The function of RPTPs in chemoattractant-mediated GPCR signaling has not previously been appreciated. In this study, we found that the two RPTPs CD45 and CD148 have critical and unique as well as redundant functions in a chemoattractant-GPCR signaling pathway. Our studies along these lines were initiated by the unexpected finding that the loss of CD45 or CD148 has distinct and opposite influences on neutrophil-dependent bacterial clearance in the skin air pouch model. We sought a mechanistic explanation for these discordant effects. The observed redundant deficits in neutrophil functions, such as in cell adhesion, phagocytosis, respiratory burst and bactericidal activity are in line with our previous observations of the redundant influence of these RPTPs in ITAM-coupled receptor signaling systems in macrophages and B cells (Zhu et al., 2008
). The functional impairments of neutrophils from Ptprc−/−
mice likely contribute to their relatively weaker host defense. However, the early increased neutrophil recruitment as well as the increased chemotatic responses and fMLF receptor signaling of neutrophils from PtprjTM−/TM−
mice was surprising, but correlated with their ability to more rapidly clear the infection. This increased responsiveness by CD148 deficient cells is unique and in marked contrast to the reduced responsiveness of Ptprc−/−
neutrophils. However, these RPTPs do have some overlap in their functional activities, as revealed in the Ptprc−/−PtpriTM−/TM−
mice. Deficiency of both of these RPTPs much more substantialy impaired overall neutrophil function. These redundant functions in regulating SFKs likely accounts for the more profound impairment in resistance to S. aureus
infection in the Ptprc−/−PtpriTM−/TM−
animals and the more profound defects observed in most of the signaling and functional assays.
The role of RPTPs in S. aureus
infection is complex. In this paper we chose to focus on a localized infection model, the air pouch, and to understand the unexpected non-redundant function of CD45 versus CD148 in the fMLF signaling pathway. Previously, we found that these RPTPs have only redundant functions in ITAM receptor-mediated signaling pathways in B cells and macrophages (Zhu et al., 2008
). Directly correlated with the differential host defense and migratory responses of CD45 versus CD148 deficient neutrophils is the differential effect of these RPTPs on GPCR-mediated signaling responses. CD148-deficient cells consistently demonstrated increased signaling responses (Ca2+
flux, Erk and Akt phosphorylation) while CD45-deficient cells were modestly impaired compared to wild-type cells. This differential effect on intracellular signaling mirrors some of the differences that have been genetically revealed in studies of the main substrates of these RPTPs; namely the SFKs. In our experiments, neutrophils from Lyn kinase-deficient mice manifested the same increased fMLF signaling response as cells from CD148-deficient animals. Loss of all three SFKs produced a signaling defect closely resembling Ptprc−/−PtpriTM−/TM−
cells. These observations suggest that at least in the GPCR pathway the preferential regulation, presumably involving dephosphorylation, of Lyn by CD148 versus CD45, accounts for the unexpected non-redundant functions for these RPTPs.
The differential function of Hck and Fgr versus Lyn kinase in GPCR pathways has been incompletely studied. Clearly, all three kinases are involved responses to end-target chemoattractants, such as fMLF, as we have shown here. Surprisingly, Hck and Fgr have the opposite function in GPCR pathways stimulated by endogenous chemokines such as MIP-1α (CCL3) and MIP2 (CXCL2) (Zhang et al., 2005
). Neutrophils lacking Hck and Fgr show increased responses to these chemokines while Lyn-deficient cells shown only a modest impairment in response to these stimuli (H. Zong and C. Lowell, unpublished). However, the signaling pathways downstream of distinct receptors involved in chemotaxis seem to differ. For instance, it has been reported that end-target chemoattractants (such as fMLF) and endogenous chemokines (such as IL-8, MIP-2) induce different signaling pathways; end-target attractants require PI3K, whereas endogenous chemokines signal independently of PI3K but activate p38 mitogen activated protein kinases (Heit et al., 2002
). We have also tested the consequences of the loss of CD45 and CD148 in neutrophils following stimulation with MIP-2, an endogenous chemokine. In preliminary studies, we found that their effect on intracellular calcium following MIP-2 were quite different than during fMLF stimulation (supp. Fig.2
), with essentially an opposite pattern of responses observed. This suggests that CD45 and CD148 may regulate exogenous- and endogenous-chemokine mediated signaling pathways in essentially opposite ways, similar to Hck and Fgr versus Lyn kinases. This further supports the notion that CD148 is primarily responsible for Lyn activation while CD45 has broader, but incomplete activity, on all three SFK.
The discovery of unique functions of CD45 and CD148 following GPCR stimulation extended our knowledge beyond their redundant roles in Fc receptor mediated pathway. To understand these unique functions, it was important to identify their substrates within the GPCR pathway. Genetic and direct biochemical evidences in this paper provide strong support to the hypothesis that CD148 and CD45 both regulate SFKs by targeting their C-terminal negative regulatory tyrosines. However, our studies here show that these two RPTPs regulate the fMLF chemoattractant GPCR induced pathway in distinct ways, namely through the preferential recognition of Lyn by CD148 versus the broader role of CD45 in activating all SFKs, The distinct functions of Hck and Fgr versus Lyn kinase reflect the somewhat unique ability of Lyn to engage inhibitory signaling pathways through specific phosphorylation of ITIM-containing proteins in the plasma membrane and their by regulate their association with cytoplasmic regulatory molecules such as the cytoplasmic tyrosine phosphatase SHP-1 or the cytoplasmic SH2 domain containing inositol lipid 5-phosphatase SHIP-1. Lyn is the primary kinase responsible for phosphorylating inhibitory receptors such as PIR-B, SIRPα, FcRγIIb or cytoplasmic molecules such as Dok1, which in turn recruit phosphatases such as SHP-1 and SHIP-1 to dampen intracellular pathways (Scapini et al., 2009
). While Lyn does function in cooperation with other SFKs to initiate activating intracellular signals, its inhibitory function is dominant. Hence, deficiency of the Lyn kinase tends to result in hyperactive immune cells. In contrast, combinatorial loss of Hck and Fgr tends to lead to loss of function (especially when combined with Lyn deficiency). This differential function of SFKs is reflected here in the fMLF signaling pathway as well as in the neutrophil integrin signaling pathway. Lyn-deficient neutrophils show exaggerated adhesion and activation following adhesion to integrin ligands such as ICAM-1, fibrinogen or fibronectin due to reduced recruitment and activation of SHP-1 (Scapini et al., 2009
). In contrast, Hck−/−Fgr−/−
neutrophils manifest a strong impairment of adhesion-induced functions, including respiratory burst and degranulation (Lowell et al., 1996
; Mocsai et al., 1999
). In our studies Hck−/−Fgr−/−
neutrophils did not show a major defect in the fMLF-mediated pathway, presumably due to the compensatory positive role that Lyn can also play. However, the fact that inducible phosphorylation of SHP-1 is reduced in PtprjTM−/TM−
neutrophils following fMLF stimulation, while it was basally hyperphosphorylated and was normally down-regulated in CD45-deficient cells, provides further biochemical support for the model in which loss of CD148 predominantly disrupts the negative regulatory function of Lyn.
The opposing roles of CD45 and CD148 through their regulation of SFKs on fMLF mediated signaling are intriguing, although it is possible that there are additional substrates for these RPTPs. For example, CD148 may have additional target other than SFK that function only in the inhibitory pathway to modulate the early events in neutrophil GPCR signaling or may influence other pathways altogether. Moreover, the great disparity in the structures of these RPTPs suggests that they may be subject to different modes of regulation yet to be revealed.