Quantification of THEMIS2 and GRB2 Binding Open Access

Abstract

The THEMIS family of proteins plays an important role in modulating antigen receptor signaling thresholds during lymphocyte development (Choi et al., 2017b). Both isoforms of THEMIS carry conserved CABIT domains, each containing a proline-rich sequence (PRS) that interacts with the GRB2 adaptor protein (Lesourne et al., 2012). THEMIS1 has been thoroughly characterized and mechanistically linked to influence thymocyte positive selection via its interaction with GRB2. However, the THEMIS2-GRB2 interaction remains less well-defined, and its binding affinity is unknown. In this study, we address this by quantitatively characterizing the interaction between the CABIT2 domain of THEMIS2 with GRB2 using fluorescence polarization assays.

Using AlphaFold models, we identified suitable THEMIS2 CABIT2 constructs that preserve the PRS. The recombinant THEMIS2 CABIT2 +PRS construct was expressed and purified. In the fluorescence polarization assays, the THEMIS2 CABIT2 +PRS protein was titrated against fluorescently labeled GRB2. Binding curves of the interaction reveal a sigmoidal increase in polarization, demonstrating the formation of the THEMIS2-GRB2 complex. Curve-fitting analyses yielded a Kd of 17.34 µM (n=1), which is comparable to the THEMIS1-GRB2 value (Kd = 18.8 ± 4.8 µM; n=3). Our results provide one of the first quantitative measurements of the THEMIS2-GRB2 interaction in vitro, further supporting a conserved adaptor-mediated mechanism across the THEMIS family.

Table of Contents

Table of Contents

Introduction 1

Immune Signaling and Lymphocyte Selection 1

The THEMIS Family of Proteins 1

Role of THEMIS1 in T Cell Development 3

Role of THEMIS2 in Immune Cell Signaling 5

Knowledge Gaps in THEMIS2 6

Methods 7

Construct Design 7

PCR and DNA Gel Extraction 8

T4 Insert Processing, Bacterial Transformation, and Cloning 9

Colony Screening, Plasmid Miniprep, and Sequence Verification 10

Protein Expression 11

Protein Purification 12

Fluorescence Polarization Assay 14

Results 15

Design of THEMIS2 CABIT2 Constructs 15

PCR 17

T4 Processing, Cloning, and Colony PCR 18

Expression and Purification of Constructs 20

Fluorescence Polarization Assays 23

Discussion 26

References 30

About this Honors Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, Molecular
Keyword	THEMIS1 GRB2 Fluorescence Polarization THEMIS2
Committee Chair / Thesis Advisor	Tyler Beyett, Emory University
Committee Members	Michal Arbilly, Emory University Xiulei Mo, Emory University

Last modified

Primary PDF

Thumbnail	Title	Date Uploaded	Actions
	Quantification of THEMIS2 and GRB2 Binding ()	2026-04-07 18:45:42 -0400	Press to Select an action Download

Supplemental Files

Thumbnail	Title	Date Uploaded	Actions