GRID: A Novel Grb-2-Related Adapter Protein That Interacts with the Activated T Cell Costimulatory Receptor CD28

Abstract

Adapter proteins such as Grb2 play a central role in the formation of signaling complexes through their association with multiple protein binding partners. These interactions are mediated by specialized domains such as the well-characterized Src homology SH2 and SH3 motifs. Using yeast three-hybrid technology, we have identified a novel adapter protein, expressed predominantly in T lymphocytes, that associates with the activated form of the costimulatory receptor, CD28. The protein is a member of the Grb2 family of adapter proteins and contains an SH3-SH2-SH3 domain structure. A unique glutamine/proline-rich domain (insert domain) of unknown function is situated between the SH2 and N-terminal SH3 domains. We term this protein GRID for Grb2-related protein with insert domain. GRID coimmunoprecipitates with CD28 from Jurkat cell lysates following activation of CD28. Using mutants of CD28 and GRID, we demonstrate that interaction between the proteins is dependent on phosphorylation of CD28 at tyrosine 173 and integrity of the GRID SH2 domain, although there are also subsidiary stabilizing contacts between the PXXP motifs of CD28 and the GRID C-terminal SH3 domain. In addition to CD28, GRID interacts with a number of other T cell signaling proteins, including SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa), p62dok, and RACK-1 (receptor for activated protein kinase C-1). These findings suggest that GRID functions as an adapter protein in the CD28-mediated costimulatory pathway in T cells.

The role of Gads in hematopoietic cell signalling

“…In this case, the binding of Gads to LAT appears to be more selective, suggesting that there may be subtle di erences in their binding speci®city. Gads has also been reported to associate with other proteins containing a pY-X-N-X motif, including Bcr ± Abl, CD28, SHP-2, and c-kit (Ellis et al, 2000;Law et al, 1999;Liu and McGlade, 1998). However, the physiological signi®cance of these interactions has not been determined.…”

Interaction of the Wiskott–Aldrich syndrome protein with sorting nexin 9 is required for CD28 endocytosis and cosignaling in T cells

“…CD28 costimulatory signals, triggered by binding to B7 ligand on antigen-presenting cells, are key to the activation of resting naïve T cells, enabling sustained activation associated with augmented production of IL-2 and other cytokines and increases in cell proliferation and survival (21). The CD28 cytosolic domain contains a YMNM motif that, when phosphorylated on tyrosine, promotes SH2 domain-mediated interactions with the Grb family adaptors Grb2, GRID, and Gads (22)(23)(24)(25) and with phosphatidylinositol (PtdIns) 3-kinase (PI3K), an interaction needed for CD28 internalization (26). Despite these findings and CD28 importance to T cell activation, the signaling pathways linking CD28 engagement to its costimulatory effects remain unclear.…”

Controlling NF-κB activation in T cells by costimulatory receptors

“…In the cytoplasmic domain of CD28, the same YXXM motif is used to recruit the SH2 domain containing adaptor protein growth-factor receptor-bound protein 2 (Grb2) and homologous proteins such as Gads (Grb2-related adaptor protein) and GRID (Grb2-related protein with insert domain). 42 The cytoplasmic domain of CD28 also allows binding of the Tec family tyrosine kinases, Itk, which employs its SH3 domain to interact with one or two PXXP sequences. The more C-terminal PYAP sequence is bound by the tyrosine kinase Lck, which phosphorylates the cytoplasmic tail and thus prevents binding of the negatively regulating serine/ threonine phosphatase PPA2.…”