RESUMO
Activation of heterotrimeric G proteins through G protein coupled receptors (GPCRs) is an ubiquitous signaling mechanism in eukaryotic biology. The three principal molecular components of this cascade are the GPCR, Gα subunit, and Gßγ subunit. Measurement of interactions between these components and their downstream effectors in live cells is paramount to understanding how cells fine tune their physiology in response to many external stimuli. Multicolor fluorescence fluctuation spectroscopy (FFS) approaches allow the sensitive detection of heteromeric interactions by using spectrally distinct fluorophores to label biomolecules of interest. We considered three imaging-FFS approaches to measuring molecular interactions from the signals produced by a spectrally resolved confocal microscopy: raster spectral image correlation spectroscopy (RSICS), spectral spatial cumulant analysis (S-SpCA), and native resolution spatial cumulant analysis (NR-SpCA). We characterized these approaches using simulation and experiments on heteromers with known stoichiometries. We found RSICS had the best sensitivity for measuring heteromeric interactions and employed it to measure G protein complexes. As measured by RSICS, interactions between G protein subunits, Gαi1 and Gß1γ2, were sensitive to stimulation of two GPCRs, the D2 dopamine receptor and the α-2A adrenergic receptor. Interactions between GPCRs and G proteins were not detectable above background, supporting a collisional model of GPCR/G protein interactions in contrast to a pre-assembly model where strong interactions would be present. These data are uniquely available by this FFS framework, which is appropriate for multiplexed measurements not only of G protein biology but for any dynamic protein complexes in the cell.
RESUMO
Suppression of glucagon hypersecretion can normalize hyperglycemia during type 1 diabetes (T1D). Activating erythropoietin-producing human hepatocellular receptor type-A4 (EphA4) on α cells reduced glucagon hypersecretion from dispersed α cells and T1D islets from both human donor and mouse models. We synthesized a high-affinity small molecule agonist for the EphA4 receptor, WCDD301, which showed robust plasma and liver microsome metabolic stability in both mouse and human preparations. In islets and dispersed islet cells from nondiabetic and T1D human donors, WCDD301 reduced glucagon secretion comparable to the natural EphA4 ligand, Ephrin-A5. In diabetic NOD and streptozotocin-treated mice, once-daily oral administration of WCDD301 formulated with a time-release excipient reduced plasma glucagon and normalized blood glucose for more than 3 months. These results suggest that targeting the α cell EphA4 receptor by sustained release of WCDD301 is a promising pharmacologic pathway for normalizing hyperglycemia in patients with T1D.
Assuntos
Diabetes Mellitus Tipo 1 , Hiperglicemia , Humanos , Animais , Camundongos , Camundongos Endogâmicos NOD , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/tratamento farmacológico , Glucagon , Receptor EphA4 , Hiperglicemia/tratamento farmacológico , Receptores da EritropoetinaRESUMO
30% of people in the United States have diabetes or pre-diabetes. Many of these individuals will develop diabetic neuropathy as a comorbidity, which is often treated with exogenous opioids like morphine, oxycodone, or tramadol. Although these opioids are effective analgesics, growing evidence indicates that they may directly impact the endocrine pancreas function in human and preclinical models. One common feature of these exogenous opioid ligands is their preference for the mu opioid receptor (MOPR), so we aimed to determine if endogenous MOPRs directly regulate pancreatic islet metabolism and hormone secretion. We show that pharmacological antagonism of MOPRs enhances glucagon secretion, but not insulin secretion, from human islets under high glucose conditions. This increased secretion is accompanied by increased cAMP signaling. mRNA expression of MOPRs is enriched in human islet α-cells, but downregulated in T2D islet donors, suggesting a link between metabolism and MOPR expression. Conditional genetic knockout of MOPRs in murine α-cells increases glucagon secretion in high glucose conditions without increasing glucagon content. Consistent with downregulation of MOPRs during metabolic disease, conditional MOPR knockout mice treated with a high fat diet show impaired glucose tolerance, increased glucagon secretion, increased insulin content, and increased islet size. Finally, we show that MOPR-mediated changes in glucagon secretion are driven, in part, by KATP channel activity. Together, these results demonstrate a direct mechanism of action for endogenous opioid regulation of endocrine pancreas.
RESUMO
The protection of the replication fork structure under stress conditions is essential for genome maintenance and cancer prevention. A key signaling pathway for fork protection involves TRPV2-mediated Ca2+ release from the ER, which is triggered after the generation of cytosolic DNA and the activation of cGAS/STING. This results in CaMKK2/AMPK activation and subsequent Exo1 phosphorylation, which prevent aberrant fork processing, thereby ensuring genome stability. However, it remains poorly understood how the TRPV2 channel is activated by the presence of cytosolic DNA. Here, through a genome-wide CRISPR-based screen, we identify TRPM8 channel-associated factor 1 (TCAF1) as a key factor promoting TRPV2-mediated Ca2+ release under replication stress or other conditions that activate cGAS/STING. Mechanistically, TCAF1 assists Ca2+ release by facilitating the dissociation of STING from TRPV2, thereby relieving TRPV2 repression. Consistent with this function, TCAF1 is required for fork protection, chromosomal stability, and cell survival after replication stress.