Absence Of Receptor Activation Research Articles

Molecular mechanism of Fast Endophilin-Mediated Endocytosis.

Endocytosis mediates the cellular uptake of micronutrients and cell surface proteins. Clathrin-mediated endocytosis (CME) is the housekeeping pathway in resting cells but additional Clathrin-independent endocytic (CIE) routes, including Fast Endophilin-Mediated Endocytosis (FEME), internalize specific cargoes and support diverse cellular functions. FEME is part of the Dynamin-dependent subgroup of CIE pathways. Here, we review our current understanding of the molecular mechanism of FEME. Key steps are: (i) priming, (ii) cargo selection, (iii) membrane curvature and carrier formation, (iv) membrane scission and (v) cytosolic transport. All steps are controlled by regulatory mechanisms mediated by phosphoinositides and by kinases such as Src, LRRK2, Cdk5 and GSK3β. A key feature of FEME is that it is not constitutively active but triggered upon the stimulation of selected cell surface receptors by their ligands. In resting cells, there is a priming cycle that concentrates Endophilin into clusters on discrete locations of the plasma membrane. In the absence of receptor activation, the patches quickly abort and new cycles are initiated nearby, constantly priming the plasma membrane for FEME. Upon activation, receptors are swiftly sorted into pre-existing Endophilin clusters, which then bud to form FEME carriers within 10 s. We summarize the hallmarks of FEME and the techniques and assays required to identify it. Next, we review similarities and differences with other CIE pathways and proposed cargoes that may use FEME to enter cells. Finally, we submit pending questions and future milestones and discuss the exciting perspectives that targeting FEME may boost treatments against cancer and neurodegenerative diseases.

Extracellular vesicle–mediated transfer of constitutively active MyD88L265P engages MyD88wt and activates signaling

AbstractThe link between inflammation and cancer is particularly strong in Waldenström macroglobulinemia (WM), a diffuse large B-cell lymphoma wherein the majority of patients harbor a constitutively active mutation in the innate immune-signaling adaptor myeloid differentiation primary response 88 (MyD88). MyD88Leu265Pro (MyD88L265P) constitutively triggers the myddosome assembly providing a survival signal for cancer cells. Here, we report detection and a functional role of MyD88 in the extracellular vesicles (EVs) shed from WM cells. MyD88L265P was transferred via EVs into the cytoplasm of the recipient mast cells and macrophages, recruiting the endogenous MyD88 that triggered the activation of proinflammatory signaling in the absence of receptor activation. Additionally, internalization of EVs containing MyD88L265P was observed in mice with an effect on the bone marrow microenvironment. MyD88-loaded EVs were detected in the bone marrow aspirates of WM patients thus establishing the physiological role of EVs for MyD88L265P transmission and shaping of the proinflammatory microenvironment. Results establish the mechanism of transmission of signaling complexes via EVs to propagate inflammation as a new mechanism of intercellular communication.

Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins

G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function.

Modulating Calcium Entry into Microvascular Endothelium by Controlling Membrane Potential During Submaximal Muscarinic Receptor Activation

Vascular smooth muscle cell relaxation and vasodilation are coupled to a rise in endothelial cell [Ca2+]i and hyperpolarization. However, the role of membrane potential (Vm) in regulating endothelial cell [Ca2+]i remains controversial. To test the hypothesis that Vm can govern Ca2+ entry into endothelial cells in accord with its electrochemical gradient, we evaluated [Ca2+]i using Fura-2 photometry (F340/F380) while controlling Vm using intracellular current injection. Intact endothelial tubes (width: 60 μm, length: ≤ 1 mm) were freshly isolated from superior epigastric arteries of C57BL/6 mice (n≥7; 32°C, pH 7.4). Endothelial cells were electrically coupled to each other through gap junctions and lacked inherent voltage-sensitive ion channel activity as indicated by linearity (R2 = 0.990±0.002) of the current-voltage relationship throughout the Vm range of ≈-80 mV to +10 mV during current injection (±0.5 to 5 nA). In the absence of receptor activation, Vm was −37±1 mV and [Ca2+]i did not change with hyperpolarization or depolarization. In contrast, during submaximal activation of muscarinic (Gq protein-coupled) receptors (GPCRs) with acetylcholine (100 nM; ∼EC50), [Ca2+]i increased with hyperpolarization to −60±1 mV and decreased with depolarization to −28±2 mV. The effect of membrane hyperpolarization on [Ca2+]i was abolished following removal of [Ca2+]o (control [Ca2+]o = 2 mM) and reduced by half in endothelium isolated from TRPV4-/- mice during Vm > −50 mV. Thus, submaximal stimulation of GPCRs enables physiological changes in Vm to modulate Ca entry through voltage-insensitive ion channels in the plasma membrane of native microvascular endothelial cells. (Support: NIH grants R37-HL041026, 5T32-AR048523, 1K99-AG047198)

New insights into the model of dopamine D1 receptor and G-proteins interactions

The details of the interaction between G-proteins and the GPCRs have been subjected to extensive investigation with structural and functional assays, but still many fundamental questions regarding this macromolecular assembly and its mechanism remain unanswered. In the context of current structural data we investigated interactions of dopamine D1 receptor with cognate G-proteins (Gαs) in living cells, emphasizing the prevalence of preassembled D1-G-protein complexes. We also tested the effect of D1 receptor presence on the dynamics of Gαs and Gαi3 in the cellular plasma membrane. Using fluorescence resonance energy transfer (FRET) detected by fluorescence lifetime imaging microscopy (FLIM) or fluorescence recovery after photobleaching (FRAP) microscopy, we did not detect constitutive preassociated complex between D1 receptor and G-protein in the absence of receptor activation. Our work suggests that D1 receptor alters the distribution of Gαs and Gαi3 subunits inside the membrane. We also find that non-activated D1 receptor and Gαs or Gαi3 are present in the cell membrane within the same membrane microdomains in the proximity of about 9–10nm.

HINT1 protein cooperates with cannabinoid 1 receptor to negatively regulate glutamate NMDA receptor activity

BackgroundG protein-coupled receptors (GPCRs) are the targets of a large number of drugs currently in therapeutic use. Likewise, the glutamate ionotropic N-methyl-D-aspartate receptor (NMDAR) has been implicated in certain neurological disorders, such as neurodegeration, neuropathic pain and mood disorders, as well as psychosis and schizophrenia. Thus, there is now an important need to characterize the interactions between GPCRs and NMDARs. Indeed, these interactions can produce distinct effects, and whereas the activation of Mu-opioid receptor (MOR) increases the calcium fluxes associated to NMDARs, that of type 1 cannabinoid receptor (CNR1) antagonizes their permeation. Notably, a series of proteins interact with these receptors affecting their responses and interactions, and then emerge as novel therapeutic targets for the aforementioned pathologies.ResultsWe found that in the presence of GPCRs, the HINT1 protein influences the activity of NMDARs, whereby NMDAR activation was enhanced in CNR1+/+/HINT1-/- cortical neurons and the cannabinoid agonist WIN55,212-2 provided these cells with no protection against a NMDA insult. NMDAR activity was normalized in these cells by the lentiviral expression of HINT1, which also restored the neuroprotection mediated by cannabinoids. NMDAR activity was also enhanced in CNR1-/-/HINT1+/+ neurons, although this activity was dampened by the expression of GPCRs like the MOR, CNR1 or serotonin 1A (5HT1AR).ConclusionsThe HINT1 protein plays an essential role in the GPCR-NMDAR connection. In the absence of receptor activation, GPCRs collaborate with HINT1 proteins to negatively control NMDAR activity. When activated, most GPCRs release the control of HINT1 and NMDAR responsiveness is enhanced. However, cannabinoids that act through CNR1 maintain the negative control of HINT1 on NMDAR function and their protection against glutamate excitotoxic insult persists.

Site-specific monoubiquitination activates Ras by impeding GTPase-activating protein function

SUMMARYCell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K–Ras to promote tumor growth. To determine the mechanism of human Ras activation we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling, and biochemical activity measurements. We established that monoubiquitination has little effect on Ras GTP binding, GTP hydrolysis, or exchange factor activation, but severely abrogates the response to GTPase activating proteins in a site–specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation.

Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells

Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the alpha2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits alpha o, beta1, and gamma2 and a number of GPCRs including M4, alpha2A, the adenosine A1 receptor, and the dopamine D2 receptor under resting conditions. The interaction between GPCRs and Galpha proteins shows specificity: there is interaction between the alpha2A receptor and Go, but little interaction between the alpha2A receptor and Gs. In contrast, the predominantly Gs-coupled prostacyclin receptor interacted with Gs, but there was little interaction between the prostacyclin receptor and Go. Inverse agonists did not change the FRET ratio, whereas the addition of agonist resulted in a modest fall. Our work suggests that GPCR dimers and the G protein heterotrimer are present at the cell membrane in the resting state in a pentameric complex.

A New Functional Role of the Fibrinogen RGD Motif as the Molecular Switch That Selectively Triggers Integrin αIIbβ3-dependent RhoA Activation during Cell Spreading

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2.

Expression of FKBP12 and ryanodine receptors (RyRs) in the spinal cord of MND patients

We investigated the FKBP12 and ryanodine receptor (RyR) immunoreactivity (IR) in the spinal cords of neurological controls and patients with motor neuron disease (MND). In the neurological controls, the cytoplasm of the spinal anterior horn neurons was stained with anti-FKBP12 antibodies and anti-RyR (type 1 and type 2) antibodies. In the MND cases, the residual neurons in the anterior horn of the spinal cord showed IR for RyR (type 1 and 2) antibodies, while weak IR for anti-FKBP12 antibodies was comparable to that of controls. The numbers of neurons recognized with the anti-FKBP 12 or anti-RyR (type 1 and 2) antibodies were counted in the anterior horn of spinal cords from the MND cases and neurological controls. Frequency of neurons stained with anti-FKBP 12 antibody was significantly decreased in the MND cases compared to that in controls (48.7+/-23.2%, 71.0+/-18.5%, respectively, mean+/-SD, p<0.0005). In the MND cases, numbers of normal-appearing, chromatolytic neurons showing IR to anti-FKBP12 (N19) antibody were significantly decreased compared to those in the controls. Immunoreactivities to anti-RyR antibodies (type 1and 2) in MND cases were present and there was no difference compared to those of the controls. Neurons in the spinal cord anterior horn of Kii-ALS cases with prolonged clinical duration were immunostained with both anti-FKBP12 and anti-RyR (type 1 and 2) antibodies similar to that in the controls. The anterior horn neurons of MND cases of short clinical duration showed absent IR to FKBP 12 antibody but present IR to RyR (type 1 and 2) antibodies. The present result suggests that FKBP12 IR was decreased in the MND cases with short clinical duration. RyR (type 1 and 2) is a major component of the intracellular calcium channel, which mediates calcium-induced calcium release. FKBP12, which is an endogenous ligand for RyR, stabilizes the calcium channels preventing calcium leakage in the absence of receptor activation. Imbalance between FKBP12 and RyR IR may play an important role in degeneration due to MND. Further study of the correlation between RyR and FKBP12 should contribute to clarifying the mechanisms of neurodegeneration in MND, including calcium-induced neuronal loss.

Dual Function for a Unique Site within the β2I Domain of Integrin αMβ2

Integrin activation has been postulated to occur in part via conformational changes in the I domain of the beta subunit (the betaI domain), especially near the F-alpha(7) loop, in response to "inside-out" signaling. However, direct evidence for a role of the F-alpha(7) loop in ligand binding and activity modulation is still lacking. Here, we report our finding that the F-alpha(7) loop (residues 344-358) within the beta(2)I domain has dual functions in ligand binding by alpha(M)beta(2). On the one hand, it supports intercellular adhesion molecule 1 (ICAM-1) binding to alpha(M)beta(2) directly as part of a recognition interface formed by five noncontiguous segments (Pro(192)-Glu(197), Asn(213)-Glu(220), Leu(225)-Leu(230), Ser(324)-Thr(329), and Glu(344)-Asp(348)) on the apex of the beta(2)I domain. On the other hand, it controls the open and closed conformation of the alpha(M)beta(2) receptor, thereby indirectly affecting alpha(M)beta(2) binding to other ligands. Switching the five constituent sequences of the ICAM-1-binding site within the beta(2)I domain to their beta(1) counterparts destroyed ICAM-1 binding but had no effect on the gross conformations of the receptor. Of the five ICAM-1 binding-defective mutants, four had normal or even stronger interaction with Fg and C3bi, as reported in our previous study. Synthetic peptides derived from the identified site inhibited alpha(M)beta(2)-ICAM-1 interaction and supported direct binding to ICAM-1. Most importantly, perturbation of the F-alpha(7) loop caused conformational changes within the beta(2)I domain, which was further propagated to other regions of alpha(M)beta(2). Altogether, our data demonstrate that inside-out signaling could modulate ligand binding directly by changing the ligand-binding pocket per se and/or indirectly by inducing multiple conformational changes within the receptor.

Helix I of β-Arrestin Is Involved in Postendocytic Trafficking but Is Not Required for Membrane Translocation, Receptor Binding, and Internalization

beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT(1)R), to promote receptor desensitization and internalization. The AT(1) R is a class B GPCR that recruits both beta-arrestin1 and beta-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. beta-Arrestins contain one amphipathic and potentially amphitropic (membrane-targeting) alpha-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of beta-arrestin1 and beta-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type beta-arrestins. After angiotensin II stimulation, both wild-type and beta-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all beta-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type beta-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not amphitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and beta-arrestin mutants retained a capacity to interact with the AT(1)R, although the interaction with the mutants was less stable. Finally, wild-type and mutant beta-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in beta-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly amphitropic.

Participation of RGS8 in the ternary complex of agonist, receptor and G-protein

The RGS (regulators of G-protein signalling) protein family sharpen signalling kinetics through heterotrimeric G-proteins by enhancing the GTPase activity of the G-protein alpha subunit. Paradoxically, they also accelerate receptor-stimulated activation. We investigated this paradox using the cloned G-protein gated K(+) channel as a reporter of the G-protein cycle, and FRET (fluorescence resonance energy transfer) between cyan and yellow fluorescent protein tagged proteins to detect physical interactions. Our results with the neuronal protein, RGS8, show that the enhancement of activation kinetics is a variable phenomenon determined by receptor type, G-protein isoform and RGS8 expression levels. In contrast, deactivation was consistently accelerated after removal of agonist. FRET microscopy revealed a stable physical interaction between RGS8-yellow fluorescent protein and G(o) alpha(A)-cyan fluorescent protein that occurred in the presence and absence of receptor activation and was not competed away by Gbetagamma overexpression. FRET was also seen between RGS8 and Ggamma, demonstrating that RGS8 binds to the heterotrimeric G-protein as well as G-protein alpha subunit-GTP and the transition complex. We propose a novel model for the action of RGS proteins on the G-protein cycle involving participation of the RGS in the ternary complex: for certain combinations of agonist, receptor and G-protein, RGS8 expression improves upon the 'kinetic efficacy' of G-protein activation.

In Situ Respiration and Bioenergetic Status of Mitochondria in Primary Cerebellar Granule Neuronal Cultures Exposed Continuously to Glutamate

Mitochondria play a central role in neuronal death during pathological exposure to glutamate (excitotoxicity). To investigate the detailed bioenergetics of the in situ mitochondria, a method is described to monitor continuously the respiration of primary cerebellar granule neuron cultures while simultaneously imaging cytoplasmic Ca(2+) and mitochondrial membrane potential. Coverslip-attached cells were perfused in an imaging chamber with upstream and downstream flow-through oxygen electrodes. The bioenergetic consequences of chronic glutamate exposure were investigated, including ATP supply and demand, proton leak, and mitochondrial respiratory capacity during chronic glutamate exposure. In 25 mM K(+) medium supplemented with 10% dialyzed serum, cells utilized 54% of their respiratory capacity in the absence of receptor activation (37% for ATP generation, 12% to drive the mitochondrial proton leak, and the residual 5% was nonmitochondrial). glutamate initially increased mitochondrial respiration from 51 to 68% of capacity, followed by a slow decline. It was estimated that 85% of this increased respiration was because of increased ATP demand, whereas 15% was attributable to a transient mitochondrial proton leak. N-Methyl-D-aspartate receptor activation was only responsible for 62% of the increased respiration. When adjusted for cell death over 3 h of glutamate exposure, respiration of the viable cells remained near basal and protonophore stimulated respiration to the same extent as control cells. Pyruvate-supplemented media protected cells from glutamate excitotoxicity, although this was associated with mitochondrial dysfunction. We conclude that excitotoxicity under these conditions is not because of an ATP deficit or uncoupling. Furthermore, mitochondria maintain the same respiratory capacity as in control cells.

Heparan Sulfate Proteoglycans Function as Receptors for Fibroblast Growth Factor-2 Activation of Extracellular Signal–Regulated Kinases 1 and 2

Fibroblast growth factor-2 (FGF2) activates the extracellular signal-regulated kinases 1 and 2 (ERK1/2) through its specific receptors. Interaction of FGF2 with cell-surface heparan sulfate proteoglycans has also been suggested to induce intracellular signals. Thus, we investigated whether FGF2 can stimulate ERK1/2 activation through heparan sulfate proteoglycans using mechanisms that do not depend on receptor activation in vascular smooth muscle cells. The activation of FGF receptors was inhibited by treating cells with 5'-deoxy-5'methyl-thioadenosine and by expressing truncated dominant-negative FGF receptors. In both cases, FGF2 was able to stimulate the phosphorylation of ERK1/2 despite the absence of detectable FGF receptor tyrosine kinase activity. The FGF2 activation of ERK1/2 in the absence of receptor activity was completely dependent on heparan sulfate, because this activity was abolished by heparinase III digestion of the cells. In contrast, heparinase III treatment of control cells, with functional FGF receptors, showed only slight changes in FGF2-mediated ERK1/2 activation kinetics. Thus, in addition to serving as coreceptors for FGF receptor activation, heparan sulfate proteoglycans might also function directly as receptors for FGF2-induced ERK1/2 activation. Activation of ERK1/2 via cell-surface proteoglycans could have significant biological consequences, potentially directing cell response toward growth, migration, or differentiation.

The Metabotropic Glutamate Receptor mGluR5 Is Endocytosed by a Clathrin-independent Pathway

Metabotropic glutamate receptors 5 (mGluR5) are members of the growing group C G protein-coupled receptor family. Widely expressed in mammalian brain, they are involved in modulation of the glutamate transmission. By means of transfection of mGluR5 receptors in COS-7 cells and primary hippocampal neurons in culture followed by immunocytochemistry and quantitative image analysis and by a biochemical assay, we have studied the internalization of mGluR5 splice variants. mGluR5a and -5b were endocytosed in COS-7 cells as well as in axons and dendrites of cultured neurons. Endocytosis occurred even in the absence of receptor activity, because receptors mutated in the glutamate binding site were still internalized as well as receptors in which endogenous activity had been inhibited by an inverse agonist. We have measured a constitutive rate of endocytosis of 11.7%/min for mGluR5a. We report for the first time the endocytosis pathway of mGluR5. Internalization of mGluR5 is not mediated by clathrin-coated pits. Indeed, inhibition of this pathway by Eps15 dominant negative mutants did not disturb their endocytosis. However, the large GTPase dynamin 2 is implicated in the endocytosis of mGluR5 in COS-7. mGluR5 is the first shown member of the group C G-protein coupled receptor family internalized by a nonconventional pathway.

Swapping Smad Partners

Members of the transforming growth factor β (TGF-β) family regulate essential processes like cell proliferation, cell death, and developmental morphogenesis. The Smad proteins carry signals from activated TGF-β receptors to the nucleus where they promote transcription of target genes. This process requires a series of protein-protein interactions that, as the details emerge, appear like a highly orchestrated dance in which the regulated Smad (R-Smads) spin deftly from partner to partner on their way from the cell membrane to the nucleus. Just how the R-Smad recognizes the appropriate partners at the correct time is clarified by the work of Qin et al. In the course of activation, Smad3 interacts with the TGF-β receptor, SARA (Smad anchor for receptor activation), and the common mediator Smad (Co-Smad) Smad4. All of these interactions occur through a single domain of Smad3 (the effector or MH2 domain). Qin et al. analyzed crystal structures of monomeric and trimeric Smad3 and of a complex of the MH2 domain of Smad3 with the Smad-binding domain of SARA. The structures revealed that monomeric Smad3, which is brought to the receptor by interaction with SARA, undergoes a conformational change when phosphorylated by the receptor to form activated Smad3 trimers. This structural change disrupted interaction with SARA but enhanced interaction of Smad3 (again through the MH2 domain) with another partner, the proto-oncogene product Ski, which functions to repress transcriptional activation by Smad3. It thus appears that in the absence of receptor activation, SARA acts to suppress Smad3 activation by favoring the monomeric state of Smad3. The activating phosphorylation by the receptor then promotes formation of Smad3 trimers, which interact in the nucleus with the negative feedback component Ski and probably with other coactivators, corepressors, and regulatory molecules. Moustakas and Heldin discuss these results. B. Y. Qin, S. S. Lam, J. J. Correia, K. Lin, Smad3 allostery links TGF-β receptor kinase activation to transcriptional control. Genes Dev. 16 , 1950-1963 (2002). [Abstract] [Full Text] A. Moustakas, C-H. Heldin, From mono- to oligo-Smads: The heart of the matter in TGF-β signal transduction, Genes Dev. 16 , 1867-1871 (2002). [Full Text]

Impact of the characteristics of patients and their clinical management on outcomes in children with homozygous familial hypercholesterolemia.

To relate clinical factors to the development of cardiovascular atherosclerosis for patients with homozygous familial hypercholesterolemia. Homozygous familial hypercholesterolemia is associated with extreme elevations in levels of cholesterol causing aggressive atherosclerosis. We reviewed 10 children, 8 of whom were male, assessed at a single institution. We found that individual characteristics, levels of lipid, cardiovascular investigations, and management were related to the activity of low density lipoprotein receptors. Activity of low density lipoprotein receptors was defined as absent, being less than 2% of normal, in 4 patients who presented at the ages of 0.3, 1.4, 1.8, and 4.5 years, respectively. The activity was minimal, representing 5%-30% of normal, in another 4 patients presenting at the ages of 6.1, 9.6, 9.9, and 12 years, and was undetermined in 2 patients who presented at the ages of 3.5, and 12.1 years. Levels of low density lipoprotein cholesterol at presentation ranged from 12.2 to 24 millimoles per litre. Plasmapheresis was performed bi-weekly in 9 patients. Patients with absence of receptor activity were less likely to have a serial decrease in the levels of low density lipoprotein cholesterol prior to plasmapheresis, and one of these patients was increased to weekly plasmapheresis. In addition, they had more aggressive cardiovascular involvement of the coronary arteries, aortic valve and aorta, requiring surgical intervention at the age of 8 and 12 years in 2 patients, with sudden death at the age of 3.1 years in one patient. In contrast, 1 patient with minimal receptor activity had surgical intervention at the age of 16.6 years and another patient died suddenly at the age of 33.6 years. Complete cardiac assessment is recommended at presentation. The frequency of plasmapheresis should be adjusted according to the activity of low density lipoprotein receptors and the individual response of the patient.

AGS3 Frees βγ Subunits

G proteins are heterotrimeric proteins that are activated when guanosine triphosphate (GTP) binds the α subunit, promoting the dissociation of the complex into a GTP-bound α subunit and a free βγ subunit, each of which can regulate downstream signaling events. Extracellular signals regulate this process through interaction with G protein-coupled receptors in the plasma membrane. However, intracellular regulators of G protein signaling are also being identified. Bernard et al. characterized biochemically how activator of G protein signaling 3 (AGS3) is able to enhance G protein signaling in the absence of receptor activation. In vitro binding assays showed that AGS3 competes with the βγ subunit for binding to the inactive, guanosine diphosphate (GDP)-bound state of Gα i . AGS3 was able to interact with multiple α subunits simultaneously; however, AGS3 was selective for binding Gα i1-3 and exhibited limited or undetectable binding to Gα s , Gα q , or Gα 0 . AGS3 inhibited binding of GTPγS to the a subunit suggesting that AGS3 may stabilize the GDP-bound or nucleotide-free form of the α subunit. This effect on the α subunit along with the ability to compete for the binding of βγ could increase the available pool of free βγ subunits that can activate downstream signaling pathways. M. L. Bernard, Y. K. Peterson, P. Chung, J. Jourdan, S. M. Lanier, Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins. J. Biol. Chem. 276 , 1585-1593 (2001). [Abstract] [Full Text]

Regulators of G Protein Signaling (RGS) Proteins Constitutively Activate Gβγ-gated Potassium Channels

Here we report novel effects of regulators of G protein signaling (RGS) on G protein-regulated ion channels. RGS3 and RGS4 induced a substantial increase in currents through the Gbeta gamma-regulated inwardly rectifying K+ channels, IK(ACh), in the absence of receptor activation. Concomitantly, the amount of current that could be activated by agonist was reduced. Pretreatment with pertussis toxin or a muscarinic receptor antagonist abolished agonist-induced currents but did not modify RGS effects. Cotransfection of cells with a Gbetagamma-binding protein significantly reduced the RGS4-induced basal IK(ACh) currents. The RGS proteins also modified the properties of another Gbeta gamma effector, the N-type Ca2+ channels. These observations strongly suggest that RGS proteins increase the availability of Gbeta gamma in addition to their previously described GTPase-activating function.