RIAM Interacts with the Hematopoietic-Specific Adaptor Protein Gads and Forms a LAT-Independent Node of Signal Integration That Regulates Activation of PLC-γ1
Bibliographic record
Abstract
Abstract TCR stimulation triggers the activation of protein tyrosine kinases resulting in phosphorylation of the adaptor protein LAT. SLP-76, interacts constitutively with PLC-γ1 and with the SH3 domain of Gads, which via its SH2 domain mediates inducible recruitment of SLP-76 and PLC-γ1 to LAT, upon T cell activation. PLC-γ1 hydrolyzes phosphatidylinositol-4, 5 bisphosphate [PI(4,5)P2], generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), second messengers responsible for mediating intracellular calcium release and activation of downstream signals. The adaptor protein RIAM constitutively interacts with PLC-γ1 and is required for PLC-γ1 activation. RIAM is a multidomain protein with a small N-terminus proline-rich region, two coiled-coiled regions, sequential Ras association (RA) and pleckstrin homology (PH) domains, and a large C-terminus proline-rich region, which interacts with PLC-γ1. The RA domain of RIAM has specificity for Rap1-GTP whereas the PH domain binds to the PLC-γ1 substrate PI(4,5)P2. The RA-PH domain region of RIAM functions as a single structural unit and mediates translocation of RIAM to the plasma membrane upon T cell activation. Previously, we determined that RIAM deficiency results in impaired activation of PLC-γ1 in spite of the formation of the PLC-γ1-SLP-76-LAT complex, suggesting perhaps somewhat paradoxically, that PLC-γ1-SLP-76-LAT signalosome is not sufficient to mediate distal signaling in the absence of RIAM. This observation indicated that RIAM mediates its effects at a level distal to SLP-76-LAT or through a signaling pathway parallel but distinct from SLP-76-Gads-LAT. Here we investigated whether RIAM forms a signalosome parallel to PLC-γ1-SLP-76-Gads and whether such pathway might be involved in the activation of PLC-γ1. Using primary T lymphocytes and Jurkat T cells stimulated via TCR/CD3 and CD28 we determined that RIAM constitutively interacted with Gads as determined by immunoprecipitation with RIAM-specific antibody followed by Gads immunoblot. To determine whether the interaction between RIAM and Gads was direct, we employed an in vitro protein association assay. Glutathione S-transferase (GST) and GST-fusion protein of Gads were coupled to glutathione-sepharose and incubated with [35S]methionine-labeled RIAM or luciferase, as negative control. Gads bound to [35S]methionine-labeled RIAM indicating that RIAM interacts directly with Gads. We further examined domain-specific interaction of RIAM with endogenous Gads using GST fusion proteins of RIAM. We determined a constitutive interaction between Gads and GST fusion proteins of full-length RIAM or C-terminus region of RIAM. Although a number of tyrosine phosphorylated proteins were associated with the RIAM-Gads complex upon T cell activation, LAT was not detected among the components of this complex as determined by immunoblot with anti-phosphotyrosine-specific or LAT-specific antibodies. Using a GST fusion protein of the RA-PH domain of RIAM we determined that, surprisingly, Gads displayed activation-dependent interaction with the RA-PH domain, which mediates the recruitment of RIAM to the plasma membrane upon T cell activation. Furthermore, in addition to Gads, SLP-76 and PLC-γ1 were recruited to the RA-PH domain of RIAM in activated T cells. To determine whether RIAM and Gads had a synergistic effect on IL-2 transcription, we performed luciferase-based reporter assays using a reporter construct driven by the entire IL-2 promoter or by NFAT binding sequences. We found that RIAM and Gads had a synergistic effect on IL-2 and on NFAT-mediated transcriptional activation, which depends on PLC-γ1. Thus, via its C-terminus region, RIAM directly and constitutively interacts with Gads. In addition, via its RA-PH domain, RIAM mediates an activation-dependent interaction with Gads and serves as a docking site recruiting the PLC-γ1-SLP-76-Gads complex to the plasma membrane in a LAT-independent manner. These findings indicate a crosstalk between RIAM and SLP-76 in the activation of PLC-γ1 and reveal a previously unidentified, alternative signaling pathway leading to Gads-SLP-76 recruitment to the plasma membrane of activated T cells in a LAT-independent manner. Disclosures No relevant conflicts of interest to declare.
Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.
How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".