The CD3 ε subunit of the TCR complex contains two defined signaling domains, a proline-rich sequence and an ITAM. We identified a third signaling sequence in CD3 ε, termed the basic-rich stretch (BRS). Herein, we show that the positively charged residues of the BRS enable this region of CD3 ε to complex a subset of acidic phospholipids, including PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and PI(4,5)P2. Transgenic mice containing mutations of the BRS exhibited varying developmental defects, ranging from reduced thymic cellularity to a complete block in T cell development. Peripheral T cells from BRS-modified mice also exhibited several defects, including decreased TCR surface expression, reduced TCR-mediated signaling responses to agonist peptide-loaded APCs, and delayed CD3 ε localization to the immunological synapse. Overall, these findings demonstrate a functional role for the CD3 ε lipid-binding domain in T cell biology.

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. While all the CD3 subunits possess at least one ITAM, CD3 ε subunit also contains a proline-rich sequence (PRS) and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. Herein, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P2, and PtdIns(3,4,5)P3 with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to stably accumulate at the immunological synapse during T cell-antigen presenting cell interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures following T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.

The activation of protein kinases is one of the primary mechanisms whereby T cell receptors (TCR) propagate intracellular signals. To date, the majority of kinases known to be involved in the early stages of TCR signaling are protein-tyrosine kinases such as Lck, Fyn, and ZAP-70. Here we report a constitutive association between the TCR and a serine/threonine kinase, which was mediated through the membrane-proximal portion of CD3 ⑀. Mass spectrometry analysis of CD3 ⑀-associated proteins identified G protein-coupled receptor kinase 2 (GRK2) as a candidate Ser/Thr kinase. Transient transfection assays and Western blot analysis verified the ability of GRK2 to interact with the cytoplasmic domain of CD3 ⑀ within a cell. These findings are consistent with recent reports demonstrating the ability of certain G protein-coupled receptors (GPCR) and G proteins to physically associate with the ␣/␤ TCR. Because GRK2 is primarily involved in arresting GPCR signals, its interaction with CD3 ⑀ may provide a novel means whereby the TCR can negatively regulate signals generated through GPCRs. The T cell receptor (TCR)3 is a multimeric complex composed of the polymorphic ␣ and ␤ subunits and the CD3 ␥, ␦, ⑀, and invariant chains (1). Extracellular interactions between the ␣/␤ subunits of the TCR and peptide/major histocompatibility complexes initiate an intricate cascade of intracellular signals, which regulate T cell proliferation, effector function, and/or programmed cell death. Signaling events triggered through TCR interactions are controlled by a conserved amino acid motif, termed the immunoreceptor tyrosine-based activation motif (ITAM), which is found in the cytoplasmic domains of each of the CD3 invariant chains (reviewed in Refs. 2 and 3). Following TCR engagement, members of the Src family of protein-tyrosine kinases (PTK) phosphorylate tyrosine residues in the invariant chain ITAMs (4, 5). Bi-phosphorylated ITAMs subsequently associate with the Syk family PTK, -associated protein of 70 kDa (ZAP-70), via its tandem Src homology 2 domains (6). Once associated, ZAP-70 is tyrosine-phosphorylated and catalytically activated. It then phosphorylates and/or interacts with additional signaling molecules, such as Vav, LAT, and SLP-76 (reviewed in Ref. 7). These initial tyrosine phosphorylation pathways are subsequently channeled into the activation of multiple serine/threonine (Ser/Thr) kinases, including MAPKs, IB kinases, and PKC family members, which ultimately regulate transcription factor activity. In addition to influencing T cell effector function or proliferation, the association of certain PTKs with the TCR invariant chains has been demonstrated to induce cross-talk between the TCR and certain chemokine receptors, thus enhancing T cell migration (8, 9).Tyrosine phosphorylation of the invariant chain ITAMs is one of the earliest detectable signaling events to occur upon TCR engagement (10). However, recent reports have identified a ligand-induced conformational change within the cytoplasmic tail of CD3 ⑀ that precedes I...

The TCR complex, when isolated from thymocytes and peripheral T cells, contains a constitutively tyrosine-phosphorylated CD3ζ molecule termed p21. Previous investigations have shown that the constitutive phosphorylation of CD3ζ results from TCR interactions with MHC molecules occurring in both the thymus and the periphery. To determine what contribution the selection environment had on this constitutive phosphorylation, we analyzed CD3ζ from several distinct class I- and II-restricted TCR-transgenic mice where thymocyte development occurred in either a selecting or a nonselecting MHC environment. Herein, we report that constitutively phosphorylated CD3ζ (p21) was present in thymocytes that developed under nonselecting peptide-MHC conditions. These findings strongly support the model that the TCR has an inherent avidity for MHC molecules before repertoire selection. Biochemical analyses of the TCR complex before and after TCR stimulation suggested that the constitutively phosphorylated CD3ζ subunit did not contribute to de novo TCR signals. These findings may have important implications for T cell functions during self-MHC recognition under normal and autoimmune circumstances.

Positron emission tomography (PET) is a molecular imaging modality that provides the opportunity to rapidly and non-invasively visualize tumors derived from multiple organs. In order to do so, PET utilizes radiotracers, such as 18F-FDG and 11C-acetate, whose uptake coincides with altered metabolic pathways within tumors. Increased expression and activity of enzymes in the fatty acid synthesis pathway is a frequent hallmark of cancer cells. As a result, this pathway has become a prime target for therapeutic intervention. Although multiple drugs have been developed that both directly and indirectly interfere with fatty acid synthesis, an optimal means to assess their efficacy is lacking. Given that 11C-acetate is directly linked to the fatty acid synthesis pathway, this probe provides a unique opportunity to monitor lipogenic tumors by PET. Herein, we review the relevance of the fatty acid synthesis pathway in cancer. Furthermore, we address the potential utility of 11C-acetate PET in imaging tumors, especially those that are not FDG-avid. Last, we discuss several therapeutic interventions that could benefit from 11C-acetate PET to monitor therapeutic response in patients with certain types of cancers.