Biomedical subjects

Susan K Lyman

Publications and source records attributed to Susan K Lyman.

3 recordsLinked to original sources

PubMed2004

Multiplex GPCR assay in reverse transfection cell microarrays.

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists.

Dose-Response Relationship, Drug↗

PubMed2003

Phosphorylation-dependent and -independent nuclear import of RS domain-containing splicing factors and regulators.

SR proteins and related RS domain-containing polypeptides are an important class of splicing regulators in higher eukaryotic cells. The RS domain facilitates nuclear import of SR proteins and mediates protein-protein interactions during spliceosome assembly; both functions appear to subject to regulation by phosphorylation. Previous studies have identified two nuclear import receptors for SR proteins, transportin-SR1 and transportin-SR2. Here we show that transportin-SR1 and transportin-SR2 are the alternatively spliced products of the same gene and that transportin-SR2 is the predominant transcript in most cells and tissues examined. While both receptors import typical SR proteins in a phosphorylation-dependent manner, they differentially import the RS domain-containing splicing regulators hTra2alpha and hTra2beta in different phosphorylation states. We suggest that differential regulation of nuclear import may serve as a mechanism for homeostasis of RS domain-containing splicing factors and regulators in the nucleus and for selective cellular responses to signaling.

Alternative Splicing↗

PubMed2002

Influence of cargo size on Ran and energy requirements for nuclear protein import.

Previous work has shown that the transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis. We now demonstrate that in the importin alpha/beta and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran. Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC. We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin beta and transportin. We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC.

Active Transport, Cell Nucleus↗