Guanine Nucleotide-dependent Regulatory Proteins Research Articles

Tyrosine Phosphorylation and Bradykinin-Induced Signaling in Endothelial Cells

It is generally accepted that in endothelial cells the occupation of bradykinin B2 receptors, which are linked to the guanine nucleotide-dependent regulatory proteins, Gi and Gq, results in the activation of phospholipase C-β1 (PLC-β1), followed by a transient increase in the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. The PLC-β1 isoform, in contrast to the γ1 isoform, is present only at a low level in cultured endothelial cells, implying that PLC-γ1 activation may play an important role in endothelial signaling pathways. In cultured human endothelial cells, bradykinin induced a rapid increase in the tyrosine phosphorylation of several Triton-soluble proteins. Immunoprecipitation of tyrosine-phosphorylated proteins from bradykinin-stimulated cells followed by Western blotting using the respective antibodies facilitated the identification of a 77 kiloDalton (kDa) protein as paxillin, a 130 kDa protein as PLC-γ1, and a 42/44 kDa doublet as mitogen-activated protein (MAP) kinase. The bradykinin-induced tyrosine phosphorylation of PLC-γ1 was relatively transient and was associated with an increase in intracellular levels of IP3. Bradykinin also induced the rapid and transient activation of phosphotyrosine phosphatases localized mainly in the Triton X-100-soluble cell fraction; this tyrosine phosphatase activity was apparently initiated after the release of Ca2+ from intracellular stores.

Regulation of effectors by G-protein α- and βγ-Subunits: Recent insights from studies of the phospholipase C-β isoenzymes

Both the α- and βγ-subunits of heterotrimeric guanine nucleotide-dependent regulatory proteins (G-proteins) couple members of the heptahelical class of cell-surface receptors to a diverse range of signal-generating effectors including retinal cyclic GMP phosphodiesterase, ion channels, adenylylcyclases, phosphoinositide 3-kinase, and members of the β-class of inositol lipid-specific phospholipases C. Although the molecular details of the G-protein-regulated phospholipase C system were elucidated comparatively recently, these enzymes have become an important model for investigations of the process of G-protein effector coupling. A combination of molecular biological, biochemical, and structural studies using the phospholipase C-β enzymes has provided some important insights into the interplay between G-proteins and their effectors and promises to reveal the mechanisms by which G-protein α- and βγ-subunits selectively associate with and activate effectors.

Ontogeny of beta-adrenergic regulation of adenylate cyclase in intrapulmonary arteries from fetal and postnatal lambs.

The enzyme adenylate cyclase is critically involved in the regulation of vasodilatation in the developing pulmonary circulation in that it mediates the vascular smooth muscle effects of beta-adrenergic agonists and vasodilatory prostaglandins. These agonists activate receptors coupled to the catalytic subunit of the enzyme by stimulatory guanine nucleotide-dependent regulatory proteins. We examined the ontogeny of the function of the adenylate cyclase system in intrapulmonary arterial segments from fetal lambs at 110-115 (F1) and 125-135 d gestation (F2) and from postnatal lambs at 7-14 (P1) and 35-56 d of age (P2). The function of the intact enzyme system and its components was assessed in incubations measuring cAMP accumulation during phosphodiesterase inhibition. beta-Adrenergic mediation of adenylate cyclase was examined because the binding characteristics of the smooth muscle receptors can be readily quantified. Nonstimulated (basal) cAMP accumulation was similar in F1 and F2, it increased 8-fold from F2 to P1, and it was equivalent in P1 and P2. cAMP accumulation with isoproterenol increased 5.9-fold from F1 to F2 and was similar in F2, P1, and P2. Radioligand binding studies revealed that the greater response to isoproterenol in F2 versus F1 is not related to an increase in beta-adrenergic receptor density or affinity. cAMP accumulation with forskolin, which activates adenylate cyclase by actions that involve both the stimulatory guanine nucleotide-dependent regulatory protein and the catalytic subunit, was similar in the four age groups, indicating that there are no maturational changes in the funciton of these enzyme system components.(ABSTRACT TRUNCATED AT 250 WORDS)

Inositol Phosphate Formation and Its Relationship to Calcium Signaling

The activation of a variety of cell surface receptors results in a biphasic increase in the cytoplasmic Ca2+ concentration due to the release or mobilization of Ca2+ from intracellular stores and to the entry of Ca2+ from the extracellular space. It is well established that phosphatidylinositol 4,5-bisphosphate hydrolysis is responsible for the changes in Ca2+ homeostasis. Stimulation of Ca2(+)-mobilizing receptors also results in the phospholipase C-catalyzed hydrolysis of the minor plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, with the concomitant formation of inositol (1,4,5) trisphosphate [1,4,5)IP3) and diacylglycerol. Analogous to the adenylyl cyclase signaling system, receptor-mediated stimulation of phospholipase C also appears to be mediated by one or more intermediary guanine nucleotide-dependent regulatory proteins. There is strong evidence that (1,4,5)IP3 stimulates Ca2+ release from intracellular stores. The Ca2(+)-releasing actions of (1,4,5)IP3 are terminated by its metabolism through two distinct pathways. (1,4,5)IP3 is dephosphorylated by a 5-phosphatase to inositol (1,4) bisphosphate; alternatively, (1,4,5)IP3 can be phosphorylated to inositol (1,3,4,5) tetrakisphosphate by a 3-kinase. Whereas the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known. A recent proposal that (1,4,5)IP3 secondarily elicits Ca2+ entry by emptying an intracellular Ca2+ pool will be considered. This review summarizes our current understanding of the mechanisms by which inositol phosphates regulate cytoplasmic Ca2+ concentrations.

Inositol phosphate formation and its relationship to calcium signaling.

The activation of a variety of cell surface receptors results in a biphasic increase in the cytoplasmic Ca2+ concentration due to the release or mobilization of Ca2+ from intracellular stores and to the entry of Ca2+ from the extracellular space. It is well established that phosphatidylinositol 4,5-bisphosphate hydrolysis is responsible for the changes in Ca2+ homeostasis. Stimulation of Ca2(+)-mobilizing receptors also results in the phospholipase C-catalyzed hydrolysis of the minor plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, with the concomitant formation of inositol (1,4,5) trisphosphate [1,4,5)IP3) and diacylglycerol. Analogous to the adenylyl cyclase signaling system, receptor-mediated stimulation of phospholipase C also appears to be mediated by one or more intermediary guanine nucleotide-dependent regulatory proteins. There is strong evidence that (1,4,5)IP3 stimulates Ca2+ release from intracellular stores. The Ca2(+)-releasing actions of (1,4,5)IP3 are terminated by its metabolism through two distinct pathways. (1,4,5)IP3 is dephosphorylated by a 5-phosphatase to inositol (1,4) bisphosphate; alternatively, (1,4,5)IP3 can be phosphorylated to inositol (1,3,4,5) tetrakisphosphate by a 3-kinase. Whereas the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known. A recent proposal that (1,4,5)IP3 secondarily elicits Ca2+ entry by emptying an intracellular Ca2+ pool will be considered. This review summarizes our current understanding of the mechanisms by which inositol phosphates regulate cytoplasmic Ca2+ concentrations.

Guanine nucleotides stimulate hydrolysis of phosphatidylinositol and polyphosphoinositides in permeabilized Swiss 3T3 cells

Hydrolysis-resistant analogues of GTP specifically stimulate the formation of [ 3H]inositol mono-, bis- and trisphosphates by saponin-permeabilized Swiss 3T3 cells prelabelled with [ 3H]inositol. Each inositol phosphate is formed largely by hydrolysis of its parent lipid and not by dephosphorylation of inositol 1,4,5-trisphosphate [(1,4,5)IP 3]. Although hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP 2) is most sensitive to guanine nucleotides, hydrolysis of phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP) is quantitatively more important. These results suggest that a guanine nucleotide-dependent regulatory protein(s) (G-protein) is involved in regulating the hydrolysis of PI and PIP, as well as PIP 2, and so may allow formation of diacylglycerol (DG) without simultaneous production of (1,4,5)IP 3 and mobilization of intracellular Ca 2+.

The role of phosphoinositide metabolism in signal transduction in secretory cells.

Activation of a variety of cell surface receptors results in a biphasic increase in the cytoplasmic Ca2+ concentration, due to the release, or mobilization, of intracellular Ca2+ stores and to the entry of Ca2+ from the extracellular space. Stimulation of these same receptors also results in the phospholipase-C-catalysed hydrolysis of the minor plasma membrane phospholipid, phosphatidylinositol 4,5-bisphosphate, with the concomitant formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol. Analogous to the adenylyl cyclase signalling system, receptor-mediated stimulation of phospholipase C also appears to occur through one or more intermediary guanine nucleotide-dependent regulatory proteins. It is well established that phosphatidylinositol 4,5-bisphosphate hydrolysis is responsible for the changes in Ca2+ homeostasis. There is strong evidence that Ins(1,4,5)P3 stimulates Ca2+ release from intracellular stores. The Ca2+-releasing actions of Ins(1,4,5)P3 are terminated by its metabolism through two distinct pathways. Ins(1,4,5)P3 is dephosphorylated by a 5-phosphatase to Ins(1,4)P2; alternatively, Ins(1,4,5)P3 can also be phosphorylated to Ins(1,3,4,5)P4 by a 3-kinase. Whereas the mechanism of Ca2+ mobilization is understood, the precise mechanisms involved in Ca2+ entry are not known; a recent proposal that Ins(1,4,5)P3 by emptying an intracellular Ca2+ pool, secondarily elicits Ca2+ entry will be considered. This review summarizes our current understanding of the mechanisms by which inositol phosphates regulate cytoplasmic Ca2+ concentrations.

Pertussis toxin blocks the inhibitory effect of adenosine on rat cerebral cortical neurons

Rat cerebral cortex was injected with 2 μg of purified pertussis toxin, which inactivates various guanine nucleotide-dependent regulatory proteins. The spontaneous firing of cortical neurons in toxin-treated animals was unresponsive to adenosine or adenosine 5′- N-ethylcarboxamide applied iontophoretically in coparison with the pronounced inhibitory effects of these purines observe on neurons in the contralateral, saline-injected, hemisphere. Excitatory effects of acetylcholine were also reduced in the toxin-treated cortex. These findings implicate guanine nucleotide-dependent regulatory proteins (G proteins) in the inhibitory actions of adenosine on transmitter release.

Formation and actions of calcium-mobilizing messenger, inositol 1,4,5-trisphosphate.

A variety of surface membrane receptors can activate a phospholipase C, which degrades phosphatidylinositol 4,5-bisphosphate liberating a calcium mobilizing second messenger, inositol 1,4,5-trisphosphate [(1,4,5)IP3]. The coupling of surface receptors to the phospholipase C involves one or more guanine nucleotide-dependent regulatory proteins that are similar but not identical to those that regulate adenylate cyclase. (1,4,5)IP3 has been shown to release Ca2+ from a portion of the endoplasmic reticulum and is believed responsible for the initial phase of Ca2+ mobilization ascribed to internal Ca2+ release. (1,4,5)IP3 acts by binding to a specific receptor that either is a component of, or regulates, a Ca2+ ion channel. The release of Ca2+ from the (1,4,5)IP3-sensitive component of the endoplasmic reticulum may secondarily activate the second phase of Ca2+ mobilization, which involves Ca2+ entry. (1,4,5)IP3 is metabolized by two pathways. One involves the action of a 5-phosphatase that degrades (1,4,5)IP3 to inositol 1,4-bisphosphate, whereas the other involves a 3-kinase that phosphorylates (1,4,5)IP3 to produce inositol 1,3,4,5-tetrakisphosphate. The significance of this dual metabolism is not known, but it may be important in rapidly extinguishing the Ca2+-releasing activity (1,4,5)IP3.

High-affinity agonist binding to rat brainstem muscarinic receptors is eliminated by low pH

The effects of hydrogen ion concentration on high-affinity agonist binding to muscarinic receptors were determined in rat brainstem membranes using [ 3H]oxotremorine-M as a probe. [ 3H]Oxotremorine-M bound with high affinity to a small subpopulation of brainstem muscarinic receptors (10% of the receptors labelled by [ 3H]methylscopolamine). [ 3H]Oxotremorine-M binding was constant between pH 7.0 and 9.0; the number of high-affinity sites decreased below pH 7.0 and at pH 5.0 no binding was detected. This decrease was irreversible; when brainstem membranes were incubated for 1 h at low pH and then returned to pH 8.0, agonist binding was not restored. In contrast, the total number of receptors, i.e. the number of [ 3H]antagonist binding sites, was not affected by prolonged incubation at low pH. Agonist affinity for the surviving [ 3H]oxotremorine binding sites and the sensitivity of agonist binding to guanine nucleotides were not altered in media of low pH (pH 5.5). These findings suggest that [ 3H]oxotremorine-M binds only to receptors which are functionally coupled to guanine nucleotide-dependent regulatory proteins and that this coupling is irreversibly inactivated in media of low pH.

A guanine nucleotide-dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland

Electrically permeabilized cells of rat parotid gland, prelabelled with [ 3H]-inositol, synthesized [ 3H]-inositol phosphates (IP 3 and IP 2) when stimulated with α 1-adrenergic, muscarinic-cholinergic, and substance P receptoragonists. Non-hydrolyzable analogues of GTP (GTPγS and GppNHp) also stimulated [ 3H]-IP 3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [ 3H]-IP 3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP 3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca 2+-mobilizing receptors to phospholipase C in parotid gland.

Effect of pH on muscarinic acetylcholine receptors from rat brainstem.

The effect of hydrogen ion concentration on ligand binding to muscarinic acetylcholine receptors was studied in membranes isolated from rat brainstem. The binding of [3H]methylscopolamine was constant between pH 7 and 10. The affinity, but not the number, of [3H]methylscopolamine binding sites decreased below pH 7; at pH 4 little binding was detected. When brainstem membranes were incubated at various pH levels from 3 to 11 for 1 h and then returned to pH 8, [3H]methylscopolamine binding affinity was restored to control levels. Carbamylcholine binding affinity was also depressed in media of low pH. However, this decrease was permanent after a 1-h incubation at pH 4 (i.e. carbamylcholine affinity was not restored on raising the pH to 8). The capacity of a guanine nucleotide to affect carbamylcholine was also abolished by a 1-h incubation at pH 4, and was not restored by raising the pH. The guanine nucleotide-dependent regulatory protein may be irreversibly inactivated or dissociated from the receptor at low pH. The receptor's binding subunit, on the other hand, appears to be much less sensitive to hydrogen ion concentration.

Receptor coupling to polyphosphoinositide turnover: a parallel with the adenylate cyclase system

Calcium-mobilizing receptors stimulate polyphosphoinositide hydrolysis, catalysed by phospholipase C, with the formation of intracellular messengers that then control the cytosolic Ca 2+ concentration and the activity of protein kinase C. Colin Taylor and Janet Merritt describe recent studies which suggest that a guanine nucleotide-dependent regulatory protein (G protein) couples these receptors to phospholipase C, a finding that presents a striking parallel with receptor regulation of adenylate cyclase.

Muscarinic acetylcholine receptors in chick heart: Influence of heat and N-ethylmaleimide on receptor conformations and interactions with guanine nucleotide-dependent regulatory proteins

Several conformations of muscarinic acetylcholine receptors in chick heart are revealed by the energetics of agonist binding. Heating cardiac membranes at 50 degrees C for 5 min decreased agonist affinity, steepened agonist binding curves and eliminated receptor sensitivity to guanine nucleotides. N-ethylmaleimide (NEM) steepened agonist binding curves and eliminated receptor sensitivity to guanine nucleotides without decreasing agonist binding affinity. NEM prevented and reversed the effect of heat on agonist affinity. Pirenzepine and N-methylscopolamine binding were unaffected by either treatment. NEM and heat stabilize muscarinic receptors in conformations unrelated to those associated with naturally occurring receptor populations.

β-Adrenergic Receptors in Pediatric Tumors: Uncoupled β<sub>1</sub>-Adrenergic Receptor in Ewing's Sarcoma<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

beta-Adrenergic receptors were demonstrated in membrane preparations from 6 human Ewing's sarcomas and compared to those from 46 other pediatric cancers with the use of the beta-adrenergic antagonist (-)-(3H)dihydroalprenolol [(-)[3H]DHA]. In contrast to the high numbers of receptor sites found in Ewing's sarcomas (55-640 fmol x mg-1 protein; dissociation constant Kd, 1-2 nM), other childhood cancers (neuroblastoma, rhabdomyosarcoma, brain tumors, lymphoma, osteosarcoma, hepatoblastoma, yolk sac, and Wilms' tumor) contained in general fewer beta-adrenergic receptor sites. Characteristics of (-)-[3H]DHA binding were therefore more fully characterized in the Ewing's tumors. Competition of (-)-[3H]DHA binding by classical catecholamine agonists, as well as by subtype selective agents metoprolol and zinterol, demonstrated the presence of a homogeneous population of beta 1-adrenergic sites in several Ewing's tumors. Adenylate cyclase activity in all Ewing's sarcomas was enhanced by GTP and NaF. However, in spite of high numbers of beta-adrenergic receptors, (-)-isoproterenol was not very effective in the activation of adenylate cyclase activity in several of the Ewing's tumors tested. Neither guanyl-5'-yl-imidophosphate nor GTP altered agonist potency for the receptor site in these catecholamine-insensitive tumors. Hill coefficients obtained from the competition experiments with (-)-isoproterenol (in the presence or absence of guanine nucleotide) were approximately 1.0. These uncoupled receptors were resistant to N-ethylmaleimide denaturation and were densensitized only 50% during culture in the presence of (-)-isoproterenol. Thus Ewing's sarcomas are relatively rich in beta-adrenergic sites, and several tumors appear to have a coupling lesion involving guanine nucleotide-dependent regulatory protein interaction with beta-adrenergic receptors and adenylate cyclase, similar in phenotype to that described in the (unc) variant of S49 mouse lymphoma.