The P2Y(2) receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides. R272 is also directly involved in binding of dinucleotides, which behaved as partial agonists. Y118A (3.37) mutation led to dramatically reduced efficacy of agonists; it is part of the entry channel as well as the triphosphate binding site. While the Y114A (3.33) mutation did not have any effect on agonist activities, the antagonist Reactive Blue 2 (6) was completely inactive at that mutant. The disulfide bridge Cys25-Cys278 was found to be important for agonist potency but neither for agonist efficacy nor for antagonist potency.

No abstract

It is well known that clonal cells can make different fate decisions, but it is unclear whether these decisions are determined during, or before, a cell's own lifetime. Here, we engineered an endogenous fluorescent reporter for the pluripotency factor OCT4 to study the timing of differentiation decisions in human embryonic stem cells. By tracking single‐cell OCT4 levels over multiple cell cycle generations, we found that the decision to differentiate is largely determined before the differentiation stimulus is presented and can be predicted by a cell's preexisting OCT4 signaling patterns. We further quantified how maternal OCT4 levels were transmitted to, and distributed between, daughter cells. As mother cells underwent division, newly established OCT4 levels in daughter cells rapidly became more predictive of final OCT4 expression status. These results imply that the choice between developmental cell fates can be largely predetermined at the time of cell birth through inheritance of a pluripotency factor.

Eight human G protein-coupled P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(12), P2Y(13), and P2Y(14)) that respond to extracellular nucleotides have been molecularly identified and characterized. P2Y receptors are widely expressed in epithelial cells and play an important role in regulating epithelial cell function. Functional studies assessing the capacity of various nucleotides to promote increases in short-circuit current (I(sc)) or Ca(2+) mobilization have suggested that some subtypes of P2Y receptors are polarized with respect to their functional activity, although these results often have been contradictory. To investigate the polarized expression of the family of P2Y receptors, we determined the localization of the entire P2Y family after expression in Madin-Darby canine kidney (MDCK) type II cells. Confocal microscopy of polarized monolayers revealed that P2Y(1), P2Y(11), P2Y(12), and P2Y(14) receptors reside at the basolateral membrane, P2Y(2), P2Y(4), and P2Y(6) receptors are expressed at the apical membrane, and the P2Y(13) receptor is unsorted. Biotinylation studies and I(sc) measurements in response to the appropriate agonists were consistent with the polarized expression observed in confocal microscopy. Expression of the G(q)-coupled P2Y receptors (P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(11)) in lung and colonic epithelial cells (16HBE14o- and Caco-2 cells, respectively) revealed a targeting profile nearly identical to that observed in MDCK cells, suggesting that polarized targeting of these P2Y receptor subtypes is not a function of the type of epithelial cell in which they are expressed. These experiments highlight the highly polarized expression of P2Y receptors in epithelial cells.