Current applications of mini G proteins to study the structure and function of G protein-coupled receptors

G protein-coupled receptors (GPCRs) regulate intracellular signalling pathways that contribute to virtually all aspects of cell function. Characterising GPCRs in each of their conformational states is key to understanding their mechanism of action, but structure determination of receptors in their active state, bound to a heterotrimeric G protein or β-arrestin, has proved challenging. A number of G protein surrogates have been developed to simplify this process, including G protein-derived peptides, nanobodies and, most recently, mini G proteins. The aim of these surrogates is to bind the receptor and stabilise its active conformation, whilst eliminating the problems inherent to native signalling proteins, namely their large size, instability and conformational dynamics. Mini G proteins are composed of a single domain from the G protein α-subunit that has been engineered to form a stable complex with GPCRs. They induce comparable pharmacological and structural changes in the receptor to those elicited by heterotrimeric G proteins, and retain their native receptor-coupling specificity. At least one member of each G protein family has been converted into a mini G protein, which means that they can be used to characterise a wide variety of GPCRs. Since their initial publication two years ago, mini G proteins have facilitated the structure determination of three different receptors in their active state and enabled the development of a methodology to thermostabilise GPCRs in their fully active conformation. They have also been used to develop a range of assays that can measure mini G protein coupling to receptors in vitro, and a sensitive cell-based assay that is capable of accurately reporting ligand efficacy and quantifying G protein coupling in vivo. This review presents an overview of the current applications of mini G proteins to study the structure and function of GPCRs

A Neural Network Method for Land Use Change Classification, with Application to the Nile River Delta

Detecting and monitoring changes in conditions at the Earth's surface are essential for understanding human impact on the environment and for assessing the sustainability of development. In the next decade, NASA will gather high-resolution multi-spectral and multi-temporal data, which could be used for analyzing long-term changes, provided that available methods can keep pace with the accelerating flow of information. This paper introduces an automated technique for change identification, based on the ARTMAP neural network. This system overcomes some of the limitations of traditional change detection methods, and also produces a measure of confidence in classification accuracy. Landsat thematic mapper (TM) imagery of the Nile River delta provides a testbed for land use change classification methods. This dataset consists of a sequence of ten images acquired between 1984 and 1993 at various times of year. Field observations and photo interpretations have identified 358 sites as belonging to eight classes, three of which represent changes in land use over the ten-year period. Aparticular challenge posed by this database is the unequal representation of various land use categories: three classes, urban, agriculture in delta, and other, comprise 95% of pixels in labeled sites. A two-step sampling method enables unbiased training of the neural network system across sites.National Science Foundation (SBR 95-13889); Office of Naval Research (N00014-95-1-409, N00014-95-0657); Air Force Office of Scientific Research (F49620-01-1-0397, F49620-01-1-042

ARTMAP Neural Network Classification of Land Use Change

The ability to detect and monitor changes in land use is essential for assessment of the sustainability of development. In the next decade, NASA will gather high-resolution multi-spectral and multi-temporal data, which could be used for detecting and monitoring long-term changes. Existing methods are insufficient for detecting subtle long-term changes from high-dimensional data. This project employs neural network architectures as alternatives to conventional systems for classifying changes in the status of agricultural lands from a sequence of satellite images. Landsat TM imagery of the Nile River delta provides a testbed for these land use change classification methods. A sequence often images was taken, at various times of year, from 1984 to 1993. Field data were collected during the summer of 1993 at88 sites in the Nile Delta and surrounding desert areas. Ground truth data for 231 additional sites were determined by expert site assessment at the Boston University Center for Remote Sensing. The field observations are grouped into classes including urban, reduced productivity agriculture, agriculture in delta, desert/coast reclamation, wetland reclamation, and agriculture in desert/coast. Reclamation classes represent land use changes. A particular challenge posed by this database is the unequal representation of various land use categories: urban and agriculture in delta pixels comprise the vast majority of the ground truth data available in the database. A new, two-step training data selection method was introduced to enable unbiased training of neural network systems on sites with unequal numbers of pixels. Data were successfully classified by using multi-date feature vectors containing data from all of the available satellite images as inputs to the neural network system.National Science Foundation Graduate Fellowship; National Science Foundation (SBR 95-13889); Office of Naval Research (N00014-95-I-409, N00014-95-0657); Air Force Office of Scientific Research (F49620-0l-1-0397)

Human adenosine A2A receptor : molecular mechanism of ligand binding and activation

Adenosine receptors (ARs) comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs). ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR), making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes

Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation

G protein-coupled receptors (GPCRs) modulate cytoplasmic signalling in response to extracellular stimuli, and are important therapeutic targets in a wide range of diseases. Structure determination of GPCRs in all activation states is important to elucidate the precise mechanism of signal transduction and to facilitate optimal drug design. However, due to their inherent instability, crystallisation of GPCRs in complex with cytoplasmic signalling proteins, such as heterotrimeric G proteins and β-arrestins, has proved challenging. Here, we describe the design of a minimal G protein, mini-Gs, which is composed solely of the GTPase domain from the adenylate cyclase stimulating G protein Gs. Mini-Gs is a small, soluble protein, which efficiently couples GPCRs in the absence of Gβγ subunits. We engineered mini-Gs, using rational design mutagenesis, to form a stable complex with detergent-solubilised β1-adrenergic receptor (β1AR). Mini G proteins induce similar pharmacological and structural changes in GPCRs as heterotrimeric G proteins, but eliminate many of the problems associated with crystallisation of these complexes, specifically their large size, conformational dynamics and instability in detergent. They are therefore novel tools, which will facilitate the biochemical and structural characterisation of GPCRs in their active conformation

Active state structures of G protein-coupled receptors highlight the similarities and differences in the G protein and arrestin coupling interfaces

G protein-coupled receptors (GPCRs) regulate cellular signalling through heterotrimeric G proteins and arrestins in response to an array of extracellular stimuli. Structure determination of GPCRs in an active conformation bound to intracellular signalling proteins has proved to be highly challenging. Nonetheless, three new structures of GPCRs in an active state have been published during the last year, namely the adenosine A2A receptor (A2AR) bound to an engineered G protein, opsin bound to visual arrestin and the m opioid receptor (mOR) bound to a G protein-mimicking nanobody. These structures have provided novel insight into the sequence of events leading to GPCR activation, and have highlighted both similarities and differences in the structure of the interface between GPCRs and different signalling proteins

Cryo-EM structure of the adenosine A2A receptor coupled to an engineered heterotrimeric G protein

The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here, we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the βγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 Å or better. Comparison with the 3.4 Å resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the β subunit of the G protein was observed

Mini G protein probes for active G protein– coupled receptors (GPCRs) in live cells

G protein–coupled receptors (GPCRs) are key signaling proteins that regulate nearly every aspect of cell function. Studies of GPCRs have benefited greatly from the development of molecular tools to monitor receptor activation and downstream signaling. Here, we show that mini G proteins are robust probes that can be used in a variety of assay formats to report GPCR activity in living cells. Mini G (mG) proteins are engineered GTPase domains of G subunits that were developed for struc- tural studies of active-state GPCRs. Confocal imaging revealed that mG proteins fused to fluorescent proteins were located diffusely in the cytoplasm and translocated to sites of receptor activation at the cell surface and at intracellular organ- elles. Bioluminescence resonance energy transfer (BRET) assays with mG proteins fused to either a fluorescent protein or luciferase reported agonist, superagonist, and inverse agonist activities. Variants of mG proteins (mGs, mGsi, mGsq, and mG12) corresponding to the four families of G subunits displayed appropriate coupling to their cognate GPCRs, allowing quantitative profiling of subtype-specific coupling to individual receptors. BRET between luciferase–mG fusion proteins and fluorescent markers indicated the presence of active GPCRs at the plasma membrane, Golgi apparatus, and endosomes. Complementation assays with fragments of NanoLuc luciferase fused to GPCRs and mG proteins reported constitutive receptor activity and agonist-induced activation with up to 20-fold increases in luminescence. We conclude that mG proteins are versatile tools for studying GPCR activation and coupling specificity in cells and should be useful for discovering and characterizing G protein sub- type–biased ligands

Protocol for DRAUP: A deimplementation programme to decrease routine chest radiographs after central venous catheter insertion

INTRODUCTION: Avoiding low value medical practices is an important focus in current healthcare utilisation. Despite advantages of point-of-care ultrasound (POCUS) over chest X-ray including improved workflow and timeliness of results, POCUS-guided central venous catheter (CVC) position confirmation has slow rate of adoption. This demonstrates a gap that is ripe for the development of an intervention. METHODS: The intervention is a deimplementation programme called DRAUP ( ETHICS AND DISSEMINATION: Approval of the study by the Human Research Protection Office has been obtained. This work will be disseminated by publication of peer-reviewed manuscripts, presentation in abstract form at scientific meetings and data sharing with other investigators through academically established means. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier, NCT04324762, registered on 27 March 2020

A mixed methods multiple case study of implementation as usual in children’s social service organizations: study protocol

Background Improving quality in children’s mental health and social service settings will require implementation strategies capable of moving effective treatments and other innovations (e.g., assessment tools) into routine care. It is likely that efforts to identify, develop, and refine implementation strategies will be more successful if they are informed by relevant stakeholders and are responsive to the strengths and limitations of the contexts and implementation processes identified in usual care settings. This study will describe: the types of implementation strategies used; how organizational leaders make decisions about what to implement and how to approach the implementation process; organizational stakeholders’ perceptions of different implementation strategies; and the potential influence of organizational culture and climate on implementation strategy selection, implementation decision-making, and stakeholders’ perceptions of implementation strategies. Methods/design This study is a mixed methods multiple case study of seven children’s social service organizations in one Midwestern city in the United States that compose the control group of a larger randomized controlled trial. Qualitative data will include semi-structured interviews with organizational leaders (e.g., CEOs/directors, clinical directors, program managers) and a review of documents (e.g., implementation and quality improvement plans, program manuals, etc.) that will shed light on implementation decision-making and specific implementation strategies that are used to implement new programs and practices. Additionally, focus groups with clinicians will explore their perceptions of a range of implementation strategies. This qualitative work will inform the development of a Web-based survey that will assess the perceived effectiveness, relative importance, acceptability, feasibility, and appropriateness of implementation strategies from the perspective of both clinicians and organizational leaders. Finally, the Organizational Social Context measure will be used to assess organizational culture and climate. Qualitative, quantitative, and mixed methods data will be analyzed and interpreted at the case level as well as across cases in order to highlight meaningful similarities, differences, and site-specific experiences. Discussion This study is designed to inform efforts to develop more effective implementation strategies by fully describing the implementation experiences of a sample of community-based organizations that provide mental health services to youth in one Midwestern city