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

摘要

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.

摘要

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.

引言

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....

方案

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....

结果

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,.......

讨论

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 .......

材料

本文使用的材料清单

姓名	公司	目录编号	评论
PBS	Lonza	17-516F	磷酸盐缓冲盐水
微量离心机	Eppendorf	5424-R	1.5 mL
胰蛋白酶-EDTA （0.25%） 酚红	Gibco	25200114
热灭活 FBS	Denville	FB5001-H	胎牛血清
青霉素/链霉素	Fisher	BP295950
不含 L-谷氨酰胺的 DMEM 高葡萄糖	Lonza	BE12-614F
SuperFect 转染试剂	Qiagen	301305
抗 SHP2	抗体 Santa Cruz	SC-280
抗–GFP-琼脂糖	MBL	D153-8
抗GFP	Roche	118144600
抗肌动蛋白	Santa Cruz	SC-1616
HEK-293	ATCC	CRL-1573
原钒酸盐	Sigma	S6508
H2O2	Sigma	216763	30%
蛋白酶抑制剂混合物	罗氏	11836170001	不含 EDTA
Tris-甘氨酸 SDS 样品缓冲液 （2x）	Invitrogen	LC2676	改良 Laemmli 缓冲液
4-20% Tris-甘氨酸小尺寸凝胶	Invitrogen	XP04205BOX	15 孔
硝酸纤维素膜	通用电气	10600004
NaCl	Sigma	S7653	钠氯化物
HEPES	Gibco	15630080	N-2-羟乙基哌嗪-N-2-乙烷磺酸
DTT	Invitrogen	D1532	二硫苏糖醇
pNPP	Sigma	20-106	对硝基苯磷酸
NaOH	Sigma	S8045	氢氧化钠
BCA	Fisher	23225	双金鸡宁酸测定