cytoplasmic portion can bind and activate heterotrimeric G proteins. The activated G-protein subunits subsequently stimulate multiple signal transduction pathways involving phospholipase Cβ (PLCβ) isoforms, phosphoinositide-3 kinases (PI3K), and various Src family kinases; the pathway details outlined in Fig. 1 vary slightly, depending on the cellular context in which the relevant receptor is expressed. For instance, the activation of PI3Kγ, but not PLCβ activity, is required for myeloid cell chemotaxis,(3) whereas PI3Kγ activation is dispensable for lymphocyte chemotaxis.(4) In neutrophils, PI3K activation results in the generation of phophatidylinositol-3,4,5-triphosphate (PIP3), and both PI3K and PIP3 translocate to the leading edge of the chemotaxing cell, where they activate the small GTPase Rac, which then induces local actin polymerization in the leading edge. Protein kinase B (PKB) also translocates to the leading edge, where it contributes to local actin polymerization.(5,6) In lymphocytes, the translocation of PI3K and PIP3 has not been documented,(7) and activation of a PI3K-independent pathway leads to Rac activation, in which the scaffold protein DOCK-2 appears to play a critical role.(8) Indeed, in DOCK-2-deficient mice, in which chemokine-induced Rac activation is severely decreased in lymphocytes but not in monocytes, lymphocyte chemotaxis is preferentially abrogated, whereas monocyte chemotaxis is uncompromised,(8) validating the idea that lymphocytes use biochemical pathways for directional cell migration that are distinct from those used by monocytes.(7) In the leading edge of monocytes, another small GTPase, Cdc42, is recruited and activated locally, and appears to be essential for directional cell migration, because the absence of Cdc42 results in non-directional cell migration.(6,9) In neutrophils, Cdc42 has an essential role in the exclusion from the leading edge of a PIP3-phosphatase, PTEN, which is a negative regulator of PIP3.(10) Thus, upon chemokine stimulation, PI3K localizes anteriorly, whereas PTEN localizes posteriorly in neutrophils,(11) and the spatial and temporal regulation of the PI3K-dependent pathways as well as of Rac and Cdc42 GTPases appears essential for determining the internal polarity (particularly the ‘frontness’) of a migrating cell.
Figure 1. The chemokine receptor signaling network. Chemokine receptors can bind and activate heterotrimeric G proteins, including Gαißγ and Gα13βγ. In a chemotaxing cell, signaling components responsible for the formation of cell polarity, directional sensing and F-actin polymerization, such as PI3K, Rac and Cdc42, are preferentially recruited to the leading edge, whereas the mediators of actomyosin contraction, which are downstream of Rho, are recruited to the trailing edge. PTEN is excluded from the leading edge, helping to restrict PI3K signaling to the leading edge. Thus, the spatially regulated localization of signaling components plays a vital role in determining the ‘frontness’ and ‘backness’ in a chemotaxing cell. The details of the chemokine signaling pathways appear to vary slightly depending on the cellular context.
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Another small GTPase, Rho, is involved in the regulation of cellular morphology through its effects on the actin cytoskeleton. Upon chemokine stimulation, Rho preferentially localizes to the trailing edge of a migrating cell and is activated by Rho guanine nucleotide exchange factors (GEF) in non-lymphoid cells.(6) Activated Rho then induces the formation of actin and myosin complexes, resulting in the retraction of the trailing edge of the cell. Thus, Rho appears to be directly involved in determining the ‘backness’ of at least non-lymphoid cells. Interestingly, the polarity signals are transmitted by different G-protein subunits in non-lymphoid cells, in which frontness is controlled by Gβγ subunits, which coordinate the activation of PI3K and Rac and of actin organization at the leading edge, whereas backness is controlled by the Gα12/13 subunit, which activates Rho, Rho-dependent kinase, and myosin II.(11) The role of Rho in lymphoid cells is currently unclear.(7)
In lymphocytes, Rap1 appears be important in both cell polarity determination and integrin activation by chemokines. Rap1 activation occurs rapidly and transiently upon chemokine stimulation in a Gαi-dependent manner.(12) Following Rap1 activation, a Rap1-binding protein (RAPL) rapidly associates with a β2 integrin (LFA-1) and translocates with LFA-1 to the leading edge,(13) which is essential for the activation of cell-surface LFA-1 and its adhesion to intercellular adhesion molecule-1 (ICAM-1). The expression of a dominant-active form of Rap1 promotes shear-resistant cell adhesion to LFA-1 ligands such as ICAM-1 and promotes a polarized morphology in lymphoid cells.(12) Inhibition of Rap1 activation by the forced expression of a Rap GTPase-activating protein, Spa-1, inhibits lymphoid chemotaxis,(12) and a lack of RAPL results in remarkably decreased levels of integrin-mediated cell adhesion and chemotaxis.(13,14) These results strongly indicate that Rap1 plays an essential role in integrin activation and cell polarity determination through the action of RAPL in lymphocytes, although the exact biochemical pathways leading to Rap1 activation remain to be fully explored.