Bridging the Gap: Modulatory Roles of the Grb2-Family Adaptor, Gads, in Cellular and Allergic Immune Responses

Abstract

Antigen receptor signaling pathways are organized by adaptor proteins. Three adaptors, LAT, Gads, and SLP-76, form a heterotrimeric complex that mediates signaling by the T cell antigen receptor (TCR) and by the mast cell high affinity receptor for IgE (FcεRI). In both pathways, antigen recognition triggers tyrosine phosphorylation of LAT and SLP-76. The recruitment of SLP-76 to phospho-LAT is bridged by Gads, a Grb2 family adaptor composed of two SH3 domains flanking a central SH2 domain and an unstructured linker region. The LAT-Gads-SLP-76 complex is further incorporated into larger microclusters that mediate antigen receptor signaling. Gads is positively regulated by dimerization, which promotes its cooperative binding to LAT. Negative regulation occurs via phosphorylation or caspase-mediated cleavage of the linker region of Gads. FcεRI-mediated mast cell activation is profoundly impaired in LAT- Gads- or SLP-76-deficient mice. Unexpectedly, the thymic developmental phenotype of Gads-deficient mice is much milder than the phenotype of LAT- or SLP-76-deficient mice. This distinction suggests that Gads is not absolutely required for TCR signaling, but may modulate its sensitivity, or regulate a particular branch of the TCR signaling pathway; indeed, the phenotypic similarity of Gads- and Itk-deficient mice suggests a functional connection between Gads and Itk. Additional Gads binding partners include costimulatory proteins such as CD28 and CD6, adaptors such as Shc, ubiquitin regulatory proteins such as USP8 and AMSH, and kinases such as HPK1 and BCR-ABL, but the functional implications of these interactions are not yet fully understood. No interacting proteins or function have been ascribed to the evolutionarily conserved N-terminal SH3 of Gads. Here we explore the biochemical and functional properties of Gads, and its role in regulating allergy, T cell development and T-cell mediated immunity.

Keywords: FcεRI; Gads; LAT; SLP-76; TCR; signal transduction; thymocyte development.

Figure 1

The Grb2 family. (A) Domain structure of the Grb2 family members: Grb2, Gads, and Grap. (B) Through its SH2 and C-terminal SH3, Gads bridges the antigen receptor-induced recruitment of SLP-76 to LAT (shown at top). Additional binding partners are listed below their relevant binding domain, with interactions of unknown functional relevance listed below the dotted line. The N-terminal SH3 has no known binding partner or regulatory function. Bait proteins that were used to identify Gads are shown in blue. 14-3-3 binds to a threonine phosphorylation site within the linker region. CD28 and BCR-Abl may interact with two Gads domains.

Figure 2

The LAT-nucleated signaling complex. Upon TCR stimulation, LAT is phosphorylated by ZAP-70 at Tyr¹³², Tyr¹⁷¹, Tyr¹⁹¹, and Tyr²²⁶ (indicated by gray arrows), triggering the SH2-mediated recruitment of key signaling proteins. PLC-γ1 binds directly to LAT at pTyr¹³². Grb2 bridges the recruitment of SOS to LAT. Gads binds cooperatively to pTyr¹⁷¹ and pTyr¹⁹¹, bridging the recruitment of SLP-76 and its associated binding partners to LAT. Nck, Vav, and Itk bind to N-terminal tyrosine phosphorylation sites on SLP-76 (Tyr¹¹³, Tyr¹²⁸, and Tyr¹⁴⁵), whereas HPK1 and ADAP (not shown) can bind to the C-terminal SH2 of SLP-76. Binding to SLP-76 is required for the catalytic activity of two SLP-76-associated kinases, Itk, and HPK1. Substrates of Itk within the signalosome are indicated by light pink arrows; Itk phosphorylates SLP-76 at Tyr¹⁷³, which is required for subsequent Itk-mediated phosphorylation of PLCγ1 at Tyr⁷⁸³. HPK1 phosphorylates SLP-76 and Gads at negatively regulatory sites, indicated by purple arrows (SLP-76 Ser³⁷⁶ and Gads Thr²⁶²).

Figure 3

Gads regulatory sites include an SH2 dimerization interface. (A) The SH2 domain of murine Gads, cocrystalized with a short LAT peptide encompassing LAT pTyr¹⁷¹ [(6); PDB file 1R1P]. The minimal asymmetric unit includes two pairs of closely associated Gads SH2 domains, each bound to a pLAT peptide. Shown is the structure of one pair of SH2 domains (cyan and green chains), each bound to the LAT peptide (red). Three amino acid side chains from the putative dimerization interface are shown, including F92 (orange), R109 (pink), and D91 (blue). (B) A space-filling representation of the structure shown in A, revealing an extensive dimerization interface measuring ~850 Ų. The dotted pink line illustrates the possibility that the two bound pTyr peptides could represent dual binding sites on a single molecule of LAT, binding cooperatively to two molecules of Gads. (C) Summary of the currently known regulatory mechanisms converging on Gads, including Gads dimerization, HPK1-mediated phosphorylation of Gads, and caspase3-mediated cleavage of Gads. Tyr⁴⁵ is a conserved TCR-inducible Gads phosphorylation site of unknown function, found within the N-terminal SH3 of Gads.

Figure 4

TCR-induced clustering of LAT. A web of multipoint, SH2-mediated interactions promotes the formation of microclusters, containing LAT, Gads, and SLP-76. ADAP-mediated oligomerization of SLP-76: Upon TCR stimulation ADAP is phosphorylated at three tyrosines that bind to the SH2 of SLP-76, leading to oligomerization of SLP-76 and its associated Gads. Cooperative binding of Gads to LAT: Gads SH2 dimerization promotes its cooperative binding to two adjacent sites on LAT, thereby recruiting ADAP-oligomerized SLP-76 to one or more LAT molecules. Grb2-SOS-mediated clustering of LAT: Each molecule of SOS can bind to two molecules of Grb2, each of which may bind to a different molecule of LAT, thereby bringing them into the growing cluster. Each of the above interactions can occur repeatedly, creating a web of interacting proteins that together stabilize a large signaling cluster.

Figure 5

An overview of thymic development. (A) The three phases of thymocyte development, known as double negative (DN, CD4⁻CD8⁻) double positive (DP, CD4⁺CD8⁺) and single positive (SP, CD4⁺, or CD8⁺). (B) A higher resolution depiction, based on additional cell surface markers. Transitions regulated by Gads are indicated by dashed arrows. The DN phase can be subdivided into DN1 (CD44^hiCD25⁻), DN2 (CD44^hiCD25⁺), DN3 (CD44^loCD25⁺), and DN4 (CD44^loCD25⁻). TCR rearrangement begins during DN3. Successful rearrangement of TCRβ results in expression of the pre-TCR, composed of TCRβ, pre-Tα and CD3. At the β-selection checkpoint, pre-TCR signaling triggers cellular proliferation and transition through DN4 and on to the DP stage. DP cells rearrange TCRα, and the resulting mature αβ TCR may recognize self-pMHC ligands. High affinity recognition of pMHC triggers cell death through negative selection. Moderate affinity recognition triggers positive selection, accompanied by increased expression of CD5, CD69, and the TCR. Positively selected cells pass through a CD4 transitional single positive stage (TSP), characterized by high expression of CD24, and then transition into the mature SP compartment (CD4⁺ or CD8⁺ and TCR^hiCD24^loCD62L^hi). (C) Schematic summary of the markers of positive selection.