Biology subjects

Pietraszewska-Bogiel, A.

Publications and source records attributed to Pietraszewska-Bogiel, A..

3 recordsLinked to original sources

bioRxiv2019

Seeing β-arrestin in action: The role of β-arrestins in Histamine 1 Receptor signaling

{beta}-arrestins regulate G protein-coupled receptor functions by influencing their signaling activity and intracellular location. Histamine is a major chemical mediator of allergic reactions, and its action is mainly mediated by the Gq/11- and Gi-coupled H1R. Contrary to accumulating insights into G protein-mediated signaling downstream of H1R, very little is known about the function of {beta}-arrestins in H1R signaling. Here, we describe dynamic, live cell measurements of {beta}-arrestin recruitment upon H1R activation in HEK293TN cells. Our observations classify H1R as a class A receptor, undergoing transient interactions with {beta}-arrestin. To investigate the relative contributions of G proteins and {beta}-arrestins to H1R signaling, we use specific G protein inhibitors, as well as {beta}-arrestin overexpression and depletion, and quantify various signaling outcomes in a panel of dynamic, live cell biosensor assays. Overall, we link {beta}-arrestins to desensitization of H1R-mediated signaling and show that ERK activation downstream of endogenous (HeLa, HUVEC) or transiently expressed (HEK293TN) H1R is largely Gq-mediated.

molecular biology↗

bioRxiv2019

Not so dry after all. DRY mutants of the AT1A receptor and H1 receptor can induce G protein-dependent signaling

GPCRs are seven transmembrane spanning receptors that regulate a wide array of intracellular signaling cascades in response to various stimuli. To do so, they couple to different heterotrimeric G proteins and adaptor proteins, including arrestins. Importantly, arrestins were shown to regulate GPCR signaling through G proteins, as well as promote G protein-independent signaling events. Several research groups have reported successful isolation of exclusively G protein-dependent and arrestin-dependent signaling downstream of GPCR activation using biased agonists or receptor mutants incapable of coupling to either arrestins or G proteins. In the latter category, the DRY mutant of the angiotensin II type 1 receptor was extensively used to characterize functional selectivity downstream of AT1AR. In an attempt to understand histamine 1 receptor signaling, we characterized the signaling capacity of the H1R DRY mutant in a panel of dynamic, live cell biosensor assays, including arrestin recruitment, heterotrimeric G-protein activation, Ca2+ signaling, protein kinase C activity, GTP binding of RhoA, and activation of ERK1/2. Here we show that both H1R DRY mutant and the AT1AR DRY mutant (used as a reference) are capable of efficient activation of G protein-mediated signaling. Therefore, contrary to common belief, they do not constitute suitable tools for dissection of arrestin-mediated, G protein-independent signaling downstream of these receptors.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC=\"FIGDIR/small/773044v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (27K):\norg.highwire.dtl.DTLVardef@3b068eorg.highwire.dtl.DTLVardef@12b6a4borg.highwire.dtl.DTLVardef@14f66a1org.highwire.dtl.DTLVardef@1dad4b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2019

Seeing cells smell: Dynamic optical measurements of Ca2+ and cAMP signaling from Olfactory Receptors transiently expressed in HEK293TN cells

Olfactory receptors (ORs) constitute the largest family of G-protein coupled receptors. They are responsible for the perception of odor (olfaction) and also play important roles in other biological processes, including regulation of cell proliferation. Their increasing diagnostic and therapeutic potential, especially for cancer research, requests the ongoing development of methodologies that would allow their robust functional expression in non-olfactory cells, and dynamic analysis of their signaling pathways. To enable realtime detection of OR activity, we use single cell imaging with genetically encoded fluorescent biosensors, Yellow Cameleon or EPAC, which are routinely used for kinetic measurements of Ca2+ or cAMP signaling downstream of various G-protein coupled receptors. We demonstrate that the co-expression of Lucy-Rho tagged variants of ORs together with an accessory protein, RTP1s, in HEK293TN cells is sufficient to detect the activity of a panel of ORs. Using this methodology, we were able to detect both Ca2+ and cAMP signaling downstream of twelve ORs within 2 minutes from the application of odorant.

molecular biology↗