trans-Interacting Plasma Membrane Proteins and Binding Partner Identification

Abstract

Plasma membrane proteins (PMPs) play critical roles in a myriad of physiological and disease conditions. A unique subset of PMPs functions through interacting with each other in trans at the interface between two contacting cells. These trans-interacting PMPs (tiPMPs) include adhesion molecules and ligands/receptors that facilitate cell-cell contact and direct communication between cells. Among the tiPMPs, a significant number have apparent extracellular binding domains but remain orphans with no known binding partners. Identification of their potential binding partners is therefore important for the understanding of processes such as organismal development and immune cell activation. While a number of methods have been developed for the identification of protein binding partners in general, very few are applicable to tiPMPs, which interact in a two-dimensional fashion with low intrinsic binding affinities. In this review, we present the significance of tiPMP interactions, the challenges of identifying binding partners for tiPMPs, and the landscape of method development. We describe current avidity-based screening approaches for identifying novel tiPMP binding partners and discuss their advantages and limitations. We conclude by highlighting the importance of developing novel methods of identifying new tiPMP interactions for deciphering the complex protein interactome and developing targeted therapeutics for diseases.

Keywords: ELISA; Plasma membrane proteins; avidity-enhanced screening; protein interactions; protein interactome; pull-down.

Figure 1.. tiPMP interactions at the interface between an APC and a T cell.

Initiation of T cell activation requires alignment of membrane by sequential interactions between adhesion molecules, starting with LFA-1-ICAM-1 and α4 integrin-VCAM interactions with large extracellular domains followed by CD2-CD58 with smaller extracellular domains. The close membrane alignment excludes transmembrane proteins with very large extracellular domains such as CD45 and enables interactions between tiPMPs with small extracellular domains including pMHC-TCR that is key to T cell antigen recognition. T cell activation is modulated by interactions between auxiliary tiPMPs including co-stimulatory CD28-B7-1 and co-inhibitory PD1-PDL1.

Figure 2.. Approaches based on avidity-enhanced screening of protein libraries for identifying low-affinity tiPMP interactions.

A. Avidity-based extracellular interaction screen (AVEXIS). Library of prey proteins anchored on streptavidin-coated plate are screened against pentameric β-lactamase-tagged bait proteins. B. Extracellular Interactome Assay (ECIA). Library of bait proteins anchored on protein A-coated plate are screened against pentameric AP-tagged prey proteins. C. Avidity-enhanced screening using IgG Fc-based multimeric preys. AP-fused bait proteins anchored on anti-AP-coated plates are used to screen Fc-tagged prey protein library. Signal outputs are detected using HPR-conjugated anti-Fc antibodies. D. Avidity-enhanced screening using bait proteins on microbeads. Bait ECD-Fc fusion proteins anchored onto protein A microbeads are used to screen a prey library in the form of a protein microarray. Fc bait proteins are labeled with Cy5 fluorescent dye.