The Role of the Cell Background in Biased Signaling

Guillermo G. Romero,    Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

Cellular signaling events are initiated by the interaction of agonists with specific cellular receptors. However, agonists do not uniformly activate all cellular signaling events in all cellular systems. This phenomenon has been termed ligand or agonist bias. Classical models to explain and measure ligand bias in cell surface receptors assume this phenomenon is the consequence of agonist-selective stabilization of specific conformational states of the receptor. However, signaling phenomena are critically influenced by the intracellular context and the tissue microenvironment. Cell surface receptors are often associated with other receptors and multiple regulatory proteins, which leads to crosstalk between different pathways and signaling switches from one signaling pathway to another. Furthermore, cells exist in close proximity to other cells, and the crosstalk between different cell types critically influences the outcome of specific signaling processes. This chapter presents an overview of the mechanisms by which the intra- and extra-cellular environments modulate responses to selected stimuli. The review focuses on two receptor superfamilies: the G protein coupled receptors and the receptor tyrosine kinases.

Keywords

cellular background; signaling bias; accessory proteins; receptor dimerization; signaling crosstalk

Outline

Biased Signaling

The concept of biased signaling commonly refers to the selectivity of receptor agonists to differentially activate alternative signaling pathways. This phenomenon has been proposed to result from agonist-selective stabilization of alternative active receptor conformations.1,2 This conformational model is an extension of the classical ligand-receptor-transducer-effector model (Figure 2.1). It implies that the activation of selective signaling pathways by different agonists is an intrinsic property of specific receptor–ligand interactions. This operational model has some limitations. Cell surface receptors do not exist in isolation, as they engage multiple cell surface and intracellular interactive partners that modify their function. Thus, the receptor interactome significantly influences signaling outcomes. Furthermore, a “conformational” model applies primarily to multiple conformations that may exist in equilibrium or under steady state conditions. However, receptors participate in multiple processes that are irreversible from a thermodynamic point of view, such as phosphorylation and compartmentalization (i.e., endocytosis). Thus, the classical ligand-receptor-transducer-effector model is a useful first approximation but its application to real biological systems must be subject to experimental verification.

An example to illustrate the relevance of the cellular physiological background in biased signaling can be found in the parathyroid hormone receptor system. The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor (GPCR) that interacts with parathyroid hormone (PTH) and a selected set of PTH analogs. When activated, the PTH1R promotes G-protein-dependent signaling (cAMP production, calcium mobilization) and arrestin-dependent signaling (activation of ERK kinases). The PTH1R ligand D-Trp(12),Tyr(34)-bPTH(7-34) [PTH(7-34)] does not activate cAMP production or calcium release from intracellular stores. However, this ligand induces endocytic traffic of the PTH1R by an arrestin-independent mechanism in kidney distal convoluted tubule (DCT) and ROS 17/2.8 cells but does not in proximal tubule cells.3 The origin of these distinct behaviors has been linked to the expression of an adaptor protein known as the Na+/H+ exchanger regulatory factor 1 (NHERF1), a protein that binds the C-terminus of the PTH1R and modulates its functions.3 Transfection of NHERF1 to DCT and ROS 17/2.8 cells inhibits PTH(7-34)-induced PTH1R endocytosis. PTH(7-34) does not stimulate β-arrestin translocation to the membrane in DCT, ROS 17/2.8,3,4 or CHO5,6 cells independently of the expression of NHERF1. No effects of PTH(7-34) on β-arrestin recruitment and ERK phosphorylation have been detected on multiple cell lines transiently or stably transfected with human PTH1R.7

In contrast, a different study reported that PTH(7-34) is a biased agonist that inhibits G protein-dependent signaling while promoting β-arrestin signaling and ERK activation in HEK293 cells.8 Furthermore, PTH(7-34) reportedly increased bone anabolism in wild-type mice9 but not in parathyroidectomized rats.10 In conclusion, the specific responses to PTH(7-34) are exquisitely dependent on the target cells and their environment. This example is by no means isolated. Because of this, the design of robust platforms to detect and study biased agonism requires reliable information regarding crosstalk between signaling pathways and the proteome of the cell models selected.

Receptor Interacting Proteins: Defining the Receptor Interactome

The idea that cellular receptors function as multimeric signaling complexes has been well established for multiple types of receptors. Nuclear and cell surface receptors interact with multiple proteins that regulate receptor localization, functional partners, and, ultimately, biological function. The yeast two-hybrid system (Y2H) first described by Fields and Song in 198911 has probably been the most widely used procedure to detect and identify receptor interacting proteins. This technology is based on the complementation of two separate fragments of a yeast transcription factor. One of the fragments is fused to a selected, specific bait gene (or gene fragment) and the second to the multiple genes included in a prey cDNA library. The fragments of the transcription factors are designed such that the isolated gene products are not functional unless the two fused genes or gene fragments (bait and prey) bind to one another.

Since the Y2H system is based on the interactions between nuclear factors, the methodology has been particularly useful in the detection of nuclear receptor partners. In fact, the technology has been adapted to high-throughput screening analysis of nuclear receptor partners.12 The results of these efforts, however, highlight the daunting complexity of protein–protein interactions: Albers et al.12 detected in excess of 1600 interaction pairs using 38 of the 48 known human nuclear receptors as bait in their screening assays. Global analyses based on the Y2H system have been published (see, for instance, ref. 13). Since interacting partners often modify function, these interactome maps have significant implications for the drug discovery process, as they point towards potential new therapeutic targets.

The application of the original Y2H screening methods to the analysis of cell surface receptors also yielded useful novel information. Numerous partners for GPCRs and receptor tyrosine kinases (RTK) were initially detected in early Y2H screens.14 However, RTKs and GPCRs are membrane proteins, such that traditional Y2H approaches do not work with the full-length protein. Thus, most efforts were concentrated on the study of the intracellular regions of the receptors. Moreover, some receptor–target interactions are dependent on post-translational modifications, such as phosphorylation, that cannot be mimicked in yeast. This led to the development of alternative two-hybrid technologies (including one-hybrid and three-hybrid systems) that can be adapted to cell surface receptors and performed in mammalian cells.15 Other alternative methodologies have been developed in recent years, including tandem affinity/pull-down methods and alternative methods for the solubilization of the targeted receptor.14,16–18

The discovery of multiple interacting proteins that play some role in specific signaling processes has led to models in which the functional units are higher order multimeric complexes containing one or more classes of receptors, scaffolds, signal transducers, and effectors (Figure 2.2). These complexes have been...