Adapters in the organization of mast cell signaling

Abstract

Mast cells are pivotal in innate immunity and play an important role in amplifying adaptive immunity. Nonetheless, they have long been known to be central to the initiation of allergic disorders. This results from the dysregulation of the immune response whereby normally innocuous substances are recognized as non-self, resulting in the production of IgE antibodies to these 'allergens'. Preformed and newly synthesized inflammatory (allergic) mediators are released from the mast cell following allergen-mediated aggregation of allergen-specific IgE bound to the high-affinity receptors for IgE (FcepsilonRI). Thus, the process by which the mast cell is able to interpret the engagement of FcepsilonRI into the molecular events necessary for release of their allergic mediators is of considerable therapeutic interest. Unraveling these molecular events has led to the discovery of a functional class of proteins that are essential in organizing activated signaling molecules and in coordinating and compartmentalizing their activity. These so-called 'adapters' bind multiple signaling proteins and localize them to specific cellular compartments, such as the plasma membrane. This organization is essential for normal mast cell responses. Here, we summarize the role of adapter proteins in mast cells focusing on the most recent advances toward understanding how these molecules work upon FcepsilonRI engagement.

Figure 1. Schematic representation of the FcεRI-stimulated assembly of macromolecular signaling complexes in mast cells (MCs)

FcεRI aggregation by cell surface-bound IgE antibody interaction with a multivalent antigen leads to the phosphorylation of the receptors β- and γ-chain immunoreceptor tyrosine-based activation motifs (ITAMs) by Lyn kinase. Fyn and Syk kinases are then recruited to the receptor and initiate complementary signals that cooperate in MC responses. Fyn and possibly Syk phosphorylate adapters like Gab2 that are essential for PI3K activation. Syk also phosphorylates the adapters LAT1 and LAT2 in several tyrosine residues. LAT1 organizes a complex that binds phospholipase C-γ (PLC-γ), SLP-76, and son-of-sevenless (SOS) via adapters such as Gads, Grb2, and Shc. Activation of PLC-γ in this complex causes the ₂₊ hydrolysis of PI(4, 5)P₂ into IP₃ and DAG, increasing intracellular Ca concentrations and activating PKC. In addition to LAT1, the adapter LAT2 is also phosphorylated after FcεRI aggregation and organizes a signaling complex by recruiting Grb2 to its several YXN motifs. Grb2 then participates in activating the PI3K pathway through its association with the adapter Gab2. Like LAT1, LAT2 is able to recruit the guanine nucleotide exchange factors Vav1 and SOS, which in turn activate MAPKs such as ERK. LAT2 may also bind PLC-γ indirectly by its association with Grb2. LAT1 is well documented to be involved in Ca²⁺ mobilization, which is crucial for MC degranulation, NFAT activation, and cytokine production. PKC activation is also essential for MC degranulation, activation of NF-κB, and cytokine production.

Figure 2. Negative regulation of Fyn kinase by Lyn-phosphorylated Cbp/PAG

The adapter Cbp is a transmembrane protein that is targeted to lipid rafts due to its palmitoylation. In mast cells (MCs), Cbp is moderately phosphorylated in resting cells by Lyn kinase and shows a moderate association with the negative regulatory kinase Csk, which phosphorylates Src PTKS and inactivates them. FcεRI stimulation greatly enhances the phosphorylation of Cbp (on Y317) in a Lyn-dependent manner. This allows additional recruitment of Csk, which phosphorylates Fyn at its C-terminal tyrosine (Y525) causing an intra-molecular folding (via interaction with its own SH2 domain) that inactivates it. This negative regulation of Fyn (and possibly of other Src PTKS) controls the responsiveness of the MC. In the absence of Lyn, MCs are hyperresponsive due to loss of this negative regulatory mechanism.

Figure 3. Adapter proteins in mast cells (MCs)

Schematic model of the adapter proteins expressed in MCs, structural motifs that allow protein–protein or protein lipid–interactions, and molecular mass. Binding partners confirmed in MCs are indicated.

Figure 4. Model of FcεRI-induced LAT1 and LAT2 multiprotein complexes in mast cells (MCs)

FcεRI stimulation causes LAT1 tyrosine phosphorylation, which allows association with Gads, Grb2, SLP-76, Btk, Vav1, SLAP-130, and phospholipase C-γ (PLC-γ). Structural motifs for interactions are shown. The small adapters Grb2 and Gads bring molecules like son-of-sevenless (SOS) and SLP-76 into the complex. SLP-76 can recruit two additional adapters, SLP-130 and most probably Nck. Other signaling molecules, like Vav1 and Btk, can bind to tyrosine-phosphorylated SLP-76 via their SH2 domains. Like LAT1, LAT2 also becomes tyrosine phosphorylated upon FcεRI stimulation and is also able to recruit Grb2 and SOS. In contrast to LAT1, the adapter LAT2 associates with Vav1 primarily via binding to Grb2. In addition, through a second Grb2-interaction site, the adapter Gab2 may be indirectly recruited to the LAT2 multiprotein complex. Grb2 mediates recruitment of Gab2 via its SH2 domain binding to LAT2 and its SH3 domain binding to the proline-rich region of Gab2. Tyrosine phosphorylation of Gab2 results in the recruitment of the regulatory PI3K subunit p85 via its SH2 domain subsequently leading to PI3K activation.

Figure 5. Negative regulation of FcεRI signaling by the SHIP-1/Dok-1/RasGAP complex

Upon co-aggregation of the FcεRI with the FcγRIIB by a multivalent antigen, SHIP-1 is recruited to the tyrosine-phosphorylated ITIM (immune receptor tyrosine-based inhibitory motif) of the FcγRIIB (via its SH2-domain) in a Lyn kinase-dependent manner. Tyrosine phosphorylated SHIP-1 becomes associated with the adapters Shc and Grb2. Grb2 binds to another C-terminal phosphorylated tyrosine in the FcγRIIB, which stabilizes the complex (197). In addition, Dok-1, which is constitutively associated with RasGAP, is also recruited to tyrosine phosphorylated SHIP-1. One known effect of recruitment of SHIP-1 to the plasma membrane is the downregulation of PIP3 levels, which leads to decreased phospholipase C-γ (PLC-γ) activity, IP₃ production, and impaired Ca²⁺ fluxes, thus inhibiting mast cell degranulation. Recruitment of RasGAP to Dok-1 inhibits son-of-sevenless (SOS) and thereby Ras activation, leading to decreased cytokine and eicosanoid production. Similar to the FcγRIIB, the tyrosine-phosphorylated ITAM (immunoreceptor tyrosine-based activation motif) sequence of the β-chain of the FcεRI has been shown to recruit SHIP-1 and Grb2, and an association of Dok-1, but not RasGAP, with the FcεRI has been demonstrated.