April 15, 2014 (Vol. 34, No. 8)

Ken Doyle, Ph.D.

Recent years have seen major advances in understanding the structure-function relationships of G protein-coupled receptors (GPCRs). This large superfamily of transmembrane receptors comprises over 800 members in humans.

GPCRs regulate a wide variety of physiological processes including sensation (vision, taste, and smell), growth, hormone responses, and regulation of the immune and autonomic nervous systems. Their involvement in multiple disease pathways makes GPCRs attractive targets for drug discovery efforts.

These multifaceted proteins will be the subject of “GPCR Structure, Function and Drug Discovery,” a Global Technology Community conference scheduled to take place May 22–23 in Boston. The conference is expected to cover a broad range of topics including biased signaling, membrane protein structures, GPCR signaling dynamics, computational approaches to disease.

According to Bryan Roth, M.D., Ph.D., Michael Hooker Distinguished Professor at the University of North Carolina, Chapel Hill, drugs that can selectively target various downstream GPCR pathways hold the most promise. Dr. Roth’s laboratory studies approximately 360 different GPCRs with therapeutic potential using massively parallel screening methods. His research focuses on “functional selectivity,” which he describes as “the ligand-dependent selectivity for certain signal transduction pathways in one and the same receptor.”

Dr. Roth notes that structural data have demonstrated that GPCRs exist in multiple conformations: “The structures of the 5-hydroxytryptamine 2B receptor and the recent high-resolution delta-opioid receptor structure have provided evidence for conformational rearrangements that contribute to functional selectivity.” Drugs that take advantage of this selectivity by preferentially stabilizing certain conformations may have unique therapeutic utility.

“Generally, we look at G protein versus arrestin-based signaling, although it’s also possible to examine how drugs activate one G protein-mediated signaling pathway versus another,” says Dr. Roth. “Alternatively, some drugs target specifically endocytic pathways and have no effect on either G-protein signaling or arrestin translocation.”

The Role of β-Arrestins

β-Arrestins constitute a major class of intracellular scaffolding proteins that regulate GPCR signaling by preventing or enhancing the binding of GPCRs to intracellular signaling molecules. Laura Bohn, Ph.D., associate professor at Scripps Florida,  studies the roles that β-arrestins play in GPCR-mediated signaling. Dr. Bohn collaborates with medicinal and synthetic chemists, focusing on two major receptor groups, opioid and serotonin 2A receptors.

Opioid receptors are a class of GPCRs that bind opiate drugs such as morphine and codeine. The serotonin (or 5-hydroxytryptamine) 2A receptor is a GPCR that mediates the action of several antipsychotic drugs.

“Our group is trying to understand how receptors signal in the endogenous setting and how diverse ligands can promote certain signaling pathways over others,” says Dr. Bohn. “Our major goal is to understand how those signaling pathways lead to physiological responses.”

Dr. Bohn notes that a particular β-arrestin can play multiple, tissue-specific roles—shutting down the signaling of a receptor in one tissue while activating signaling in another. “One agonist may elicit the dampening effects of β-arrestins while another agonist may promote facilitative signaling interactions,” adds Dr. Bohn.

Dr. Bohn’s laboratory studies functional selectivity of GPCR activation by multiple drug candidates. Ultimately the hope is to improve therapeutic action while minimizing side-effects. Dr. Bohn notes that functional selectivity will be highly influenced by the complement of signaling components available within a given cell.

“In a nutshell, different ligands can direct GPCR signaling to different effectors, which could result in different physiological effects,” comments Dr. Bohn. “Our challenge is in determining what signaling pathways to harness to promote certain effects, while avoiding others.”

Cells that express fluorescently tagged β-arrestins and a GPCR of interest can be used to visualize the interactions that follow stimulation with agonists. This image, provided by Scripps Florida, shows U2OS cells expressing the human κ-opioid receptor after they have been treated with U69,593, a known κ-opioid receptor agonist. The tagged β-arrestin translocates from a diffuse distribution throughout the cell and concentrates in punctae on the cell surface when engaging with the receptor.

Using Designer Proteins

The multifunctional signaling abilities of β-arrestins has prompted large-scale study of their properties. Vsevolod Gurevich, Ph.D., professor of pharmacology at Vanderbilt University, studies the structure, function, and biology of arrestin proteins.

According to Dr. Gurevich, β-arrestins have three main functions. First, they prevent the coupling of GPCRs to G proteins, thereby blocking further G protein-mediated signaling (a process known as desensitization). Second, the binding of a GCPR releases the β-arrestin’s carboxy-terminal “tail” and promotes internalization of the receptor. Third, receptor-bound β-arrestins bind other signaling proteins, resulting in a second wave of arrestin-mediated signaling.

Dr. Gurevich’s laboratory studies β-arrestin biology through the use of three types of specially designed mutants—enhanced phosphorylation-dependent, receptor-specific, and signaling-biased mutants.

“We showed that an enhanced mutant of visual β-arrestin-1 partially compensates for defects of rhodopsin phosphorylation in vivo,” states Dr. Gurevich. “Several congenital disorders are caused by mutant GPCRs that cannot be normally phosphorylated because they have lost GPCR kinase (GRK) sites. Enhanced super-active arrestins have the potential to compensate for these defects, bringing the signaling closer to normal.”

Dr. Gurevich explains the strategy involved in creating designer β-arrestins: “We identify residues critical for individual β-arrestin functions by mutagenesis, using limited structural information as a guide. We also work on getting more structural information. In collaboration with different crystallographers, we solved the crystal structures of all four vertebrate β-arrestin subtypes in the basal state, as well as the structure of the arrestin-1-rhodopsin complex.”

Dr. Gurevich believes that designer β-arrestins “are the next step in research and therapy, moving way beyond what small molecules can achieve. The difference in capabilities between redesigned signaling proteins, including β-arrestins, and conventional small molecule drugs is about the same as that between airplanes and horse-driven carriages.”

Dr. Gurevich observes that redesigned signaling proteins face considerable obstacles in terms of gene delivery, but that the efforts are worth it. “Using designer signaling proteins, we can tell the cell what to do in a language it cannot disobey,” asserts Dr. Gurevich.

Because proteins respond to biological feedback, he continues, “they are less likely to overdo whatever they are doing, whereas many [small molecule] drugs cause unwanted side-effects by doing exactly what they were designed to do.” According to Dr. Gurevich, the potential of designer proteins can help compensate for what many pharmaceutical companies are discovering: small molecule drug discovery efforts are reaching their limits.

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling and facilitates receptor internalization due to arrestin interactions with clathrin and AP2. The arrestin-receptor complex also serves as a nucleus of signalosome, recruiting various signaling proteins and scaffolding mitogen-activated protein kinase cascades, among other pathways. [Vanderbilt University]

Dynamics of GPCR Oligomerization

Structure-function studies of GPCRs increasingly rely on biophysical research by scientists such as Marta Filizola, Ph.D., a tenured associate professor of structural and chemical biology at Mount Sinai Hospital’s Icahn School of Medicine. Dr. Filizola’s group studies the molecular determinants underlying ligand binding, activation, and functional selectivity of GPCRs.

By deploying an impressive arsenal of computational techniques including “molecular modeling, bioinformatics, chemoinformatics, molecular dynamics simulation, and rational drug design approaches,” Dr. Filizola examines how oligomerization of GPCRs within the cell membrane modulates their functional properties.

Dr. Filizola’s in silico analysis complements conventional wet lab techniques such as binding assays, functional assays, and technologies in experimental biophysics, including single-molecule imaging, for studying GPCR-ligand, GPCR-GPCR, and GPCR-protein interactions. Dr. Filizola says that “with the current availability of multiple GPCR crystal structures, in silico methods have recently proven to be very successful in expediting the drug discovery process via their more rational approach.”

“We have recently invested considerable effort in the development and validation of computational strategies that use molecular dynamics-based enhanced sampling algorithms, in combination with atomistic or coarse-grained representations of the receptor and its lipid-water environment,” elaborates Dr. Filizola.

Finally, Dr. Filizola notes that her research reveals molecular details of receptor-receptor interactions that cannot be obtained through experimentation but can be tested iteratively.

GPCR-Agonist Structure Determination

Another dimension to studying GPCR structure and function is provided by Reinhard Grisshammer, Ph.D., investigator at the Membrane Protein Structure and Function Unit of the National Institute of Neurological Disorders and Stroke, National Institutes of Health. Dr. Grisshammer’s group focuses on understanding the structural changes that occur upon activation of GPCRs, in particular neurotensin receptor 1 (NTS1), by x-ray crystallography. Citing a parallel task, Dr. Grisshammer says, “We conduct functional studies, using nanodisc technology, to complement our structural studies, exploring the behavior and determinants of NTS1 signaling.” Nanodiscs are self-assembled discs of phospholipid bilayers that are stabilized by membrane scaffold proteins.

Dr. Grisshammer’s goal is to obtain a complete picture of the NTS1 signaling mechanism that will provide broad insights into GPCR signaling.

Considerable challenges can hamper the study of the structure of a GPCR bound to its agonist. According to Dr. Grisshammer, in addition to the normal obstacles inherent in expressing and purifying large quantities of a membrane protein, there is the problem posed by wild-type NTS1, which is unstable in many detergents used for crystallization.

Dr. Grisshammer’s laboratory has implemented a number of cutting-edge approaches. For example, in collaboration with Chris Tate (a group leader at the MRC Laboratory of Molecular Biology), it has used conformational thermostabilization. This approach, says Dr. Grisshammer, yielded a mutant NTS1 that shows “greatly improved stability and is locked into an agonist-binding, active-like conformation.” Also, in collaboration with Nagarajan Vaidehi (a professor of immunology at the City of Hope National Medical Center), the laboratory has used computational approaches to study how thermostabilizing mutations affect receptor dynamics.

Dr. Grishammer’s laboratory carries out structural studies. (They revealed, for the first time, how a peptide agonist binds to a GPCR.) It also uses classic pharmacology, such as radioligand-binding assays and nucleotide exchange assays with heterotrimeric G proteins. Finally, it looks forward to implementing emerging technologies that Dr. Grishammer believes hold great promise in the study of GPCR structure and function.

“Mini-beams at synchrotrons are quite advanced in the analysis of small crystals with dimensions in the tens of microns,” points out Dr. Grisshammer. “However, obtaining well-ordered crystals of those dimensions is still challenging.”

One technique that can overcome this problem, is “femtosecond crystallography using an X-ray free-electron laser.” According to Dr. Grisshammer, this technique has recently resulted in the structure of a human serotonin receptor.