# Proteomics Mapping of CD28 Costimulation

> **NIH NIH R21** · UNIVERSITY OF ROCHESTER · 2020 · $231,000

## Abstract

Project Summary / Abstract
In spite of clear functional importance of CD28 in T cell activation, we still do not have a complete
understanding of how CD28 transduces proximal signals. CD28 costimulation has been associated with a
variety of downstream signaling pathways, but most of these overlap with TCR pathways initiated by TCR and
CD28 is often thought of as an amplifier of TCR signaling. Thus, it has been difficult to assign a specific
function to CD28. Most of the focus has been on the short cytosolic tail domain (CTD) of CD28 that contains
two well established protein binding motifs. However, mutation of these motifs in the endogenous CD28 locus
have only partial effects on T cell activation, even when both sites are mutated together. These data indicate
that additional signaling motifs/pathways are required to account for the full effect of CD28 costimulation.
Therefore, we are proposing an unbiased proteomic approach to identify any proteins that are selectively
colocalized with CD28 during T cell costimulation. This approach does not depend on direct or stable
interaction with CD28 and has the potential to identify novel proteins that have not previously been implicated
in CD28 costimulation. We will achieve this goal through two Specific Aims:
1. Identify the proteome colocalized with CD28 during T cell costimulation. A major contribution to the
magnitude, specificity, and regulation of cell signaling is mediated through colocalization of receptors with
proximal and downstream signaling components. Proximity labeling is a relatively new technique that has been
developed to identify critical colocalization events that are not detectable by conventional biochemical
approaches. The use of APEX2 affords both a spatial and temporal resolution that allows for the identification
of proteins that are colocalized with the target protein, without requiring stable physical association. In this Aim
we will apply this technology in combination with SILAC (Stable Isotope Labeling by Amino acids in Cell
culture), LS-MS/MS to quantify and sequence peptides, and established search programs and protein
databases to identify proteins that are colocalized with CD28 during T cell activation and costimulation.
2. Determine the role of colocalized proteins in CD28 costimulation. In this Aim we will determine the
relative contribution of candidate proteins that we have found to be colocalized with CD28 during costimulation
through a combination of biochemical, molecular, cellular and immunological assays.
1

## Key facts

- **NIH application ID:** 9937643
- **Project number:** 5R21AI147503-02
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **Principal Investigator:** JIM F Miller
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $231,000
- **Award type:** 5
- **Project period:** 2019-06-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9937643

## Citation

> US National Institutes of Health, RePORTER application 9937643, Proteomics Mapping of CD28 Costimulation (5R21AI147503-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9937643. Licensed CC0.

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