Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Summary

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Abstract

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Introduction

Catalytic receptors and receptor tyrosine kinases are transmembrane proteins in which binding of an extracellular ligand causes enzymatic activity on the intracellular side¹. Some receptors possess both receptor and enzymatic functions, while others recruit specific enzymes such as kinases and phosphatases to their cytoplasmic tails. Recruitment of an enzyme to the receptor's tail and the subsequent catalytic action of this enzyme are two separate processes that are not always regulated by the same protein domains². Unfortunately, there are no specific tools to assess both the interaction and enzymatic activity simul....

Protocol

1. Transfection of Cells

1. Seed HEK 293T cells into twelve 10 cm plates (5 x 10⁶ cells per plate) the day before transfection (Figure 1). Perform the cell counting using a hemocytometer. For each plate, use 10 mL of DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Incubate the cells at 37 °C in 5% CO₂.
2. Once the cells are 80-90% confluent, transfect 5 HEK 293T plates using a lipid-based transfection reagent with one of the following plasmids: a SHP2-expressing vector, a PD-1-GFP-expressing vector [a wild type (WT) version of PD-1], an ITIM-mutated version (Y223F) of a PD-1-GFP-ex....

Results

While the contribution of the ITSM of PD-1 to SHP2 binding is established, the role of the ITIM of PD-1 is less clear. Because SHP2 has two SH2 domains that can bind to two sequential phosphotyrosines on PD-1 (one tyrosine in the ITSM and another in the ITIM), we hypothesized that the ITSM of PD-1 anchors SHP2 to PD-1, while the ITIM of PD-1 facilitates SHP2 activity by stabilizing its open conformational state^11,14. To test this,.......

Discussion

Receptor-enzyme interactions are crucial for intracellular signaling. Many enzymes are recruited to receptors through SH2 domains binding to phosphorylated tyrosines that decorate tails of the same receptors. However, enzymes are often folded into closed inactive conformations, and activation requires a conformational change¹¹ that can be mediated by other domains of the same receptor. The assay described here measures the interactions between receptors and enzymes as well as the activity induced .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PBS	Lonza	17-516F	Phosphate Buffered Saline
Microcentrifuge	Eppendorf	5424-R	1.5 mL
Trypsin-EDTA (0.25%) Phenol Red	Gibco	25200114
Heat Inactivated FBS	Denville	FB5001-H	Fetal bovine serum
Penicillin / Streptomycin	Fisher	BP295950
DMEM high glucose without L-glutamine	Lonza	BE12-614F
SuperFect Transfection Reagent	Qiagen	301305
Anti-SHP2	Santa Cruz	SC-280
Anti–GFP-agarose	MBL	D153-8
Anti-GFP	Roche	118144600
Anti-Actin	Santa Cruz	SC-1616
HEK-293 cells	ATCC	CRL-1573
Orthovanadate	Sigma	S6508
H2O2	Sigma	216763	30%
Protease inhibitor cocktail	Roche	11836170001	EDTA-free
Tris-Glycine SDS Sample Buffer (2x)	Invitrogen	LC2676	Modified Laemmli buffer
4-20% Tris-Glycine Mini Gels	Invitrogen	XP04205BOX	15-well
Nitrocellulose membranes	General Electric	10600004
NaCl	Sigma	S7653	Sodium chloride
HEPES	Gibco	15630080	N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid
DTT	Invitrogen	D1532	Dithiothreitol
pNPP	Sigma	20-106	p-Nitrophenyl Phosphate
NaOH	Sigma	S8045	Sodium hydroxide
BCA	Fisher	23225	Bi Cinchoninic Acid assay