Tom Wehrman

Protein-protein interactions monitored in mammalian cells via complementation of β-lactamase enzyme fragments

We have defined inactive α and ω fragments of β-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the α fragment increased complemented enzyme activity by up to 4 orders of magnitude. β-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date.

Restriction enzyme-generated siRNA (REGS) vectors and libraries.

Small interfering RNA (siRNA) technology facilitates the study of loss of gene function in mammalian cells and animal models, but generating multiple siRNA vectors using oligonucleotides is slow, inefficient and costly. Here we describe a new, enzyme-mediated method for generating numerous functional siRNA constructs from any gene of interest or pool of genes. To test our restriction enzyme–generated siRNA (REGS) system, we silenced a transgene and two endogenous genes and obtained the predicted phenotypes. REGS generated on average 34 unique siRNAs per kilobase of sequence. REGS enabled us to create enzymatically a complex siRNA library (>4 × 105 clones) from double-stranded cDNA encompassing known and unknown genes with 96% of the clones containing inserts of the appropriate size.

A system for quantifying dynamic protein interactions defines a role for Herceptin in modulating ErbB2 interactions.

The orphan receptor tyrosine kinase ErbB2 is activated by each of the EGFR family members upon ligand binding. However, difficulties monitoring the dynamic interactions of the membrane receptors have hindered the elucidation of the mechanism of ErbB2 activation. We have engineered a system to monitor protein–protein interactions in intact mammalian cells such that different sets of protein interactions can be quantitatively compared. Application of this system to the interactions of the EGFR family showed that ErbB2 interacts stably with the EGFR and ErbB3, but fails to spontaneously homooligomerize. The widely used anti-cancer antibody Herceptin was found to effectively inhibit the interaction of the EGFR and ErbB2 but not to interfere with the interaction of ErbB2–ErbB3. Treatment of cells expressing EGFR and ErbB2 with Herceptin results in increased EGFR homooligomerization in the presence of EGF and a subsequent rapid internalization and down-regulation of the EGFR. In summary, the protein interaction system described here enabled the characterization of ErbB2 interactions within the biological context of the plasma membrane and provides insight into the mechanism of Herceptin action on cells overexpressing ErbB2.

Screening β-Arrestin Recruitment for the Identification of Natural Ligands for Orphan G-Protein–Coupled Receptors:

A variety of G-protein–coupled receptor (GPCR) screening technologies have successfully partnered a number of GPCRs with their cognate ligands. GPCR-mediated β-arrestin recruitment is now recognized as a distinct intracellular signaling pathway, and ligand-receptor interactions may show a bias toward β-arrestin over classical GPCR signaling pathways. We hypothesized that the failure to identify native ligands for the remaining orphan GPCRs may be a consequence of biased β-arrestin signaling. To investigate this, we assembled 10 500 candidate ligands and screened 82 GPCRs using PathHunter β-arrestin recruitment technology. High-quality screening assays were validated by the inclusion of liganded receptors and the detection and confirmation of these established ligand-receptor pairings. We describe a candidate endogenous orphan GPCR ligand and a number of novel surrogate ligands. However, for the majority of orphan receptors studied, measurement of β-arrestin recruitment did not lead to the identification of cognate ligands from our screening sets. β-Arrestin recruitment represents a robust GPCR screening technology, and ligand-biased signaling is emerging as a therapeutically exploitable feature of GPCR biology. The identification of cognate ligands for the orphan GPCRs and the extent to which receptors may exist to preferentially signal through β-arrestin in response to their native ligand remain to be determined.

Enzymatic detection of protein translocation.

Fundamental to eukaryotic cell signaling is the regulation of protein function by directed localization. Detection of these events has been largely qualitative owing to the limitations of existing technologies. Here we describe a method for quantitatively assessing protein translocation using proximity-induced enzyme complementation. The complementation assay for protein translocation (CAPT) is derived from β-galactosidase and comprises one enzyme fragment, ω, which is localized to a particular subcellular region, and a small complementing peptide, α, which is fused to the protein of interest. The concentration of α in the immediate vicinity of ω correlates with the amount of enzyme activity obtained in a dose- and time-dependent manner, thus acting as a genetically encoded biosensor for local protein concentration. Using CAPT, inducible protein movement from the cytosol to the nucleus or plasma membrane was quantitatively monitored in multiwell format and in live mammalian cells by flow cytometry.

Biased Agonism as a Mechanism for Differential Signaling by Chemokine Receptors

Background: Chemokines have been thought to act in a redundant fashion through their shared receptors. Results: Chemokines can display different efficacies for G proteins and β-arrestins, resulting in different chemotactic profiles. Conclusion: Chemokines can behave as biased agonists at their receptors, leading to functionally distinct, not redundant, responses. Significance: Biased agonism plays an important role in biological signaling. Chemokines display considerable promiscuity with multiple ligands and receptors shared in common, a phenomenon that is thought to underlie their biochemical “redundancy.” Their receptors are part of a larger seven-transmembrane receptor superfamily, commonly referred to as G protein-coupled receptors, which have been demonstrated to be able to signal with different efficacies to their multiple downstream signaling pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands, and the biologic prevalence and importance of such signaling are unclear. Here, to assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a detailed pharmacologic analysis of a set of chemokine receptors with multiple endogenous ligands using assays for G protein signaling, β-arrestin recruitment, and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein- or β-arrestin-mediated signaling or receptor internalization. This ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common and likely evolutionarily conserved biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.

Discovery of positive allosteric modulators and silent allosteric modulators of the μ-opioid receptor

μ-Opioid receptors are among the most studied G protein-coupled receptors because of the therapeutic value of agonists, such as morphine, that are used to treat chronic pain. However, these drugs have significant side effects, such as respiratory suppression, constipation, allodynia, tolerance, and dependence, as well as abuse potential. Efforts to fine tune pain control while alleviating the side effects of drugs, both physiological and psychological, have led to the development of a wide variety of structurally diverse agonist ligands for the μ-opioid receptor, as well as compounds that target κ- and δ-opioid receptors. In recent years, the identification of allosteric ligands for some G protein-coupled receptors has provided breakthroughs in obtaining receptor subtype-selectivity that can reduce the overall side effect profiles of a potential drug. However, positive allosteric modulators (PAMs) can also have the specific advantage of only modulating the activity of the receptor when the orthosteric agonist occupies the receptor, thus maintaining spatial and temporal control of receptor signaling in vivo. This second advantage of allosteric modulators may yield breakthroughs in opioid receptor research and could lead to drugs with improved side-effect profiles or fewer tolerance and dependence issues compared with orthosteric opioid receptor agonists. Here, we describe the discovery and characterization of μ-opioid receptor PAMs and silent allosteric modulators, identified from high-throughput screening using a β-arrestin–recruitment assay.

A Homogeneous Enzyme Fragment Complementation-Based β-Arrestin Translocation Assay for High-Throughput Screening of G-Protein-Coupled Receptors

G-protein-coupled receptors (GPCRs) represent one of the largest gene families in the human genome and have long been regarded as valuable targets for small-molecule drugs. The authors describe a new functional assay that directly monitors GPCR activation. It is based on the interaction between β-arrestin and ligand-activated GPCRs and uses enzyme fragment complementation technology. In this format, a GPCR of interest is fused to a small (~4 kDa), optimized α fragment peptide (termed ProLink™) derived from β-galactosidase, and β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (termed the enzyme acceptor [EA]). Upon activation of the receptor, the β-arrestin-EA fusion protein binds the activated GPCR. This interaction drives enzyme fragment complementation, resulting in an active β-galactosidase enzyme, and thus GPCR activation can be determined by quantifying β-galactosidase activity. In this report, the authors demonstrate the utility of this technology to monitor GPCR activation and validate the approach using a Gαi-coupled GPCR, somatostatin receptor 2. Potential application to high-throughput screens in both agonist and antagonist screening modes is exemplified. (Journal of Biomolecular Screening 2008:737-747)

A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization

G‐protein‐coupled receptor (GPCR) signaling is involved in a wide range of physiological processes and diseases, and around one‐half of currently used drugs target GPCRs. Assays for the signaling of GPCRs have suffered from drawbacks, including low signal‐to‐noise, temporally transient signals, and difficulty in applying a single assay to a wide range of GPCRs. We have developed a set of assays for G‐protein‐coupled receptor signaling based on β‐galacto‐sidase enzyme complementation in live mammalian cells. We previously described an assay for GPCR activation by monitoring the binding of β‐arrestin to the receptor. Here we describe a second assay that monitors the internalization of GPCRs to endosomes, an event that follows receptor activation and is critical in desensitizing and resensitizing the receptor. We show that both assays display high signal‐to‐noise ratios with low variability and are quantitative for a wide range of GPCRs. EC50s obtained with these assays closely match results reported in the literature. Finally, we show that these assays are readily adapted to high‐throughput chemical screens. Thus, these two assays for monitoring G‐protein‐coupled receptor activation and internal‐ization should prove valuable in basic biological studies as well as in high‐throughput screens.— Hammer M. M., Wehrman, T. S., Blau H. M. A novel enzyme complementation‐based assay for monitoring G‐protein‐coupled receptor internalization. FASEB J. 21, 3827–3834 (2007)

Measurements of β-Arrestin Recruitment to Activated Seven Transmembrane Receptors Using Enzyme Complementation

The recruitment of arrestins to activated 7TMRs results in the activation of alternative signaling pathways, quenching of G-protein activation, and coupling to clathrin-mediated endocytosis. The nearly ubiquitous involvement of arrestin in 7TMR signaling has spurred the development of several methods for monitoring this interaction in mammalian cells. Nonetheless, few maintain the reproducibility and precision necessary for drug discovery applications. Enzyme fragment complementation technology (EFC) is an emerging protein-protein interaction technology based on the forced complementation of a split enzyme that has proven to be highly effective in monitoring the formation of GPCR-arrestin complexes. In these systems, the target proteins are fused to two fragments of an enzyme that show little or no spontaneous complementation. Interaction of the two proteins forces the complementation of the enzyme, resulting in an enzymatic measure of the protein interaction. This chapter discusses the utility and methods involved in using the PathHunter β-galactosidase complementation system to monitor arrestin recruitment and the advantages of exploiting this pathway in the characterization of 7TMR function.