Cooperative assembly of a four-molecule signaling complex formed upon T cell antigen receptor activation

Abstract

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.phospholipase C | protein complex | signal transduction | T cell antigen receptor | tyrosine phosphorylation T hree decades of investigation have resulted in a description of T cell antigen receptor (TCR) engagement and the biochemical events that follow. The binding of the TCR ligand, an antigenic peptide presented by a protein encoded by the major histocompatibility complex, leads to the activation of associated and recruited protein tyrosine kinases (PTKs) (1). Critical substrates of these PTKs include the adapter molecules, linker for activation of T cells (LAT), and Src homology 2 (SH2) domaincontaining leukocyte protein of 76kDa (SLP-76) (2, 3). The phosphorylation of tyrosine residues on these adapters initiates binding of many other adapters and enzymes to generate molecular complexes of great heterogeneity (4). In this manner enzymes such as phospholipase C-γ1 (PLC-γ1) and son of sevenless homolog 1 (SOS1) are recruited to the plasma membrane where they encounter their substrates. The products that are a consequence of these enzyme activations-diacylglycerol, elevated intracellular calcium, and activated Ras-have complex intracellular effects during the process of T cell activation (5, 6).Sites of protein-protein interactions between these v...

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

“…Assembly of the LAT signalosome appears to be driven by multiple cooperative binding events, including, but not limited to Gads dimerization. A recent study highlighted the cooperative assembly of LAT, Gads, SLP-76, and PLC-γ1 into a tetrameric complex centered around LAT Tyr 132 and Tyr 171 (46). Upon in vitro reconstitution of this binding complex, elimination of any one of the above components reduced the binding interactions between the other three.…”

“…One possible compensatory mechanism is the Gads-independent recruitment of SLP-76 to LAT by other Grb2-family members, Grb2 or Grap. Both Gads and Grb2 can bind via their C-terminal SH3 domains to a non-canonical RxxK motif found in SLP-76; however, Gads binds this motif with high affinity, measured at 9-20 nM (8, 10, 46), whereas Grb2 binds with ~500-fold lower affinity (10, 14, 15). Due to its low affinity, Grb2 cannot interact with SLP-76 in the presence of competing Gads (18); however, the absence of Gads may permit weak, Grb2-mediated recruitment of SLP-76 to LAT.…”

“…Any such compensatory recruitment may be cooperatively stabilized by additional interactions occurring within the LAT-nucleated signalosome. For example, binding of the SH3 domain of LAT-bound PLC-γ1 to a proline-rich motif in SLP-76 can substantially stabilize the recruitment of SLP-76 to LAT (46, 98). While consistent with previously reported protein-protein interactions, neither Grb2- nor Grap-mediated recruitment of SLP-76 to LAT has been directly demonstrated.…”

PLCγ1 promotes phase separation of the T cell signaling clusters

“…PLC1 is a multi-domain lipase. Besides its catalytic core, PLC1 also contains two SH2 domains and one SH3 domain that serve structural or regulatory roles (Braiman et al, 2006;Manna et al, 2018). Its N-terminal SH2 domain (nSH2) binds specifically to phosphor-tyrosine (Y132) on LAT (Braiman et al, 2006) whereas its C-terminal SH2 domain (cSH2) is involved in releasing autoinhibition of PLC1 (Gresset et al, 2010;Hajicek et al, 2019).…”

“…The SH3 domain of PLC1 directly interacts with the proline-rich motifs on Sos1 (Kim et al, 2000), a RasGEF which is also enriched in the LAT microclusters. The multiple binary interactions between PLC1 and other components in the LAT complex mediate a synergistic assembly of the LAT complex (Braiman et al, 2006;Hartgroves et al, 2003;Manna et al, 2018). However, because of the complex protein-protein interactions involved, the exact mechanism of how PLC1 regulates LAT microcluster formation remains elusive.…”