Guanine Nucleotide-binding Proteins Function Research Articles

Fluorescence Intensity-Based eIF2B's Guanine Nucleotide-Exchange Factor Activity Assay.

Guanine nucleotide-exchange factors (GEFs) activate the function of guanine nucleotide-binding proteins (G-proteins) by promoting the exchange of GDP for GTP on the latter. Here, we describe a protocol for in vitro measurements of the GEF activity of eukaryotic translation initiation factor 2B, eIF2B, toward its substrate eIF2. This protocol provides a relatively simple method for determining the eIF2B's GEF activity in crude cell extracts. The eIF2 heterotrimeric substrate, with phosphorylated or unphosphorylated eIF2α, is prepared by immunoprecipitation, following subsequent loading of a fluorescent BODIPY-FL dye-attached GDP. The exchange of the bound fluorescent GDP molecule for an unlabeled one on eIF2 promoted by eIF2B is monitored kinetically using a fluorescence microplate reader.

ArfGAP1 function in COPI mediated membrane traffic: currently debated models and comparison to other coat-binding ArfGAPs.

The ArfGAPs are a family of proteins containing an ArfGAP catalytic domain that induces the hydrolysis of GTP bound to the small guanine nucleotide binding-protein ADP-ribosylation factor (Arf). Functional models for Arfs, which are regulators of membrane traffic, are based on the idea that guanine nucleotide-binding proteins function as switches: Arf with GTP bound is active and binds to effector proteins; the conversion of GTP to GDP inactivates Arf. The cellular activities of ArfGAPs have been examined primarily as regulatory proteins that inactivate Arf; however, Arf function in membrane traffic does not strictly adhere to the concept of a simple switch, adding complexity to models explaining the role of ArfGAPs. Here, we review the literature addressing the function Arf and ArfGAP1 in COPI mediated transport, focusing on two critical and integrated functions of membrane traffic, cargo sorting and vesicle coat polymerization. We briefly discuss other ArfGAPs that may have similar function in Arf-dependent membrane traffic outside the ER-Golgi.

Characterization of a GDP Dissociation Inhibitory Region of ADP-ribosylation Factor Domain Protein ARD1

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and later recognized as critical components in intracellular vesicular transport and phospholipase D activation. ARF domain protein 1 (ARD1) is a member of the ARF family that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We previously reported that this extension acts as a GTPase-activating protein for the ARF domain of ARD1 (Vitale, N., Moss, J., and Vaughan, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1941-1944). Both GTP binding and GTP hydrolysis are necessary for physiological function of guanine nucleotide-binding proteins, and the rates of GDP/GTP exchange and GTPase activity are critical in the activation/deactivation cycle. Dissociation of GDP from the ARF domain of ARD1 was faster than from ARD1 itself (both proteins synthesized in Escherichia coli). Using deletion mutations, it was demonstrated that the 15 amino acids directly preceding the ARF domain were responsible for decreasing the rate of GDP dissociation but not guanosine 5-[gamma-thio]triphosphate dissociation. By site-specific mutagenesis it was shown that hydrophobic amino acids in this region were particularly important in stabilizing the GDP-bound form of ARD1. It is suggested that, like the amino-terminal segment of ARF, the equivalent region in ARD1, located between the GTPase-activating protein and ARF domains, may act as a GDP dissociation inhibitor.

Inhibition of DNA synthesis in living cells by microinjection of Gi2 antibodies.

Heterotrimeric guanine nucleotide binding proteins function in the coupling of a diverse span of cell surface receptors to a variety of intracellular signaling pathways, some of which stimulate cellular proliferation. With the recent discovery that mutated forms of G proteins are present in specific tumors, there has been an increased interest in the determination of the role of specific subtypes of G proteins in the regulation of cellular growth. We have attempted to determine which subtypes of G proteins are directly involved in serum-stimulated DNA synthesis through microinjection of inhibitory antibodies into living cells. Inhibitory rabbit polyclonal antibodies directed against specific Gi alpha subunits were introduced into living Balb/c 3T3 fibroblasts by microinjection, and the effect upon serum-stimulated DNA synthesis was examined. Results of these experiments indicate that Gi2 plays a direct role in serum-stimulated DNA synthesis in living cells and suggest that G proteins may function in a variety of mitogenic signaling pathways initiated by serum growth factors.

Hyperfunctional G proteins in mononuclear leukocytes of patients with mania

In a recent study, we found that lithium inhibits the function of guanine nucleotide-binding proteins, implicating G proteins as the common site for both the antimanic and antidepressant therapeutic effects of lithium. These findings may also suggest that an altered G protein function is of pathophysiological importance in bipolar affective disorder. In the present study, the coupling of both muscarinic-cholinergic receptors and beta-adrenergic receptors to pertussis toxin-sensitive G proteins or cholera toxin-sensitive G proteins was compared among untreated manic patients, lithium-treated euthymic bipolar patients, and healthy volunteers using mononuclear leukocyte (MNL) membrane preparations. Hyperactive function of G proteins was detected in untreated manic patients. Both isoproterenol-induced and carbamylcholine-induced increases in Gpp(NH)p binding capacity were twofold to threefold higher than the increases observed in healthy volunteers. On the other hand, lithium-treated euthymic bipolar patients showed G protein responses to agonist activation that were no different from the healthy volunteers. Altered G protein function may be of pathophysiological importance in bipolar affective disorder.

Interleukin 1 and tumor necrosis factor inhibit cardiac myocyte beta-adrenergic responsiveness.

Reversible congestive heart failure can accompany cardiac allograft rejection and inflammatory myocarditis, conditions associated with an immune cell infiltrate of the myocardium. To determine whether immune cell secretory products alter cardiac muscle metabolism without cytotoxicity, we cultured cardiac myocytes in the presence of culture supernatants from activated immune cells. We observed that these culture supernatants inhibit beta-adrenergic agonist-mediated increases in cultured cardiac myocyte contractility and intracellular cAMP accumulation. The myocyte contractile response to increased extracellular Ca2+ concentration is unaltered by prior exposure to these culture supernatants, as is the increase in myocyte intracellular cAMP concentration in response to stimulation with forskolin, a direct adenyl cyclase activator. Inhibition occurs in the absence of alteration in beta-adrenergic receptor density or ligand binding affinity. Suppressive activity is attributable to the macrophage-derived cytokines interleukin 1 and tumor necrosis factor. Thus, these observations describe a role for defined cytokines in regulating the hormonal responsiveness and function of contractile cells. The effects of interleukin 1 and tumor necrosis factor on intracellular cAMP accumulation may be a model for immune modulation of other cellular functions dependent upon cyclic nucleotide metabolism. The uncoupling of agonist-occupied receptors from adenyl cyclase suggests that beta-receptor or guanine nucleotide binding protein function is altered by the direct or indirect action of cytokines on cardiac muscle cells.