The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac ...response depends on β-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic upregulation of Ca2+ currents.
G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a ...broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.
Cancers represent complex autonomous systems, displaying self‐sufficiency in growth signaling. Autonomous growth is fueled by a cancer cell's ability to “secrete‐and‐sense” growth factors (GFs): a ...poorly understood phenomenon. Using an integrated computational and experimental approach, here we dissect the impact of a feedback‐coupled GTPase circuit within the secretory pathway that imparts secretion‐coupled autonomy. The circuit is assembled when the Ras‐superfamily monomeric GTPase Arf1, and the heterotrimeric GTPase Giαβγ and their corresponding GAPs and GEFs are coupled by GIV/Girdin, a protein that is known to fuel aggressive traits in diverse cancers. One forward and two key negative feedback loops within the circuit create closed‐loop control, allow the two GTPases to coregulate each other, and convert the expected switch‐like behavior of Arf1‐dependent secretion into an unexpected dose–response alignment behavior of sensing and secretion. Such behavior translates into cell survival that is self‐sustained by stimulus‐proportionate secretion. Proteomic studies and protein–protein interaction network analyses pinpoint GFs (e.g., the epidermal GF) as key stimuli for such self‐sustenance. Findings highlight how the enhanced coupling of two biological switches in cancer cells is critical for multiscale feedback control to achieve secretion‐coupled autonomy of growth factors.
Synopsis
A combined computational modeling and experimental approach is used to dissect a Golgi‐localized GTPase circuitry in eukaryotes and to understand how it empowers cancer cells to achieve self‐sufficiency in growth factor signaling.
Coupling of two classes of GTPases enables closed‐loop feedback and mutual control.
Coupling generates dose‐response alignment behavior of sensing and secretion of growth factors.
Coupling is critical for multiscale feedback control to achieve secretion‐coupled autonomy.
A combined computational modeling and experimental approach is used to dissect a Golgi‐localized GTPase circuitry in eukaryotes and to understand how it empowers cancer cells to achieve self‐sufficiency in growth factor signaling.
AT1 angiotensin receptor plays important physiological and pathophysiological roles in the cardiovascular system. Renin-angiotensin system represents a target system for drugs acting at different ...levels. The main effects of ATR1 stimulation involve activation of Gq proteins and subsequent IP3, DAG, and calcium signaling. It has become evident in recent years that besides the well-known G protein pathways, AT1R also activates a parallel signaling pathway through β-arrestins. β-arrestins were originally described as proteins that desensitize G protein-coupled receptors, but they can also mediate receptor internalization and G protein-independent signaling. AT1R is one of the most studied receptors, which was used to unravel the newly recognized β-arrestin-mediated pathways. β-arrestin-mediated signaling has become one of the most studied topics in recent years in molecular pharmacology and the modulation of these pathways of the AT1R might offer new therapeutic opportunities in the near future. In this paper, we review the recent advances in the field of β-arrestin signaling of the AT1R, emphasizing its role in cardiovascular regulation and heart failure.
DHHC proteins catalyze the reversible S-acylation of proteins at cysteine residues, a modification important for regulating protein localization, stability, and activity. However, little is known ...about the kinetic mechanism of DHHC proteins. A high-performance liquid chromatography (HPLC), fluorescent peptide-based assay for protein S-acylation activity was developed to characterize mammalian DHHC2 and DHHC3. Time courses and substrate saturation curves allowed the determination of Vmax and Km values for both the peptide N-myristoylated-GCG and palmitoyl-coenzyme A. DHHC proteins acylate themselves upon incubation with palmitoyl-CoA, which is hypothesized to reflect a transient acyl enzyme transfer intermediate. Single turnover assays with DHHC2 and DHHC3 demonstrated that a radiolabeled acyl group on the enzyme transferred to the protein substrate, consistent with a two-step ping-pong mechanism. Enzyme autoacylation and acyltransfer to substrate displayed the same acyl-CoA specificities, further supporting a two-step mechanism. Interestingly, DHHC2 efficiently transferred acyl chains 14 carbons and longer, whereas DHHC3 activity was greatly reduced by acyl-CoAs with chain lengths longer than 16 carbons. The rate and extent of autoacylation of DHHC3, as well as the rate of acyl chain transfer to protein substrate, were reduced with stearoyl-CoA when compared with palmitoyl-CoA. This is the first observation of lipid substrate specificity among DHHC proteins and may account for the differential S-acylation of proteins observed in cells.
DHHC protein acyltransferases catalyze palmitoylation by a poorly characterized mechanism.
The acyl group covalently attached to the enzyme is transferred to substrate. Long chain acyl-CoAs are better substrates for DHHC2 than DHHC3.
DHHC proteins share a two-step ping-pong mechanism but display different acyl-CoA substrate specificity.
Acyl-CoA analogs and mechanism-based inhibitors represent possible strategies for DHHC inhibition.
Emerging evidence implicates novel roles for small G protein GDP dissociation stimulator (smgGDS) in G protein activation and subsequent targeting to relevant subcellular compartments for effector ...regulation. Given the well-established roles of small G proteins in insulin secretion, we undertook this investigation to determine the putative roles of smgGDS in insulin secretion. Immunoblotting studies revealed that both splice variants of smgGDS are expressed in human islets, rat islets and INS-1 832/13 cells. A significant inhibition (-52%) of glucose-stimulated insulin secretion (GSIS) was observed in INS-1 832/13 cells following siRNA-mediated depletion of smgGDS. In addition, insulin secretion elicited by a membrane depolarizing concentration of KCl (via increased calcium influx), forskolin (via increased cAMP generation) or IBMX (via inhibition of phosphodiesterase) was inhibited by -49%, -27%, and -28%, respectively. Subcellular distribution studies revealed no significant alterations in the abundance of smgGDS in the cytosolic and membrane fractions during the 45-min exposure of INS-1 832/13 cells to an insulinotropic concentration of glucose. Together, we present the first evidence of expression of smgGDS in human islets, rodent islets, and clonal β-cells. We also demonstrate novel regulatory roles of these proteins in insulin secretion derived from glucose metabolic events, including calcium- and cAMP-dependent signaling steps.
The filamentous fungus
decomposes lignocellulosic biomass to generate soluble sugars as carbon sources. In this study, we investigated a role for heterotrimeric G-protein signaling in cellulose ...degradation. Loss of the Gα subunit genes
and
, the Gβ subunit genes
and
, the Gγ gene
, or the gene for downstream effector adenylyl cyclase (
) resulted in loss of detectable cellulase activity. This defect was also observed in strains expressing a constitutively active version of
-
(
). We found that GNA-1 levels are greatly reduced in Δ
-
, Δ
, and Δ
strains, likely contributing to cellulase defects in these genetic backgrounds. The observation that
Δ
strains exhibit cellulase activity, despite greatly reduced levels of GNA-1 protein, is consistent with positive control of cellulase production by GNA-3 that is manifested in the absence of
Expression patterns for five cellulase genes showed that Δ
, Δ
, and Δ
mutants produce less cellulase mRNA than the wild type, consistent with transcriptional regulation. Δ
mutants had wild-type levels of cellulase transcripts, suggesting posttranscriptional control. In contrast, results for Δ
mutants support both transcriptional and posttranscriptional control of cellulase activity by cAMP signaling. Cellulase activity defects in Δ
mutants were fully remediated by cAMP supplementation, consistent with GNA-3 operating upstream of cAMP signaling. In contrast, cAMP addition only partially corrected cellulase activity defects in Δ
and Δ
mutants, suggesting participation of GNA-1 and GNB-1 in additional cAMP-independent pathways that control cellulase activity.
Filamentous fungi are critical for the recycling of plant litter in the biosphere by degrading lignocellulosic biomass into simpler compounds for metabolism. Both saprophytic and pathogenic fungi utilize plant cell wall-degrading enzymes to liberate carbon for metabolism. Several studies have demonstrated a role for cellulase enzymes during infection of economically relevant crops by fungal pathogens. Especially in developing countries, severe plant disease means loss of entire crops, sometimes leading to starvation. In this study, we demonstrate that G-protein signaling is a key component of cellulase production. Therefore, understanding the role of G-protein signaling in the regulation of the unique metabolism of cellulose by these organisms can inform innovations in strain engineering of industrially relevant species for biofuel production and in combatting food shortages caused by plant pathogens.
Mast cells are tissue-resident immune cells that have numerous cytoplasmic granules which contain preformed pro-inflammatory mediators. Upon antigen stimulation, sensitized mast cells undergo ...profound changes to their morphology and rapidly release granule mediators by regulated exocytosis, also known as degranulation. We have previously shown that Rho GTPases regulate exocytosis, which suggests that cytoskeleton remodeling is involved in granule transport. Here, we used live-cell imaging to analyze cytoskeleton remodeling and granule transport in real-time as mast cells were antigen stimulated. We found that granule transport to the cell periphery was coordinated by de novo microtubule formation and not F-actin. Kinesore, a drug that activates the microtubule motor kinesin-1 in the absence of cargo, inhibited microtubule-granule association and significantly reduced exocytosis. Likewise, shRNA knock-down of Kif5b, the kinesin-1 heavy chain, also reduced exocytosis. Imaging showed granules accumulated in the perinuclear region after kinesore treatment or Kif5b knock-down. Complete microtubule depolymerization with nocodazole or colchicine resulted in the same effect. A biochemically enriched granule fraction showed kinesin-1 levels increase in antigen-stimulated cells, but are reduced by pre-treatment with kinesore. Kinesore had no effect on the levels of Slp3, a mast cell granule cargo adaptor, in the granule-enriched fraction which suggests that cargo adaptor recruitment to granules is independent of motor association. Taken together, these results show that granules associate with microtubules and are driven by kinesin-1 to facilitate exocytosis.
beta2 adrenergic receptor (ADRB2) agonists mainly participate in regulation of airway function through the ADRB2-G protein-adenylyl cyclase (AC) signaling pathway; however, the key genes associated ...with this pathway and the spatiotemporal changes in the expression spectrum of some of their subtypes remain unclear, resulting in an insufficient theoretical basis for formulating the dose and method of drug administration for neonates. We performed sampling at different developmental time points in rhesus monkeys, including the embryo stage, neonatal stage, and adolescence. The MiSeq platform was used for sequencing of key genes and some of their subtypes in the ADRB2 signaling pathway in lung tissues, and target gene expression was normalized and calculated according to reads per kilobase million. At different lung-developmental stages, we observed expression of phenylethanolamine N-methyltransferase (PNMT), ADRB2, AC, AKAP and EPAC subtypes (except AC8, AKAP4/5), and various phosphodiesterase (PDE) subtypes (PDE3, PDE4, PDE7, and PDE8), with persistently high expression of AC6, PDE4B, and AKAP(1/2/8/9/12/13, and EZR) maintained throughout the lung-developmental process, PNMT, ADRB2, AC(4/6), PDE4B, and AKAP(1/2/8/9/12/13, EZR, and MAP2)were highly expressed at the neonatal stage. During normal lung development in rhesus monkeys, key genes associated with ADRB2-G protein-AC signaling and some of their subtypes are almost all expressed at the neonatal stage, suggesting that this signaling pathway plays a role in this developmental stage. Additionally, AC6, PDE4B, and AKAP(1/2/8/9/12/13, and EZR) showed persistently high expression during the entire lung-developmental process, which provides a reference for the development and utilization of key gene subtypes in this pathway.
•Rac1 and Rac3 have opposite roles in radial sorting and peripheral myelination during development.•Rac3 acts via PAK1 activation, lamellipodia formation and Schwann cell differentiation to compete ...with Rac1.•Neuronal Rac3 is not necessary for axonal integrity.•Rac3 only antagonizes Rac1 action in a precise time window, with a global predominance of Rac1 in Schwann cell function.
Small Rho GTPases such as Cdc42 and Rac1 regulate peripheral myelination during development. Deletion of Rac1 in Schwann cell conditional knockout mice causes a delay in the process of radial sorting, followed by hypomyelination as well as defective PAK1 activation and high number of immature Oct6+ Schwann cells. Rac3 has been shown to have redundant, specific and even opposite functions to Rac1 depending on the cell type, age and other factors. In neuronal cells, evidence suggests that Rac3 may oppose Rac1 by disrupting PAK1-GIT1-Paxillin signaling thus preventing cell differentiation and extension of lamellipodia. Therefore, we tested if these Rho GTPases have similar or opposite functions in Schwann cells, by deleting the genes for both proteins in mice during peripheral myelination. At P30, global deletion of Rac3 alleviates the developmental defects on axonal sorting and hypomyelination that are caused by Schwann cell conditional ablation of Rac1. Moreover, Rac3 deletion also reverses the arrest of Schwann cells at the Oct6+ stage and ameliorates the defects in PAK1 phosphorylation observed in Rac1 deficient mice. This partial rescue of the phenotype declines later on with aging. Since double transgenic animals showed dysmyelination without axonal degeneration at P60, we postulate that this deterioration is not likely due to loss of Rac3 in neurons, but it seems to be a Schwann cell-specific defect in the maintenance of myelin.
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