Research

G protein-coupled receptors (GPCRs) are a class of ubiquitous cell-surface receptors that regulate many physiological functions, such as cardiovascular, neuronal, endocrine and immune functions. On the cell surface, GPCRs act as extracellular biosensors that are triggered by a variety of stimuli including photons, protons, cations, small molecules, lipids, carbohydrates, peptides, and proteins. Upon stimulation by extracellular cues, GPCRs communicate with the interior of the cell by transmitting a molecular signal that leads to an appropriate cellular response. Due to their location on the cell surface, ability to bind smaller molecules, and involvement in most aspects of physiology, GPCRs have become popular drug targets with 30 percent to 50 percent of all prescribed drugs affecting them.

Classically, GPCR signaling has been thought to occur strictly at the plasma membrane and is initiated by a ligand that binds to and stabilizes active conformations of the receptor. These active GPCR conformations activate intracellular heterotrimeric G proteins, causing downstream signaling throughout the cell. In order to terminate G protein signaling, cells have devised a specialized desensitization mechanism that includes phosphorylation of receptors by GPCR kinases and subsequent recruitment of β-arrestins to the phosphorylated receptor. β-arrestins engage the receptor at the same receptor transmembrane core region that G proteins bind, and thus β-arrestin recruitment to the receptor sterically blocks further G protein activation. In addition, β-arrestins scaffolds proteins involved in endocytosis, such as clathrin and adapter protein-2, to promote internalization of GPCRs, thereby removing them from the source of the activating ligand.

Over the past decade, it has been observed that some GPCRs continue to activate G protein following β-arrestin-mediated internalization into cellular compartments called endosomes. This endosomal signaling has been difficult to incorporate within the aforementioned classical understanding of GPCR signaling, since the GPCR–β-arrestin interaction was thought to uncouple G protein from the receptor. However, we recently discovered and delineated a new signaling paradigm whereby GPCRs bind β-arrestins in a specific manner; in this conformation, β-arrestin only interacts with the receptor C-terminal tail thereby permitting the receptor transmembrane core to bind with G proteins simultaneously to form a “megaplex.” The receptor in these megaplexes still maintains its ability to activate G protein, even while being internalized by β-arrestins.

From these recent discoveries, a number of new and fundamental questions have emerged that are the focus of the Thomsen Laboratory. Our approach to addressing these questions is interdisciplinary, and involves understanding biological mechanisms all the way from the level of single atoms to the perspective of cells/whole organisms. The ultimate goal of the Thomsen Laboratory is to use our scientific discoveries to inspire the development of novel therapeutic strategies to combat diseases with unmet needs.

Mechanistic and Structural Studies of Endosomal GPCR Signaling

We only recently began to understand the paradox of endosomal GPCR signaling by the discovery of GPCR–G protein–βarrs megaplexes. However, other major mechanistic aspects of endosomal GPCR signaling remain completely unknown. For instance, it has been proposed that signaling proteins involved in endosomal signaling might exist in much larger and highly organized multi-protein signalosomes, and that these signalosomes may be confined to specific locations on the endosomal surface where they generate ‘signaling hot spots.’ Thus, to gain a better understanding of endosomal GPCR signaling, we are currently leveraging cutting-edge cryo-electron microscopy and proteomics to investigating the existence and structural organization of GPCR signalosomes at the endosomal surface.

Mechanisms of GPCR Desensitization

GPCR signaling termination or desensitization is a highly regulated process that serves to avoid inappropriate and sometimes lethal overstimulation of the cell. β-arrestins are master regulators of this process and do so through several well-characterized mechanisms including competing with G protein for access at the receptor and by removing receptors from the plasma membrane via endocytosis. However, recent data suggest that not all GPCRs are regulated similarly by β-arrestins and that there exist additional desensitization mechanisms that have not been explored yet. Therefore, a central goal of the Thomsen Laboratory is to identify novel molecular avenues that promote desensitization of GPCR signaling and understand these mechanisms at cell biological, biochemical, and structural levels.

Physiological Functions of Endosomal GPCR Signaling

Although it is well-established that certain GPCRs stimulate G protein signaling after having been internalized into endosomes, knowledge about physiological functions of this endosomal GPCR signaling is generally lacking. Therefore, our laboratory is using a variety of advanced pharmacological and biosensor approaches to detect and uncover roles of endosomal GPCR signaling in physiological relevant models. In particular, we are interested in chemokine receptors and how their signaling from endosomes may regulate proliferation, differentiation, and cell migration in both immune and cancer cells. Fleshing out specific physiological functions of endosomal signaling from cell surface signaling not only provides novel fundamental knowledge of GPCR signaling processes but may also assist in designing novel therapeutic strategies.

Funding