Cerebral muscarinic acetylcholine receptors interact with three kinds of GTP-binding proteins in a reconstitution system of purified components

A new GTP-binding protein, which serves as a substrate for pertussis toxin, was prepared from porcine brain. The new G protein was separated from other GTP-binding proteins, Gi and Go, by an anion-exchange column chromatography. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the alpha subunit of the new G protein was between those of alpha subunits of Gi and Go. Evidence that the alpha subunit is not a proteolytic fragment of the alpha subunit is not a proteolytic fragment of the alpha subunit of Gi or Go was provided by experiments involving partial hydrolysis of these G proteins with thermolysin and their interaction with an antibody raised against the amino terminal peptide of the alpha subunit of Gi. In addition, the gamma subunit of the new G protein was indicated to be different from the gamma subunits of Gi and Go, because the latter were found to be phosphorylated by protein kinase C but the former was not. GTP-sensitive high affinity binding of muscarinic receptors with acetylcholine was observed when muscarinic receptors purified from porcine cerebrum were reconstituted in phospholipid vesicles with the new G protein as well as with Gi or Go. The proportion of the high affinity sites increased with the concentrations of the G proteins, the potency of the new G protein being similar to that of Gi but a little lower than that of Go. This GTP-sensitive high affinity binding was not observed when each G protein was pretreated with pertussis toxin and then reconstituted with muscarinic receptors. Acetylcholine accelerated the dissociation of [3H]GDP from the new G protein as well as from Gi and Go, which were reconstituted with muscarinic receptors. These results indicate that muscarinic receptors interact with at least the above three kinds of G proteins, in a pertussis toxin-sensitive manner.     

Effects of sulfhydryl-modifying reagents, 3-nitro-2-pyridinesulfenyl compounds, on the coupling between inhibitory receptors and GTP-binding proteins Gi/Go in rat brain membranes

To gain insight into the coupling mechanism of inhibitory receptors, 5-hydroxytryptamine1A receptors and alpha 2-adrenoceptors, with GTP-binding proteins (G proteins) in the central nervous system, we examined the effects of two 3-nitro-2-pyridinesulfenyl compounds, S-(3-nitro-2-pyridinesulfenyl)-L-cysteine [Cys(Npys)] and N-t-butoxy-carbonyl-S-(3-nitro-2-pyridinesulfenyl)-L-cysteine [Boc-Cys(Npys)], on 1) specific binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (5-hydroxytryptamine1A agonist) and [3H]clonidine (alpha 2-agonist) to rat brain membranes, 2) [35S]guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) binding, and 3) pertussis toxin (islet-activating protein) (IAP)-catalyzed ADP-ribosylation of purified Go (an IAP-sensitive G protein present in abundance in the mammalian brain). Treatment with Cys(Npys) led to decreased [3H]8-OH-DPAT and [3H]clonidine binding, similar to the inhibitory effects of IAP and N-ethylmaleimide (NEM) on such binding. However, further treatment of Cys(Npys)-pretreated membranes with dithiothreitol completely abolished the inhibitory effect of Cys(Npys) on the binding of both ligands. On the other hand, treatment with Boc-Cys(Npys) inhibited the effect of several GTP analogs (GTP gamma S, guanylyl-imidodiphosphate, guanylyl)-(beta, gamma-methylene)-diphosphate, and GTP) on [3H]8-OH-DPAT and [3H]clonidine binding. Dithiothreitol and mercaptoethanol treatment of Boc-Cys(Npys)-pretreated membranes did not lead to a recovery of the effect of GTP analogs on agonist binding. Regardless of the presence or absence of GTP gamma S, agonist binding to Boc-Cys(Npys)-pretreated membranes was decreased by further addition of NEM or Cys(Npys). Cys(Npys) blocked [35S]GTP gamma S binding as well as IAP-catalyzed ADP-ribosylation in purified Go. In contrast, Boc-Cys(Npys) partially inhibited ADP-ribosylation and did not affect [35S]GTP gamma S binding. These results suggested that Cys(Npys) modifies the receptor-coupling domain in G proteins, followed by the uncoupling of inhibitory receptors from G proteins, similar to the effects of NEM and IAP. Boc-Cys(Npys), however, seems to stabilize the coupling state between the receptors and G proteins, thus abolishing the GTP gamma S effect.     

Immunological localization of the GTP-binding protein Go in different tissues of vertebrates and invertebrates

Bovine brain contains two GTP-binding proteins, Gi and Go, which are substrates for ADP ribosylation by pertussis toxin. The Gi protein mediates hormone and GTP inhibition of adenylate cyclase, but the function and the precise tissue distribution of Go are unknown. To immunologically probe the localization of Go, we have purified the Go alpha and G beta, gamma subunits of Go and have raised antibodies against them. The polyclonal anti-Go alpha antibodies obtained were very selective for Go alpha compared to Gi alpha or Gs alpha. The positive Go alpha and G beta, gamma immunoreactivities were investigated in different tissues of vertebrates and invertebrates on immunoblots after gel electrophoresis of the crude membranes. The anti-G beta, gamma antibodies recognized a 35-36-kDa protein in brain of vertebrates such as mammals (rat), avians (pigeon), amphibians (frog), fish (trout), and reptiles (turtle) but not in the invertebrates such as molluscs (snail) and insects (locust). With the anti-Go alpha antibodies a high level of immunoreactivity was detected at molecular weights of 39,000-40,000 in the brain of invertebrates as well as in the central nervous system of vertebrates. Moreover, ADP ribosylation with pertussis toxin occurred in the nervous system of invertebrates. These results suggest that the GTP-binding proteins of invertebrates either are devoid of G beta, gamma subunit or, more probably, possess immunologically different G beta, gamma subunits when compared to those of vertebrates. In the vertebrates, Go alpha immunoreactivity was also present in the peripheral nervous system in areas such as the superior cervical ganglia and sciatic nerve. When examined with the anti-Go alpha antibodies, the neuro-and adenohypophysis exhibited a similar immunoreactivity which was about 6 times lower than in brain. Our antibodies also recognized a 40-kDa protein in human adipocytes but at a concentration 17 times lower than that recognized in brain. Taken together, these data show that the Go alpha subunit is well conserved through evolution and, furthermore, confirm that Go alpha is not strictly limited to the nervous system. This suggests that the protein Go ensures a function required for neuronal activity but also present in some other non-nervous tissues.     

Inhibitory GTP-binding regulatory protein Gi3 can couple angiotensin II receptors to inhibition of adenylyl cyclase in hepatocytes

Angiotensin II can inhibit hormone-stimulated adenylyl cyclase in intact hepatocytes or in hepatic membrane preparations. Because the response can be blocked by pertussis toxin, the object of the present study was to determine which of the known variants of Gi can couple angiotensin II receptors to inhibition of adenylyl cyclase. The potential candidates were identified by probing RNA isolated from rat hepatocytes with cDNAs specific for the alpha subunits of known toxin-sensitive guanine nucleotide-binding regulatory proteins (G proteins). Hepatocytes contained no detectable RNA for the Go or Gi1 alpha subunits and similar levels of RNA coding for the Gi2 and Gi3 alpha subunits. To determine whether Gi3 could couple angiotensin receptors to inhibition of cyclase, membranes were prepared from hepatocytes whose G proteins were fully ADP-ribosylated with pertussis toxin, and the Gi3 holoprotein purified from rabbit liver was reconstituted into the membranes. The nature of the Gi3 reconstituted into the membrane was assessed by immunoblotting with antibodies specific for the Gi alpha subunits. Reconstitution of 6-10 pmol of Gi3/mg of membrane protein into the toxin-treated membranes restored the ability of 10 nM angiotensin II to inhibit adenylyl cyclase. Because pertussis toxin has nonspecific effects, an assay was developed to measure the interaction of the angiotensin receptor with reconstituted G proteins in normal membranes. In the presence of Mg2+, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) caused a reduction of the affinity of the angiotensin II receptor for 125I-angiotensin II that was stable to washing and the detergents used to reconstitute G proteins into the membranes. Using this protocol to activate G proteins and "uncouple" receptors, the ability of the GDP-liganded form of Gi to restore high affinity binding was examined. Reconstitution of about 10-15 pmol of oligomeric Gi3/mg of membrane protein restored both the high affinity state of the angiotensin II receptor and the ability of GTP gamma S to shift the affinity to a lower state. The same shift in receptor affinity could be accomplished by reconstituting the Gi3 alpha subunit, resolved free of beta gamma subunits, into the membranes. Reconstitution of up to 50 pmol of Gs/mg of membrane protein had no effect on angiotensin II receptor affinity. The results suggest that a major form of Gi in hepatocytes is Gi3 and that it can couple angiotensin receptors to inhibition of adenylyl cyclase.     

Phosphorylation of Gi protein by cyclic AMP-dependent protein kinase inhibits its dissociation into alpha-subunits and beta gamma-subunits by Mg2+ and GTP gamma S

Pretreatment of partially purified inhibitory GTP-binding protein (Gi, 41 kDa) with activated cyclic AMP-dependent protein kinase (PKA) decreases its ADP-ribosylation by islet-activating protein (pertussis toxin, IAP). We examined whether this decrease was associated with dissociation of the trimer of alpha beta gamma-subunits of Gi protein into alpha-subunits and beta gamma-subunits. Results showed that phosphorylation of the Gi protein by PKA impaired its dissociation into alpha-subunits and beta gamma-subunits by 50 mM Mg2+ and 100 microM GTP gamma S. The results suggested that phosphorylation of the Gi protein by PKA possibly caused a conformational change of the trimer Gi protein.     

Differential inactivation and G protein reconstitution of subtypes of [3H]5-hydroxytryptamine binding sites in brain

The sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide (NEM) inactivate high affinity [3H]serotonin [( 3H]5-HT) binding to bovine and rat brain membranes in a concentration-dependent manner. In both species, 15-25% of total specific high affinity [3H]5-HT binding is relatively insensitive to NEM. This study examines the NEM sensitivity of the various high affinity [3H]5-HT binding subtypes, using selective ligands, tissues, and pharmacological masks to study each subtype. Reconstitution of NEM-inactivated binding by addition of GTP-binding proteins (G proteins, Gi and Go) is also described. Agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites in rat brain and to 5-HT1A and 5-HT1D sites in bovine brain is sensitive to NEM. Binding of [3H]dihydroergotamine and [125I]iodocyanopindolol, both of which are weak partial agonists to 5-HT1B sites is relatively insensitive to NEM. Binding of [3H]5-HT to 5-HT1C sites in rat and bovine brain and choroid plexus is relatively insensitive to NEM. In the presence of spiperone to mask binding of 5-HT2 sites, binding of antagonist [( 3H]mesulergine) to 5-HT1C sites is also insensitive to NEM. Likewise, binding of the agonist [3H]4-bromo-2,5-dimethoxyphenylisopropylamine and of the antagonist [3H]ketanserin to 5-HT2 sites is not inhibited by NEM treatment of membranes. These findings suggest that agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites is sensitive to NEM alkylation. Binding of neither agonist nor antagonist to 5-HT1C and 5-HT2 sites is sensitive to NEM. Inability of high concentrations of a variety of ligands to protect the sensitive binding sites against NEM inactivation indicates that the critical sulfhydryl group(s) are not located in the ligand binding domain of the NEM-sensitive binding sites. When membranes are treated with NEM, displacement of [125I]iodocyanopindolol by 5-HT is no longer sensitive to 5'-guanylyl imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity of agonist displacement of 5-HT1B binding to NEM-treated membranes is restored by addition of purified guanine nucleotide binding proteins (Gi plus Go). In addition, NEM-inactivated binding to 5-HT1A and 5-HT1D sites can be restored by addition of Gi plus Go. These data suggest that NEM exerts its effects on 5-HT1A, 5-HT1B, and 5-HT1D binding sites by inactivating the G protein(s) associated with the 5-HT receptor subtypes.     

Effects of lithium ion on ADP ribosylation of inhibitory GTP-binding protein by pertussis toxin, islet-activating protein

Studies were made on the effects of Li+ on ADP ribosylation of inhibitory GTP-binding (Gi) protein by islet-activating protein (IAP), pertussis toxin. The ADP ribosylation of 40-41 kDa proteins of the membranes of rat cardiac ventricular cells by IAP was reduced by the addition of a nonhydrolyzable analog of guanine nucleotide, GTP gamma S, indicating that these proteins included Gi protein. The addition of LiCl (0.5-10 mM) to the membrane fractions of the cells attenuated the ADP ribosylation of the Gi protein of the cell membranes by IAP dose-dependently. The effects of LiCl were reversible. Of the monovalent ions tested, Li+ inhibited the ADP ribosylation of the protein by IAP most strongly. The effects of LiCl (2 mM) were observed even in the presence of 150 mM KCl. Moreover, LiCl decreased the ADP ribosylation of purified Gi protein by IAP. These results support that Gi proteins are one of the targets for the therapeutic effects of lithium.     

Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins.

The muscarinic acetylcholine receptors in heart and cerebellum form a stable association with guanine nucleotide-binding regulatory proteins (G proteins) in the presence of receptor agonists. This has been confirmed by purification of the muscarinic receptor-G protein complexes using an immunoprecipitation protocol. The isolated complexes were subjected to Western blotting to identify the G protein subunits present in the complexes. At saturating concentrations of carbachol, the muscarinic receptors in atrial membranes co-purified exclusively with Go, whereas in cerebellar and ventricular membranes an association with both Gi and Go was demonstrated. Further characterization of the G protein subunits allowed identification of the species of Gi alpha subunits present in the complexes of muscarinic receptor and G protein; in ventricle Gi alpha 2 was the only subtype present, whereas in cerebellum both Gi alpha 1 and Gi alpha 2 were present. These results demonstrate that a single muscarinic receptor subtype, depending on the tissue studied, is capable of interacting with more than one G protein subtype. The concentrations of agonist required to promote receptor-G protein association in atrial and ventricular membranes correlated with the high affinity component of receptor occupancy by agonist, as measured in equilibrium binding assays. Furthermore, incubation of cardiac membranes with saturating concentrations of pilocarpine or McN A343 resulted in reduced amounts of receptor-G protein complexes, compared with carbachol. Overall, our results suggest that the specificity of cellular effects of muscarinic agonists may relate, in part, to the selective interaction of receptor with G proteins.     

Agonist binding to rat brain somatostatin receptors alters the interaction of the receptors with guanine nucleotide-binding regulatory proteins.

To investigate the interaction of guanine nucleotide-binding regulatory proteins (G proteins) with the agonist-bound brain somatostatin (SRIF) receptor, rat brain SRIF receptor/G protein complexes were solubilized and immunoprecipitated with peptide-directed antisera selective for the different subtypes of G protein alpha subunits (G alpha). In the absence of agonist, solubilized SRIF receptor/G proteins complexes could be immunoprecipitated by antiserum 8730, which is directed against the carboxyl-terminal region of Gi alpha and recognizes all Gi alpha subtypes, and by antiserum 3646, which selectively interacts with internal regions of Gi alpha 1. In contrast, antiserum 1521, which is directed against an internal region of Gi alpha 2, and antiserum 9072, which is directed against the carboxyl-terminal region of Go alpha, did not immunoprecipitate the SRIF receptor. After the binding of agonist to solubilized SRIF receptors, antisera 9072 and 1521, as well as antisera 8730 and 3646, were able to immunoprecipitate the agonist-bound SRIF receptor/G protein complexes, indicating that agonist interaction with SRIF receptors maintained receptor association with Gi alpha 1 and promoted receptor association with Go alpha and, to a lesser extent, Gi alpha 2. Antiserum 1518, which is directed against Gi alpha 3, uncoupled SRIF receptors from Gi alpha and did not immunoprecipitate the agonist-bound or agonist-free brain SRIF receptor. These findings indicate that differences exist in the interaction of the agonist-free and agonist-bound SRIF receptors with G proteins. The binding of agonists to SRIF receptors promotes the association of the receptor with Go alpha and, to a lesser extent, Gi alpha 2, indicating that these G proteins, along with Gi alpha 1 and Gi alpha 3, may be involved in coupling SRIF receptors to cellular effector systems.     

The interaction between D-2 dopamine receptors and GTP-binding proteins

D-2 dopamine receptors were solubilized from porcine striatal membranes with 0.3% sodium cholate/1 M NaCl and separated from the bulk of the guanine nucleotide-binding regulatory proteins (G-proteins) by Ultrogel AcA34 gel filtration chromatography. The partially purified D-2 receptors were reconstituted in phospholipid vesicles with Gi or Go purified from porcine brain. The dissociation constant (Kd) of the D-2 receptors in the reconstituted vesicles for [3H]spiperone binding was 82-89 pm, which was not affected by the presence or absence of G-proteins. The displacement curve for [3H]spiperone/dopamine was analyzed, assuming that there are two populations of binding sites. The Kd values for the binding sites with high affinity for agonists (HAS) and that for the binding sites with low affinity for agonists (LAS) were approximately 1 microM and 100 microM, respectively. The proportion of HAS was 8% when the receptor preparation was reconstituted into phospholipid vesicles without G-proteins, but it increased to 58-64% with increasing G-protein concentrations. The potency of Go was a little higher than that of Gi. The proportion of HAS in the presence of G-proteins decreased to about 11% on addition of GTP. When G-proteins were treated with islet-activating protein, GTP-sensitive HAS were not observed. These results indicate that at least 50% of the partially purified D-2 receptors interact with both Gi and Go.     

Peptide inhibitors of ADP-ribosylation by pertussis toxin are substrates with affinities comparable to those of the trimeric GTP-binding proteins.

Pertussis toxin (PTX) ADP-ribosylates alpha subunits of GTP-binding proteins (G proteins) when they are in association with beta gamma dimers, and free alpha subunits are thought not to be substrates under standard assay conditions. We now report the rather unexpected discovery that synthetic peptides encompassing the last 10-20 amino acids of alpha subunits of PTX-sensitive G proteins are substrates for PTX by themselves and in the absence of beta gamma dimers. As determined for G13, the Km of PTX for the 20-amino acid carboxyl-terminal peptide is 10-fold higher than that for the trimeric G protein. Interestingly, PTX ADP-ribosylates the free full length alpha 13 subunit with a Km not different from that of the trimer but with a Vmax that is only 1% of that with which it ADP-ribosylates the trimer. It follows that the primary role of beta gamma dimers in ADP-ribosylation of G proteins is one of increasing the Vmax of the reaction without affecting the Km of the substrate for the toxin. Mutant peptides lacking the ADP-ribose acceptor site act as competitive inhibitors.     

The mechanism of changes in adrenoceptor-mediated responses

Quantitative and qualitative changes in adrenoceptors under various conditions were studied by binding experiments. Chronic treatment with reserpine increased the level of alpha 2-adrenoceptors in rat vas deferens and hypoxia increased the level of alpha 1-adrenoceptors in rat cardiomyocytes. Adenosine receptor agonists increased the affinity of the alpha 2-adrenoceptor in rat vas deferens for the agonist with an increase in receptor-mediated responses. Thus two types of changes in receptor binding sites were observed. Next, changes in the GTP-binding (G) protein were studied. Activation of cyclic AMP-dependent protein kinase (PKA) decreased the ADP-ribosylation of Gi (41 K) protein by islet-activating protein (pertussis toxin, IAP). Purified Gi protein was phosphorylated by the enzyme. IAP-sensitive G protein-mediated coupling responses such as phosphatidylinositol turnover in differentiated HL-60 cells were also modified under this condition. These results indicated that phosphorylation of Gi by PKA caused a qualitative change of Gi. Lithium ions also decreased the ADP-ribosylation of Gi by IAP. Then it determined if the decrease was accompanied with a dissociation of the subunits of Gi. Phosphorylation of Gi by PKA impaired the dissociation of the subunits of Gi caused by Mg2+ and GTP gamma S, whereas lithium ions did not have any effect on their dissociation. Thus some conditions caused a functional change in the so-called "qualitative change" of Gi.     

Identification of three separate guanine nucleotide-binding proteins that interact with the delta-opioid receptor in NG108-15 neuroblastoma x glioma hybrid cells.

Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.     

Treatment with N-ethylmaleimide selectively reduces adenosine receptor-mediated decreases in cyclic AMP accumulation in rat hippocampal slices

N-ethylmaleimide (NEM) has been reported to interact with the GTP-binding Ni-protein; we have examined its effect on adenosine receptor binding in feline cortical membranes and on adenosine-receptor mediated effects on cyclic AMP accumulation in rat hippocampal slices. Treatment of cortical membranes with NEM (100 microM for 5 min) altered the binding of [3H]-phenylisopropyladenosine (PIA) from being almost exclusively to a single class of high affinity sites (KD = 1.65 nM) to binding at two classes of sites (KDH = 2.1 nM, KDL = 102 nM). The total number of binding sites was similar (825-845 fmol mg-1 in control membranes, 944-1428 fmol mg-1 in NEM-treated membranes). In rat hippocampal slices treated with forskolin (0.3 microM) L-PIA produced a biphasic effect on cyclic AMP accumulation: an inhibition at 0.03 to 1 microM and at higher concentrations, a stimulation. Treatment with 50 microM NEM selectively inhibited the inhibitory phase, causing stimulation at lower concentrations of L-PIA. At 50 microM, NEM did not alter basal or forskolin-stimulated cyclic AMP accumulation but at higher concentrations inhibition was observed. It is concluded that NEM can, in certain doses, selectively block adenosine A1-receptor-mediated effects without affecting A2-receptor-mediated actions in the same tissue. It is suggested that this is due to NEM affecting the Ni guanine nucleotide binding protein.     

Lithium does not alter ADP-ribosylation of Gi/Go catalyzed by pertussis toxin in rat brain.

The influences of lithium in vitro and ex vivo on the ADP-ribosylation of Gi/Go catalyzed by pertussis toxin (islet-activating protein, IAP) were investigated in cerebral cortical and hippocampal membranes from rats. Incorporation of [32P]ADP-ribose into 40-41 kDa band catalyzed by IAP was markedly reduced by the addition of non-hydrolyzable GTP analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or guanosine 5'-(beta, gamma-imido)triphosphate [Gpp(NH)p], in the presence of MgCl2 but not in the absence of MgCl2. The amounts of IAP-catalyzed ADP-ribosylation of Gi/Go in the presence of 100 microM guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) and 50 mM EDTA and in the absence of MgCl2 were in proportion to the protein contents between 30 and 60 micrograms/tube, suggesting that the determination of [32P]ADP-ribosylation could be used quantitatively within this limited range. Addition of LiCl in vitro did not affect the IAP-mediated ADP-ribosylation of Gi/Go up to the concentration of 5 mM. The values of ADP-ribosylation of Gi/Go in the presence of 100 microM GTP gamma S were reduced by MgCl2 concentration-dependently. However, this inhibitory effect of MgCl2 was not influenced by 2 mM LiCl in vitro. Furthermore, chronic treatment with a diet containing 0.2% lithium carbonate did not alter the [32P]ADP-ribosylation of Gi/Go catalyzed by IAP.     

Effects of GABA and various allosteric ligands on TBPS binding to cloned rat GABA(A) receptor subtypes.

1. [35S]t-butylbicyclophosphorothionate (TBPS) is a high affinity ligand for the picrotoxin site of GABA(A) receptors. Here we examined TBPS binding to the cloned receptors made of alpha 1, alpha 3 or alpha 6 in combination with beta 2 or beta 2 and gamma 2 subunits, in the presence of GABA and several allosteric ligands (diazepam, methyl 6,7-dimethoxy-4-methyl-beta-carboline-3-carboxylate (DMCM), 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (5 alpha-THDOC), pentobarbitone and Zn). The cloned receptors were transiently expressed in SF-9 insect cells by infecting with recombinant baculoviruses. 2. In alpha beta subtypes, GABA at nanomolar concentrations enhanced TBPS binding but inhibited binding at micromolar concentrations. Half maximal GABA concentrations for enhancement or inhibition of TBPS binding were correlated with high and low affinity GABA binding sites, respectively, in individual subtypes. The maximal enhancement of binding also varied according to the alpha isoform (alpha 3 beta 2 >> alpha 1 beta 2). In alpha beta gamma subtypes, TBPS binding was unaffected by GABA at nanomolar concentrations, but was inhibited by GABA at micromolar concentrations. Addition of gamma 2 thus appeared to abolish conformational coupling between high affinity GABA sites and TBPS sites, and also altered low affinity GABA sites; in particular, the half maximal GABA concentration for inhibition of TBPS binding changed from > 100 (alpha 6 beta 2) to 1 microM (alpha 6 beta 2 gamma 2). 3. Allosteric ligands also altered TBPS binding to sensitive GABA(A) receptor subtypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of an endogenous beta3 subunit on recombinant GABA(A) receptor assembly and pharmacology in WSS-1 cells and transiently transfected HEK293 cells

Cell lines are commonly used for studying recombinant heterooligomeric ion channels with defined subunit composition. Such studies often ignore the contribution of endogenous proteins in the assembly of mature channels. We examined whether an endogenous subunit was required for the functional expression of gamma-aminobutyric acid type A (GABA(A)) receptors in WSS-1 cells, HEK293 cells stably expressing recombinant alpha1 and gamma2 subunits. Our pharmacological and RT-PCR analyses of GABA(A) receptors and their mRNAs in WSS-1 cells confirm the presence of alpha1 and gamma2 subunits and suggest the existence of an endogenous beta3 subunit. Whole-cell GABA-evoked currents recorded from untransfected WSS-1 cells were blocked by bicuculline methiodide and enhanced by anesthetics and anticonvulsants including the subunit-selective compounds diazepam and loreclezole. These data suggest that, in addition to the gamma2 subunit, WSS-1 cell receptors also contain beta2/3 subunits. RT-PCR revealed that WSS-1 cells and parental HEK293 cells contain beta3 mRNA. We examined the contribution of the beta3 subunit in the function of receptors formed by expression of alpha1 and gamma2S subunits. Untransfected HEK293 cells were unresponsive to GABA. Cells transfected with alpha1 and gamma2S cDNAs displayed small diazepam and loreclezole responsive GABA-activated currents. By contrast, the expression of alpha1 and gamma2S cDNAs in the neuroblastoma NB41A3 cell line, that lacks beta subunit mRNAs, failed to produce functional receptors. These data reaffirm that alpha1 and gamma2S subunits alone do not form functional GABA(A) receptors and that receptors of WSS-1 cells contain alpha1, beta3 and gamma2S subunits.     

Barbiturate interactions at the human GABAA receptor: dependence on receptor subunit combination.

1. Human GABAA receptors containing different alpha and beta subunits with a gamma 2s subunit were expressed in Xenopus oocytes and the effects of pentobarbitone on these subunit combinations were examined by electrophysiological recording of GABA currents with the two-electrode voltage-clamp method. 2. Pentobarbitone has previously been shown to have three actions on GABAA receptors: a potentiation of GABA responses, a direct activation of GABAA receptors and, at high concentrations, a block of the GABA chloride channel. In this study pentobarbitone activity consisted of the above mentioned three components on all the subunit combinations tested. However, the affinities and efficacies varied with receptor subtype. 3. Potentiation of GABA by pentobarbitone occurred over the same concentration-range for all the subunits with affinities in the range of 20-35 microM. The degree of potentiation obtained, however, varied from 236% of GABA EC20 on alpha 1 beta 2 gamma 2s to 536% on alpha 6 beta 2 gamma 2s. 4. Examination of the direct effect of pentobarbitone revealed that the type of alpha subunit present determines both the degree of affinity and efficacy obtained. Receptors containing an alpha 6 subunit produced maximum direct responses to pentobarbitone larger than that obtainable with maximum GABA (150% to 170% of maximum GABA). The maximum direct pentobarbitone response obtainable with other alpha subunits ranged between 45% of maximum GABA for alpha 5 beta 2 gamma 2s to 82% for alpha 2 beta 2 gamma 2s. The affinity of the direct action of pentobarbitone on alpha 6 beta 2 gamma 2s was 58 microM compared to affinities for the other alpha subunits ranging from 139 microM on alpha 2 beta 2 gamma 2s to 528 microM on alpha 5 beta 2 gamma 2s. 5. The type of beta subunit present did not influence the direct action of pentobarbitone to the same extent as the alpha subunit. There were no significant differences between affinity or efficacy on oocytes expressing alpha 6 and gamma 2s with beta 1, beta 2 or beta 3. Affinities and efficacies on oocytes expressing alpha 1 and gamma 2s with beta 1, beta 2 or beta 3 were significantly different with pentobarbitone having a higher affinity and efficacy on alpha 1 beta 3 gamma 2s followed by alpha 1 beta 2 gamma 2s and then alpha 1 beta 1 gamma 2s. 6. The direct effect of pentobarbitone was blocked by picrotoxin but not by competitive antagonists, such as bicuculline or SR95531, indicating that the direct agonist activity of pentobarbitone was not mediated via the GABA binding site. 7. For the first time the influence of the various alpha and beta subunits on the effects of pentobarbitone were demonstrated. The results indicate that GABAA receptors containing alpha 6 subunits have both a higher affinity and efficacy for direct activation by pentobarbitone, and reveal that pentobarbitone binds to more than one site on the GABAA receptor, and these are dependent on receptor subunit composition.     

Mutation of the GABAA receptor M1 transmembrane proline increases GABA affinity and reduces barbiturate enhancement

All GABA(A) receptor (GABAR) subunits include an invariant proline in a consensus motif in the first transmembrane segment (M1). In receptors containing bovine alpha1, beta1 and gamma2 subunits, we analyzed the effect of mutating this M1 proline to alanine in the alpha1 or beta1 subunit using 3 different expression systems. The beta1 subunit mutant, beta1(P228A), reduced the EC(50) for GABA about 10-fold in whole cell recordings in HEK293 cells and L929 fibroblasts. The corresponding alpha1 subunit mutant (alpha1(P233A)) also reduced the GABA EC(50) when expressed in Xenopus oocytes; alpha1(P233A)beta1gamma2S receptors failed to assemble in HEK293 cells. Binding of [(3)H]flumazenil and [(3)H]muscimol to transfected HEK293 cell membranes showed similar levels of receptor expression with GABARs containing beta1 or beta1(P228A) subunits and no change in the affinity for [(3)H]flumazenil; however, the affinity for [(3)H]muscimol was increased 6-fold in GABARs containing beta1(P228A) subunits. In L929 cells, presence of the beta1(P228A) subunit reduced enhancement by barbiturates without affecting enhancement by diazepam or alfaxalone. Single channel recordings from alpha1beta1gamma2S and alpha1beta1(P228A)gamma2L GABARs showed similar channel kinetics, but beta-mutant containing receptors opened at lower GABA concentrations. We conclude that the beta1 subunit M1 segment proline affects the linkage between GABA binding and channel gating and is critical for barbiturate enhancement. Mutation of the M1 proline in the alpha1 subunit also inhibited receptor assembly.     

Structural and functional characterization of guanyl nucleotide-binding proteins using monoclonal antibodies to the alpha-subunit of transducin

Transducin, the GTP-binding protein of the retinal light-sensitive phosphodiesterase system, and Gs and Gi, regulatory proteins of the hormone-sensitive adenylate cyclase, are members of a family of guanyl nucleotide-binding proteins termed G proteins that are important in signal transduction. To probe relationships within this family of G proteins, monoclonal antibodies were prepared against the alpha-subunit of bovine transducin (T alpha). Three of four monoclonal antibodies were specific for T alpha and did not cross-react with other G proteins. One, MAB1, cross-reacted strongly with the alpha-subunit of Gi (Gi alpha) purified from rabbit liver and, to a lesser extent, with the alpha-subunit of Go (Go alpha) purified from bovine brain and the proto-oncogene product H-ras p21. All four monoclonal antibodies recognized epitopes on a 23-kDa tryptic peptide fragment of T alpha which is derived from the N-proximal region. The three monoclonal antibodies that recognized only T alpha inhibited rhodopsin-stimulated GTP binding and hydrolysis by transducin, whereas MAB1 had no significant effect in these assays. These studies demonstrate that, within the 23-kDa tryptic peptide of T alpha, there is a domain(s) unique to T alpha that is involved in GTP binding and hydrolysis and another domain which is highly conserved in T alpha and to a lesser extent in other G proteins. Prior studies have identified regions involved in nucleotide binding and hydrolysis that are homologous in all G proteins. The observations reported here are consistent with the conclusion that the G proteins may have in addition unique regions involved in these functions.