Since its discovery in 1994 (25
), CXCR4 has been shown to be one of the widest-expressed and most biologically critical of all the chemokine receptors. CXCR4 mediates a diverse array of signaling pathways in a large number of cell types, regulating critical developmental, immunological, and pathological cues by activating a wide range of signaling molecules (1
). One strategy that CXCR4 utilizes to diversify its signal transduction mechanisms among these different cell types is heterodimerization with other types of cell-surface receptors; in T cells, CXCR4 signals by heterodimerizing with the T cell antigen receptor (TCR) (8
). In addition, we demonstrate here that the CXCR4-TCR heterodimer further diversifies its downstream signaling within T cells by utilizing distinct PLC isoforms in order to mediate different signal transduction outcomes.
We show here that upon ligation by SDF-1, the CXCR4-TCR heterodimer signals to activate one PLC isozyme for calcium mobilization and ERK activation, another PLC for migration, and yet another PLC for internalization of CXCR4. We found that ERK activation, migration, and internalization of CXCR4 in response to SDF-1 are inhibited by a PLC inhibitor drug, indicating that PLC activity is required for all of these events. We further showed that PLC-β3 expression is required for SDF-1-mediated calcium mobilization, activation of N-Ras and K-Ras, as well as for activation of ERK, while also showing that PLC-γ1 expression is not required for these events. In contrast, we showed that PLC-γ1 expression is required for T cell migration in response to SDF-1 while depletion of PLC-β3 actually enhances migration. Moreover, we found that neither PLC-β3 nor PLC-γ1 was required for SDF-1 treatment to induce the internalization and downregulation of CXCR4 from the cell-surface, indicating a role for other PLCs. Finally, we showed that PLC-γ1 mediates migration in response to SDF-1 in a manner that is independent of LAT, thus indicating that the CXCR4-TCR heterodimer couples to PLC-γ1 in a manner distinct from that arising from direct ligation of the TCR. Our results presented here, combined with previous work, provide further insight into the molecular mechanisms that permit the CXCR4-TCR heterodimer to integrate the signaling of traditional GPCR-linked and traditional TCR-linked signaling mediators. Both pertussis toxin-sensitive Gi-type G proteins and ZAP-70 have been shown to be required for calcium mobilization, ERK activation, and migration in response to SDF-1 treatment of T cells (6
). Thus, our results indicate that, in response to SDF-1, both PLC-β3 and PLC-γ1 require upstream Gi proteins and ZAP-70 for their regulation in T cells.
SDF-1-induced calcium mobilization and ERK activation require some of the same signaling molecules, the TCR, ZAP-70, and SLP-76 (6
), that the direct ligation of the TCR requires to activate these pathways (27
). We therefore investigated if, like the TCR, the CXCR4-TCR heterodimer signals via PLC-γ1. Yet we found that PLC-β3, not PLC-γ1, is required for SDF-1 to induce calcium mobilization and ERK activation, a result indicating that PLC-β3 is able to integrate signaling from ZAP-70 and SLP-76 as well as G proteins. The Gβγ subunit of Gi proteins has been shown to bind directly to and mediate the activation of PLC-β3 downstream of GPCR signaling (10
). PLC-β3 has also been shown to bind various PDZ-containing scaffold proteins, including Nherf-2 and Shank-2 that mediate the interactions of numerous signaling molecules (11
). Nherf family members are implicated in regulating T cell activation (31
), suggesting that Nherf or related molecules may mediate interactions between PLC-β3 and TCR signaling molecules.
Similarly, the requirement for PLC-γ1 in mediating migration in response to SDF-1 suggests that PLC-γ1 is able to integrate signals from Gi proteins and possibly other G proteins (32
) as well as traditional TCR signaling molecules such as ZAP-70. However, SDF-1 clearly regulates PLC-γ1 in a manner independent of other key mediators that are required for signal transduction arising from direct TCR stimulation. Following direct ligation of the TCR, LAT and SLP-76 are both required for the recruitment and activation of PLC-γ1 (27
). In contrast, we show here that SDF-1-mediated migration requires PLC-γ1 but occurs independently of LAT expression, and others have shown that SDF-1-dependent T cell migration also does not involve SLP-76 (24
). GPCR kinase interacting protein-1 (GIT1) has been shown to regulate PLC-γ1 signaling downstream of other GPCRs, and is also activated by the TCR in a manner independent of SLP-76 (33
). SDF-1 signaling via the CXCR4-TCR heterodimer may therefore utilize GIT1 as an alternative mechanism of PLC-γ1 activation in order to regulate T cell migration. Furthermore and as we show here, PLC-γ1 can mediate cellular functions without the commonly-seen inducible phosphorylation of its tyrosines (35
). Bach et al. have shown that depletion of both PLC-β2 and PLC-β3 in mouse T cells partially inhibits T cell migration in response to SDF-1 (14
). PLC-γ1 may therefore function to regulate migration via a mechanism that integrates its signals with PLC-β2, possibly via forming a PLC dimer as has been seen for other PLC isoforms (36
). Thus, CXCR4-TCR signaling utilizes a distinct mechanism to integrate the functions of GPCR-associated and TCR-associated signaling molecules in order to mediate PLC-γ1-dependent migration.
Internalization of CXCR4 has been extensively studied because the functions of CXCR4 critically depend on its cell-surface expression levels. A key mechanism regulating this pathway includes the phosphorylation of CXCR4 by G-protein-coupled receptor kinases (GRKs) and PKC followed by the recruitment ofβ-arrestins (38
). A role for PLC activity in the internalization of CXCR4 has not previously been described. Inhibition of Gi proteins (23
) or the depletion of ZAP-70 (unpublished observations) failed to inhibit internalization of CXCR4 in response to SDF-1, consistent with neither PLC-γ1 nor PLC-β3 being required for this process. Further studies will be required to determine which PLC isoforms are critically required for the internalization of CXCR4 in response to SDF-1.
Together, the results in this paper indicate that multiple PLCs are able to integrate signaling from the CXCR4-TCR heterodimer in order to act as key mediators of SDF-1-mediated T cell functions. The PLC-dependent T cell functions mediated by SDF-1 described here are critical for many immunological events. Both PLC-β3 deficient mice and mice specifically deficient in T cell PLC-γ1 display spontaneous autoimmune phenotypes involving mononuclear infiltrates particularly into the skin and ear (12
). Furthermore, T cell specific PLC-γ1 deficient mice were characterized by a paucity of peripheral T cells, possibly arising from abnormal T cell migration, as well as by the impaired development and function of regulatory T cells (12
). We previously demonstrated that SDF-1 costimulates IL-10 production in regulatory T cells by activating prolonged ERK and AP-1-mediated transcription (7
). Thus, it seems possible that the SDF-1 signaling that is impaired by the lack of PLC-γ1 may inhibit the appropriate migration of regulatory T cells, while SDF-1 signaling impaired by the lack of PLC-β3 inhibits the costimulation of IL-10 production by regulatory T cells. In addition, both CXCR4 and PLC-γ1 have been implicated as regulators of the metastasis of various cancer cell types (2
). Our results therefore raise the possibility that PLC-γ1 helps mediate the SDF-1-induced metastasis of these tumoral cells. The novel integration of multiple PLCs with distinct functions downstream of SDF-1 mediated signaling described here may thereby be responsible for the diverse roles CXCR4 plays in immunology and pathology.