We have uncovered a novel function for the G protein GAP Rgs13 in allergic responses. The physiological importance of Rgs13 as negative regulator of IgE-mediated MC reactivity was suggested by the markedly enhanced degranulation of Rgs13-deficient MCs and anaphylaxis of Rgs13−/− mice in response to Ag. Rgs13 appeared to regulate MC degranulation by physically interacting with the p85α subunit of PI(3)K and impairing Ag-induced PI(3)K activation.
Several lines of evidence indicate that Rgs13 directly inhibited PI(3)K signaling in MCs independent of its GAP activity. Rgs13 bound p85α directly, and the interaction required only the Rgs13 amino-terminus (1-51), which does not possess GAP activity (Xie et al.
MS submitted). Amplification of MC degranulation by GPCR agonists such as adenosine occurs through activation of the PI(3)Kγ isoform by Gβγ released from Gαi-GTP15
. PI(3)Kγ does not associate with p85 subunits27
, suggesting that Rgs13 would not be expected to regulate PI(3)Kγ directly. In our study, inactivation of Gαi proteins by PTX did not significantly reduce Ag-evoked BMMC degranulation, nor did it diminish the differential response of WT and Rgs13
−/− BMMCs to Ag. Lastly, a mutant RGS13 protein devoid of GAP activity inhibited Ag-evoked degranulation similar to WT RGS13 ().
Whereas Ag-evoked degranulation was strongly increased in Rgs13
−/− BMMCs, cytokine production by MCs did not appear to be affected by the loss of Rgs13. Ag-induced cytokine synthesis is controlled by calcium-NFAT and NFκB pathways in addition to MAPKs. Although Ag-elicited PLCγ phosphorylation and Ca++
release were increased in the absence of Rgs13, MAPK/Erk activation was not affected by Rgs13 deficiency. Preliminary evidence also suggests that neither Jun kinase (JNK) phosphorylation nor NFκB activation were significantly different in WT or Rgs13
−/− BMMCs stimulated with Ag (data not shown). MAPKs are regulated by intracellular Ca++
concentration, through activation of PLCγ and diacylglycerol (DAG), which in turn activate protein kinase C (PKC). Importantly, however, there are at least four isoforms of PKC identified in MCs that may both positively (PKCβ28
) and negatively (PKCδ29
) regulate MAPK activity. MAPKs can be also be activated by PKC (and therefore Ca++
For these reasons, enhanced Ag-evoked PLCγ activity and the resultant increase in Ca++
mobilization in Rgs13
−/− BMMCs could lead to increased degranulation without significantly affecting MAPK activation and cytokine synthesis. Dissociation of cytokine production and degranulation in MCs has been observed in diacylglycerol kinase (DGK) ζ-deficient BMMCs31
. β-hexosaminidase release was reduced by ~50% in DGKζ −/− BMMCs. In contrast, the production of IL-6 was enhanced in response to Ag stimulation. In these cells, decreased Ag-evoked Ca++
mobilization resulting in reduced PKCβII membrane recruitment may have accounted for the suppressed degranulation, while enhanced MAPK/ERK and Akt activation might explain the increased IL-6 production.
In addition to MAPKs, Akt is also a critical mediator of Ag-induced cytokine production32
/calmodulin was recently shown to interact with the pleckstrin homology (PH) domain of Akt, which competes with PIP3 for binding to Akt33
. Calmodulin inhibits membrane recruitment of Akt and impairs its subsequent activation. Several members of R4 subfamily of RGS proteins have been shown to interact with calmodulin through a conserved motif in the RGS domain34, 35
. Since Rgs13 contains this sequence, the absence of Rgs13 in BMMCs could result in increased amounts of calmodulin available to inhibit Akt, thus counterbalancing the increased PI3K catalytic activity. Collectively, these results suggested that Rgs13 might be more important in regulating type I immediate hypersensitivity reactions rather than innate immune functions of MCs involving cytokine synthesis and release.
It is believed that while cytosolic p85/p110 complexes are catalytically inactive, recruitment of SH2 domains in p85 subunits to phosphotyrosine residues in receptors or adaptor proteins activates PI(3)K by positioning the p110 subunit in proximity to membrane lipid substrates27
. Notably, deletion of the p85α isoform resulted in only minor defects in IgE-mediated degranulation of BMMCs while Rgs13-deficiency profoundly affected degranulation and anaphylaxis. We attribute this discrepancy to the fact that the p85β isoform, whose expression is clearly upregulated in p85α knockout MCs36
, may compensate for the loss of p85α in these cells. In contrast, Rgs13 may bind more than one p85 isoform. The increased amount of p85α bound to Gab2 in Rgs13
−/− MCs suggests that Rgs13 inhibits PI(3)K activity by restricting accessibility of p85 to its FcεRI-associated scaffold complex after receptor stimulation, resulting in diminished Ag-induced MC degranulation ().
A model for the regulation of FcεRI-mediated MC degranulation by Rgs13
Rgs13 binding to p85α and inhibition of PI(3)K signaling required only the 51 amino-terminal amino acids of Rgs13. Recent studies have suggested the amino-terminus of R4 RGS proteins, which adopts an α-helical conformation in vivo37
but is disordered in the crystal structure of recombinant RGS4 complexed with Gαi138
may collaborate with diverse signaling molecules to integrate pathways elicited by various cell surface receptors39
. Such interactions have been proposed to stabilize the amino-terminus of RGS proteins in distinct conformations depending on the binding partner39
. For example, some RGSs may selectively regulate a subset of GPCRs by binding additional proteins such as spinophilin40, 41
or the third intracellular loop of the receptor itself42
Rgs13 was previously shown to be expressed primarily in human and murine B lymphocytes. Our microarray analysis of human cells and β-galactosidase staining of LacZ
knock-in mice indicated that Rgs13 indeed displays very limited tissue expression restricted to lymphocytes, MCs, and endocrine cells of the thymus, GI, and respiratory tracts. Thus, the increased allergic responses of Rgs13
−/− mice were not likely to due enhanced vascular permeability induced by MC vasoactive mediators on end-organ targets. This explanation could account for the observed phenotype if, for example, Rgs13 were highly expressed in endothelial cells and loss of Rgs13 expression in these cells enhanced signaling to GPCR ligands such as histamine. We did detect binding of other R4 RGSs including RGS1, 4, 5, and 16 to p85α, suggesting that this interaction was common to this subfamily of RGS proteins (data not shown
). Since Rgs1 and Rgs13 are abundant in B cells43, 44
and Rgs16 is enriched in activated T cells45, 46
, RGS proteins may regulate PI(3)K activation evoked by Ag receptors in lymphocytes, which could significantly impact other adaptive immune responses.
In B lymphocytes, Rgs13
is induced by anti-CD40 and IL-4 stimulation44
. Here, we showed that Rgs13 expression in MCs is strikingly upregulated by Ag stimulation. RGS13
expression was recently found to be increased in human basophils treated with IgE/Ag47
. Thus, Ag-induced expression of Rgs13 could potentially restrict or prevent tissue damage from continual MC degranulation due to recurring Ag exposure, which may occur in beekeepers or in patients undergoing immunotherapy. Conversely, loss of RGS function due to reduced expression or inactivating mutation(s) could underlie or contribute to the pathogenesis of disorders characterized by increased MC degranulation, such as idiopathic anaphylaxis.
In summary, we have elucidated a new link between GPCR signaling pathways and the predominantly tyrosine kinase-dependent signaling elicited by immune receptor activation in MCs. Rgs13 regulation of PI(3)K activity resulted in increased MC degranulation and allergic responses. Given the ubiquity of RGS proteins, the evidence presented here for the direct mitigation of PI(3)K signaling by an R4 RGS family member portends a novel and common mechanism by which cells may integrate or specify diverse and/or conflicting extracellular cues. Since PI(3)K also has a critical function in cancer progression and glucose metabolism, further investigation into RGS regulation of PI(3)K signaling in these and other systems is warranted in light of this study.