GPCR-induced dissociation of G-protein subunits in early stage signal transduction

Leifert, Wayne R.; Aloia, Amanda L.; Bucco, Olgatina; McMurchie, Edward J.

doi:10.1080/09687860500370604

Cited by 18 publications

(16 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…1). Upon agonist binding to the receptor, the inactive a subunit, which is associated with the bg subunits, is activated through exchange of GDP for GTP at the guanine nucleotide binding site (9). This results in dissociation of the active a subunit from the receptor and the bg subunits and enables interaction with specific effector proteins (as indicated below).…”

Section: G Proteinmediated Signalingmentioning

confidence: 98%

GPCR Signaling: Understanding the Pathway to Successful Drug Discovery

Williams

Hill

2009

Methods in Molecular Biology

View full text Add to dashboard Cite

Modulators of G protein-coupled receptors (GPCRs) form a key area for the pharmaceutical industry, representing approximately 27% of all Food and Drug Administration (FDA)-approved drugs. Consequently, there are a wide variety of in vitro plate-based screening technologies that enable the measurement of compound affinity, potency, and efficacy for almost every type of GPCR. However, to maximize success it is prudent to ensure that (i) the most suitable assay formats are identified, (ii) they are configured optimally to detect the desired compound activity, and (iii) that they form a basis for predicting clinical effects. To achieve this, an understanding of the pathways and mechanisms of receptor activation relevant to the disease mechanism, as well as the benefits and/or limitations of the specific techniques, is key.

show abstract

Section: G Proteinmediated Signalingmentioning

confidence: 98%

GPCR Signaling: Understanding the Pathway to Successful Drug Discovery

Williams

Hill

2009

Methods in Molecular Biology

View full text Add to dashboard Cite

show abstract

“…For signalling and FRET assays, proteins (α 2A adrenergic receptor, Gα i1 , Gβ 4 and Gγ 2 ) were expressed using the baculovirus-Sf9 system, and purified as per published methods [4]. For surface plasmon resonance (SPR) studies, Gα i1 ICONN 2006 1-4244-0453-3/06/$20.00  2006 IEEE subunits with 6-histidine or 16-histidine residue tags (denoted h6_Gα i1 and h16_Gα i1 , respectively) were bacterially expressed using the pQE30 vector (Qiagen).…”

Section: A Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

“…Ligand-mediated signalling of reconstituted receptor:Gprotein complexes was measured by the widely used [ 35 S]GTPγS-binding assay [4].…”

Section: B [ 35 S]gtpγs Reconstitution Signalling Assaymentioning

confidence: 99%

“…As such, apart from comprising a significant proportion (>50%) of pharmaceutical drug targets [3], GPCRs represent a "tool-box" of diverse specific molecular detectors of their various ligands. For all GPCRs (which are integral membrane proteins), ligand-binding is transduced into a cellular signal through dissociation or rearrangement of the G-protein heterotrimer [4,5], whose components interact directly with cellular effectors upon receptor activation [2]. By producing modified recombinant Gproteins (and utilizing their inherent affinity), it should be possible to produce a generic "molecular switch" based on heterotrimer rearrangement, which could be attached to defined surfaces.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cell-free receptor-based biosensors

Glatz

Leifert

Bailey

et al. 2006

2006 International Conference on Nanoscience and Nanotechnology

Self Cite

View full text Add to dashboard Cite

The ability to express and purify modified recombinant signalling proteins such that they retain their biological function in a cell-free context, has provided a basis for production of molecular biosensors. Here we utilise G-protein Coupled Receptors (GPCRs) and their G-proteins to detect various binding partners in a cell-free environment. Molecular biology approaches were employed to express these proteins using baculovirus and bacteria, and to alter their characteristics to improve surface-attachment and fluorescent labelling capabilities. Ligand-mediated signalling of a GPCR could be measured (using [ 35 S]GTPγS-binding assays) in a reconstituted system with recombinant proteins either free in solution or attached to Ni 2+ -coated beads. Affinity of histidine-tagged proteins for a Ni 2+ -coated surface was significantly enhanced by addition of extra histidine residues to the tag, as determined by surface plasmon resonance. This was due to the longer tag occupying, on average, a greater number of available histidinebinding sites. Further, a novel homogeneous fluorescence resonance energy transfer (FRET)-based assay has been developed to detect rearrangements in the G-protein heterotrimer. Investigation of small peptides that can be fused to G-protein subunits, allowing for site-specific fluorescent labelling, was undertaken in order to improve the resolution of the "first generation" FRET assay. By utilizing this improved Gprotein heterotrimer "molecular switch", we are developing a generic technology such that a range of GPCRs could be assayed for ligand-mediated activation while attached to surfaces (e.g. on beads or as microarrays) or in solution (e.g. multi-well plates), with increased throughput.

show abstract

“…The Gα and Gβγ subunits then dissociate from each other and relay signals to downstream effectors, such as second messenger-generating enzymes and ion channels, ultimately initiating signaling cascades that mediate a variety of cellular functions. 1 Following agonist activation, GPCRs undergo desensitization and internalization, limiting the duration and magnitude of the response despite continued agonist availability and thereby preventing overstimulation of the receptor.…”

Section: Introductionmentioning

confidence: 99%

Integrating High-Content Analysis into a Multiplexed Screening Approach to Identify and Characterize GPCR Agonists

et al. 2014

View full text Add to dashboard Cite

G protein-coupled receptors (GPCRs) are one of the most popular and proven target classes for therapeutic intervention. The increased appreciation for allosteric modulation, receptor oligomerization, and biased agonism has led to the development of new assay platforms that seek to capitalize on these aspects of GPCR biology. High-content screening is particularly well suited for GPCR drug discovery given the ability to image and quantify changes in multiple cellular parameters, to resolve subcellular structures, and to monitor events within a physiologically relevant environment. Focusing on the sphingosine-1-phosphate (S1P1) receptor, we evaluated the utility of high-content approaches in hit identification efforts by developing and applying assays to monitor β-arrestin translocation, GPCR internalization, and GPCR recycling kinetics. Using these approaches in combination with more traditional GPCR screening assays, we identified compounds whose unique pharmacological profiles would have gone unnoticed if using a single platform. In addition, we identified a compound that induces an atypical pattern of β-arrestin translocation and GPCR recycling kinetics. Our results highlight the value of high-content imaging in GPCR drug discovery efforts and emphasize the value of a multiassay approach to study pharmacological properties of compounds of interest.

show abstract

GPCR-induced dissociation of G-protein subunits in early stage signal transduction

Cited by 18 publications

References 29 publications

GPCR Signaling: Understanding the Pathway to Successful Drug Discovery

GPCR Signaling: Understanding the Pathway to Successful Drug Discovery

Cell-free receptor-based biosensors

Integrating High-Content Analysis into a Multiplexed Screening Approach to Identify and Characterize GPCR Agonists

Contact Info

Product

Resources

About