Integrated amplification microarray system in a lateral flow cell for warfarin genotyping from saliva.

BACKGROUND: Genetic polymorphisms in the CYP2C9 and VKORC1 genes have been linked to sensitivity of the anticoagulant drug warfarin. The aim of this study is to demonstrate a method for warfarin sensitivity genotyping using gel element microarray technology in a simplified workflow from sample collection to analysis and detection. METHODS: We developed an integrated amplification microarray system combining PCR amplification, target labeling, and microarray hybridization within a single, closed-amplicon "lateral flow cell" for genotyping three single nucleotide polymorphisms (SNPs) that influence warfarin response. We combined nucleic acid extraction of saliva using the TruTip technology together with the lateral flow cell assay and with fully automated array detection and analysis. RESULTS: The analytical performance of the assay was tested using 20 genotyped human genomic DNA samples and found to be sensitive down to 330 input genomic copies (1 ng). A follow-up pre-clinical evaluation was performed with 65 blinded saliva samples and the genotyping results were in agreement with those determined by bidirectional sequencing. CONCLUSIONS: Combined with the use of non-invasive saliva samples, rapid DNA extraction, the lateral flow cell, automatic imaging and data analysis provides a simple and fast sample-to-answer microarray test for warfarin sensitivity genotyping.

Insight into the mechanism of dopamine D1-like receptor activation. Evidence for a molecular interplay between the third extracellular loop and the cytoplasmic tail.

A chimeric D1A dopaminergic receptor harboring the cytoplasmic tail (CT) of the D1B subtype (D1A-CTB) has been used previously to show that CT imparts high dopamine (DA) affinity and constitutive activity to the D1B receptors. However, the D1A-CTB chimera, unlike the D1B subtype, exhibits a significantly lower DA potency for stimulating adenylyl cyclase and a drastically lower maximal binding capacity (Bmax). Here, using a functional complementation of chimeric D1-like receptors, we have identified the human D1B receptor regions regulating the intramolecular relationships that lead to an increased DA potency and contribute to Bmax. We demonstrate that the addition of variant residues of the third extracellular loop (EL3) of the human D1B receptor into D1A-CTB chimera leads to a constitutively active mutant receptor displaying an increased DA affinity, potency, and Bmax. These results strongly suggest that constitutively active D1-like receptors can adopt multiple active conformations, notably one that confers increased DA affinity with decreased DA potency and Bmax and another that imparts increased DA affinity with a strikingly increased DA potency and Bmax. Overall, we show that a novel molecular interplay between EL3 and CT regulates multiple active conformations of D1-like receptors and may have potential implications for other G protein-coupled receptor classes.

Homologous regulation of the heptahelical D1A receptor responsiveness: specific cytoplasmic tail regions mediate dopamine-induced phosphorylation, desensitization and endocytosis.

In the present study, we investigate the role of specific cytoplasmic tail (CT) regions of the D1A receptor in mediating dopamine (DA)-induced phosphorylation, desensitization and endocytosis. Results obtained in human embryonic kidney (HEK) cells expressing the wild-type (WT) or truncation forms (Delta425, Delta379 and Delta351) of the D1A receptor show that sequences located downstream of Gly379 regulate DA-mediated phosphorylation-dependent desensitization of D1A receptors. However, the longer truncation mutant Delta351 failed to undergo detectable DA-induced phosphorylation while exhibiting DA-induced desensitization features similar to the shorter truncation mutant Delta379. These data potentially suggest a novel role for a receptor phosphorylation-independent process in the DA-promoted D1A subtype desensitization. Our immunofluorescence data also suggest that sequences located between Cys351 and Gly379 play an important role in DA-mediated receptor endocytosis. Additionally, time-course studies were done in intact cells expressing WT or truncation receptors to measure the observed rate constant for adenylyl cyclase (AC) activation or k(obs), a parameter linked to the receptor-G protein coupling status. In agreement with the desensitization data, Delta425- and Delta379-expressing cells exhibit an increase of kobs in comparison with WT-expressing cells. Nevertheless, Delta351-expressing cells, which harbor similar desensitization features of Delta379-expressing cells, display no change in k(obs) when compared with WT-expressing cells. Our results suggest that a defective DA-induced endocytosis may hamper Delta351 resensitization and concomitant increase in k(obs). Thus, our study showing that specific D1A receptor CT sequences regulate DA-induced phosphorylation, desensitization, and endocytosis highlights the underlying molecular complexity of signaling at dopaminergic synapses.