Other important clues with regards to molecules rele vant for Epo induced signaling have come from disrup tions of genes for Jak2, SHP2, PLC, STAT5a b, and GATA 1 and 2. Even though these knockout studies have supplied considerable insight into essential players in Epo induced signaling, mice are usually not a perfect method for extensive biochemical analyses simply because the number of erythroid progenitors that may be readily obtained from them is not enough. Therefore, most biochemical research aiming to unravel the detailed molecular mechanisms of EpoR signaling have so far been carried out with cell lines expressing an endog enous or stably transfected EpoR. Moreover, quite a few of these experiments have been carried out with pathophysiological concentrations of Epo above 1 U ml.
In the a lot of cell lines analyzed, a plethora selleckchem of diverse sig naling molecules appears to become vital for Epo signaling. It really is obvious that most discrepancies within the crucial sign aling proteins reported reflect the genomic instability of the different cancer cell lines, too as distinct pre set wir ing diagrams of EpoR transfected hematopoietic progeni tor cells. Therefore, these findings are essential in defining candidate pathways potentially involved in vivo, but they usually do not necessarily represent actual signals induced upon Epo stimulation of main human erythroid progenitors. Consequently, despite the fact that EpoR signaling has been intensely studied, lots of of its aspects are nevertheless unknown or remain puzzling.
One example is, it is particular that Prasugrel a large complex of signaling proteins is assembled on the EpoR upon Epo stimulation of different Epo responsive cell lines also as in vivo, and it is actually also known that quite a few compo nents of this complicated straight associate with phosphor ylated tyrosines of the EpoR. On the other hand, these tyrosines could be spatially separated in the Jak2 binding web-site around the EpoR or even abolished without apparent serious disruption of EpoR function. Phosphatidylinositol three kinases have been implicated in EpoR signaling by precipitation experi ments which focussed around the formation of complexes between the p85 p110 PI3Ks plus the EpoR and by the usage of inhibitors like LY294002 and wortmannin, which are mechanis tically and structurally distinct but lack prominent selectivity for certain class I PI3K loved ones members. The outcomes from these experiments haven’t yet provided clear evidence of an critical function for p85 p110 PI3Ks in the activation of MEKs and Erks upon stimulation of cells with Epo.
Only recently, caffeine has been described as an inhibitor of PI3K activity having a clear preference for cer tain PI3K loved ones members in vitro. In mixture with isoform certain PI3K activity assays, this now makes it possible for one to look at distinct PI3K proteins a lot more closely. For many years it was tough to carry out in depth bio chemical studies with PEPs due to the fact solutions for their effi cient in vitro expansion had been very limited.