The G-protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho.

Lysophosphatidic acid (LPA) utilizes a G-protein-coupled receptor to activate the small GTP-binding protein Rho and to induce rapid remodeling of the actin cytoskeleton. We studied the signal transduction from LPA receptors to Rho activation. Analysis of the G-protein-coupling pattern of LPA receptors by labeling activated G-proteins with [alpha-32P]GTP azidoanilide revealed interaction with proteins of the Gq, Gi, and G12 subfamilies. We could show that in COS-7 cells, expression of GTPase-deficient mutants of Galpha12 and Galpha13 triggered Rho activation as measured by increased Rho-GTP levels. In Swiss 3T3 cells, incubation with LPA or microinjection of constitutively active mutants of Galpha12 and Galpha13 induced formation of actin stress fibers and assembly of focal adhesions in a Rho-dependent manner. Interestingly, the LPA-dependent cytoskeletal reorganization was suppressed by microinjected antibodies directed against Galpha13, whereas Galpha12-specific antibodies showed no inhibition. The tyrosine kinase inhibitor tyrphostin A 25 and the epidermal growth factor (EGF) receptor-specific tyrphostin AG 1478 completely blocked actin stress fiber formation caused by LPA or activated Galpha13 but not the effects of activated Galpha12. Also, expression of the dominant negative EGF receptor mutant EGFR-CD533 markedly prevented the LPA- and Galpha13-induced actin polymerization. Coexpression of EGFR-CD533 and activated Galpha13 in COS-7 cells resulted in decreased Rho-GTP levels compared with expression of activated Galpha13 alone. These data indicate that in Swiss 3T3 cells, G13 but not G12 is involved in the LPA-induced activation of Rho. Moreover, our results suggest an involvement of the EGF receptor in this pathway.

Receptors couple to L-type calcium channels via distinct Go proteins in rat neuroendocrine cell lines.

1. The present study examines the hypothesis of G protein subtype selectivity in receptor-induced inhibition of calcium channel currents (ICa) in the insulin-secreting RINm5F and pituitary GH3 rat cell lines. Specificity of receptor coupling to G proteins was studied by infusion of purified G alpha isoforms into cells via a patch pipette. 2. In RINm5F cells, the neuropeptide galanin inhibited dihydropyridine (DHP)- and omega-conotoxin-sensitive components of ICa and slowed down their activation kinetics. In GH3 cells, DHP-sensitive ICa was inhibited by galanin, as well as by somatostatin and carbachol. Agonist-induced ICa inhibition was suppressed by pertussis toxin (PTX) pretreatment of the cells. In PTX-pretreated cells of either cell line, the response to galanin was restored only by the G alpha o1 subunit. Following PTX treatment of GH3 cells, only the G alpha o1 subunit restored carbachol-induced inhibition of ICa, whereas only the G alpha o2 subunit restored somatostatin-induced inhibition of ICa. G(i) subtypes had no effect on ICa inhibition. 3. Both cell lines expressed two distinct immunoreactive Go proteins. Whereas in RINm5F cell membranes Go1 was found to be the predominant isoform, we detected more Go2 than Go1 in GH3 cell membranes. Nevertheless, all agonists stimulated incorporation of the photoreactive GTP analogue [alpha-32P]GTP azidoanilide into both G(o) isoforms. 4. The results indicate that the same Go subtype, i.e. Go1, mediates galanin-induced inhibition of ICa in both cell lines and that the Go subtype specificity of receptor-G protein coupling is confined to intact cells.

Interaction of G protein Gbetagamma dimers with small GTP-binding proteins of the Rho family.

Gbetagamma dimers of heterotrimeric G proteins have been shown to be important for the translocation of cytosolic proteins to membranes. The involvement of Gbetagamma in those signaling processes mediated by small GTP-binding proteins of the Rho family was studied using purified proteins. We showed specific binding of bovine brain Gbetagamma to immobilized GST-Rho fusion proteins. In addition, brain Gbetagamma, but not transducin Gbetagamma, was able to inhibit GTPgammaS binding to GST-Rho in a concentration-dependent manner. GTPgammaS binding to GST-Rac was also decreased by brain Gbetagamma whereas nucleotide binding to GST-Cdc42 was not changed. We conclude that Gbetagamma dimers may participate in the process of membrane attachment and/or other regulations of Rho and Rac.

Direct activation of trpl cation channels by G alpha11 subunits.

G proteins of the Gq/11 subfamily functionally couple cell surface receptors to phospholipase C beta (PLC beta) isoforms. Stimulation of PLC beta induces Ca2+ elevation by inositol 1,4,5-trisphosphate (InsP3)-mediated Ca2+ release and store-dependent 'capacitative' Ca2+ entry through Ca(2+)-permeable channels. The Drosophila trp gene, as well as some human trp homologs, code for such store-operated channels. The related trp-like (trpl) gene product also forms a Ca(2+)-permeable cation channel, but is not activated by store depletion. Co-expression of the constitutively active Gq subfamily member G alpha 11 (G alpha 11) with trpl enhanced trpl currents 33-fold in comparison with co-expression of trpl with other G alpha isoforms or G beta gamma complexes. This activation could not be attributed to signals downstream of PLC beta. In particular, InsP3 infusion, modulation of protein kinase C activity or elevation of intracellular calcium concentration failed to induce trpl currents. In contrast, purified G alpha 11 (but not other G protein subunits) activated trpl channels in inside-out patches. We conclude that trpl is regulated by G11 proteins in a membrane-confined manner not involving cytosolic factors. Thus, G proteins of the Gq subfamily may induce Ca2+ entry not only indirectly via store-operated mechanisms but also by directly stimulating cation channels.

Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of G alpha 12 purified from rat brain.

G12 and G13 are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of G alpha 12 from rat brain membranes. G alpha 12 was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified G alpha 12 bound guanosine 5'-[gamma-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active G alpha 12, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G12 subfamily revealed differences in the affinity of G alpha 12 and G alpha 13 for G beta gamma. G alpha 12 required a higher Mg2+ concentration for AlF4- -induced dissociation from immobilized G beta gamma than did G alpha 13. In addition, the G12 subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G12 to form supramolecular structures in comparison to G13 and other G-proteins. These G13 structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active G alpha 12. Despite these distinct biochemical characteristics of G12 and G13, both purified G-proteins coupled to a recombinant thromboxane A2 (TXA2) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G12 subfamily, and (2) their specific coupling to TXA2 receptors.

Species- and tissue-dependent diversity of G-protein beta subunit phosphorylation: evidence for a cofactor.

We previously reported that, in the membranes of HL-60 cells during activation of G-proteins, a phosphate transfer reaction occurs which involves transient G-protein beta subunit (G beta) phosphorylation [Wieland, Nürnberg, Ulibarri, Kaldenberg-Stasch, Schultz and Jakobs (1993) J. Biol. Chem. 268, 18111-18118]. Here, the generality of this phenomenon is evaluated by studying membranes of various tissues obtained from different mammalian species. All membranes tested expressed at least G beta 1 and G beta 2 subunits. Cell membranes from bovine and porcine brain and liver, rat brain and human blood cells exhibited predominantly G beta 1 or both subtypes at roughly equal concentrations. In contrast, significantly more G beta 2 immunoreactivity was detected in membranes from human placenta. Bovine and porcine liver membranes exhibited weak, G beta-specific immunoreactive signals. Conversely, these membranes showed the highest levels of G beta phosphorylation after incubation with [gamma-32P]GTP or 35S-labelled guanosine 5'-[gamma-thio]triphosphate. Interestingly, G beta-specific phosphorylation of membranes from human erythrocytes and platelets was very weak. G beta phosphorylation was confirmed by immunoprecipitation with G beta-specific antibodies, and the target amino acid was identified as histidine. On SDS/PAGE, phosphorylated or thiophosphorylated G beta-proteins differed in their apparent molecular size from unmodified G beta-proteins. Moreover, phosphorylated G beta-proteins differed in a species-dependent fashion in their electrophoretic mobility. Solubilization of membrane proteins with detergent did not abolish G beta phosphorylation. In contrast, reconstituted purified Gi/Go proteins showed no G beta phosphorylation. From these experiments we conclude that: (i) G beta phosphorylation represents a general phenomenon occurring in the cells of various species to different degrees, (ii) phosphorylated G beta-proteins exhibit species-dependent diverse electrophoretic mobilities, and (iii) G beta phosphorylation requires a membrane-associated cofactor(s) which is lost during routine G-protein purification.

Cationic-amphiphilic arpromidine-derived guanidines and a histamine trifluoromethyl-toluidide derivative may activate pertussis toxin-sensitive G-proteins by a receptor-independent mechanism.

Formyl peptides activate superoxide anion (O2-) formation in human neutrophils and in HL-60 cells via pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins), and histamine (HA) mediates inhibition of O2- formation via H2-receptors. We have studied the effects of lipophilic arpromidine-derived guanidines, which are potent, full H2-receptor agonists in the guinea pig atrium, on O2- formation and on activation of G-proteins in HL-60 membranes and on purified G-proteins. We have also studied the effects of a HA trifluoromethyl-toluidide derivative (HTMT), a cationic-amphiphilic HA derivative which activates O2- formation in HL-60 cells through a mechanism which is independent of known HA receptor subtypes, on G-protein activation. Guanidines, at concentrations, up to 30 mumol/l inhibited and, at concentrations above 30 mumol/l, enhanced formyl peptide-induce O2- formation in neutrophils. In HL-60 cells, guanidines per se activated O2- formation. The stimulatory effects of guanidines on O2- formation were not inhibited by H1- or H2-receptor antagonists. In HL-60 membranes, guanidines and HTMT, activated high-affinity GTPase in a PTX-sensitive manner. These substances also increased GTP hydrolysis effected by transducin and Gi/G(o)-proteins. Our data suggest that lipophilic guanidines and HTMT may act as receptor-independent activators of PTX-sensitive G-proteins, resulting in stimulation of O2- formation.

A non-ionic vesicle lipid enhances mastoparan-stimulated GTPase activity of heterotrimeric G-proteins.

Isolated heterotrimeric G-proteins exhibit full biological activity when reconstituted into liposomes. Here, we investigated the non-ionic surfactant macrogol-260-cetylstearylether (TA 6) as an efficient vehicle for the reconstitution of G-proteins. Reconstitution efficiency of G-proteins was recorded by GTP gamma S-binding. Their biological potency was measured as basal and mastoparan-stimulated GTPase-activity. G-proteins were fully active when associated with liposomes. On the other hand, G-proteins solubilized by TA 6 micelles or reconstituted into pure TA 6 vesicles exhibited impaired biological activity. However, vesicles containing different ratios of azolectin and non-ionic TA 6 showed about 50% higher reconstitution efficiency as compared to pure liposomes. In addition, basal and mastoparan-stimulated GTPase-activity in vesicles containing an axolectin/TA 6 ratio of 3:1 were 2.7 and 9.1 fold higher than in pure liposomes, respectively. These data emphasize that the composition of the lipid membranous environment significantly influences G-protein activity.

Histamine receptor-dependent and/or -independent activation of guanine nucleotide-binding proteins by histamine and 2-substituted histamine derivatives in human leukemia (HL-60) and human erythroleukemia (HEL) cells.

In dibutyryl cAMP-differentiated human leukemia (HL-60) cells, the potent histamine H1-receptor agonist, 2-(3-chlorophenyl)histamine, activates pertussis toxin (PTX)-sensitive guanine nucleotide-binding proteins (G-proteins) of the Gi-subfamily by a mechanism which is independent of known histamine receptor subtypes (Seifert et al. Mol Pharmacol 45: 578-586, 1994). In order to learn more about this G-protein activation, we studied the effects of histamine and various 2-substituted histamine derivatives in various cell types and on purified G-proteins. In HL-60 cells, histamine and 2-methylhistamine increased cytosolic Ca2+ concentration ([Ca2+]i) in a clemastine-sensitive manner. Phenyl- and thienyl-substituted histamines increased [Ca2+]i as well, but their effects were not inhibited by histamine receptor antagonists. 2-Substituted histamines activated high-affinity GTPase in HL-60 cell membranes in a PTX-sensitive manner, with the lipophilicity of substances increasing their effectiveness. Although HEL cells do not possess histamine receptors mediating rises in [Ca2+]i, 2-(3-bromophenyl)histamine increased [Ca2+]i in a PTX-sensitive manner. It also increased GTP hydrolysis by Gi-proteins in HEL cell membranes. All these stimulatory effects of 2-substituted histamine derivatives were seen at concentrations higher than those required for activation of H1-receptors. In various other cell types and membrane systems, 2-substituted histamine derivatives showed no or only weak stimulatory effects on G-proteins. 2-Substituted histamine derivatives activated GTP hydrolysis by purified bovine brain Gi/Go-proteins and by pure Gi2 (the major PTX-sensitive G-protein in HL-60 and HEL cells). Our data suggest the following: (1) histamine and 2-methylhistamine act as H1-receptor agonists in HL-60 cells; (2) incorporation of bulky and lipophilic groups results in loss of H1-agonistic activity of 2-substituted histamine derivatives in HL-60 cells but causes a receptor-independent G-protein-stimulatory activity; (3) the effects of 2-substituted histamine derivatives on G-proteins are cell-type specific.

The class III antiarrhythmic drug amiodarone directly activates pertussis toxin-sensitive G proteins.

The class III antiarrhythmic drugs amiodarone and bretylium tosylate are cationic/amphiphilic, and various substances with these physico-chemical properties are known to directly activate heterotrimeric regulatory G proteins. We asked the question of whether class III antiarrhythmic drugs are also direct G protein activators, using HL-60 leukemic cells and purified bovine brain G proteins as model systems. In HL-60 cell membranes, aminodarone increased high affinity GTP hydrolysis with an EC50 of 7.5 microM. The stimulatory effect of amiodarone on GTP hydrolysis was inhibited by pertussis toxin. Amiodarone stimulated binding of guanosine-5'-O-(3-thio)triphosphate to, and incorporation of GTP azidoanilide into, Gi protein alpha subunits in HL-60 membranes. The drug increased the cytosolic Ca2+ concentration in HL-60 cells in the presence but not in the absence of extracellular Ca2+. Amiodarone-induced increases in the cytosolic Ca2+ concentration were reduced by pertussis toxin and by a blocker of non-selective cation channels, SK&F 96365. Amiodarone activated the GTPase of reconstituted Gi/G(o) proteins and G12 with EC50 values of 20 microM and 50 microM, respectively. Bretylium tosylate did not increase GTP hydrolysis in HL-60 membranes or with Gi/G(o) proteins. Our data suggest that amiodarone but not bretylium tosylate is a direct activator of Gi and G(o) proteins and that amiodarone activates nonselective cation channels in HL-60 cells via Gi proteins and independently of Ca2+ mobilization from intracellular stores. Future studies will have to test the hypothesis that direct G protein activation by amiodarone contributes to its toxic and/or therapeutic effects.

Mastoparan may activate GTP hydrolysis by Gi-proteins in HL-60 membranes indirectly through interaction with nucleoside diphosphate kinase.

The wasp venom, mastoparan (MP), activates reconstituted pertussis toxin (PTX)-sensitive G-proteins in a receptor-independent manner. We studied the effects of MP and its analogue, mastoparan 7 (MP 7), on G-protein activation in HL-60 cells and a reconstituted system and on nucleoside diphosphate kinase (NDPK)-catalysed GTP formation. MP activated high-affinity GTP hydrolysis in HL-60 membranes with an EC50 of 1-2 microM and a maximum at 10 microM. Unlike the effects of the formyl peptide receptor agonist, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), on GTPase, those of MP were only partially PTX-sensitive. MP-induced rises in cytosolic Ca2+ concentration and superoxide-anion formation in intact HL-60 cells were also only incompletely PTX-sensitive. N-Ethylmaleimide inhibited MP-stimulated GTP hydrolysis to a greater extent than that stimulated by fMet-Leu-Phe. Unlike the latter, MP did not enhance incorporation of GTP azidoanilide into, and cholera toxin-catalysed ADP-ribosylation of, Gi-protein alpha-subunits in HL-60 membranes. By contrast to fMet-Leu-Phe, MP did not or only weakly stimulated binding of guanosine 5'-[gamma-thio]triphosphate to Gi-protein alpha-subunits. MP 7 was considerably more effective than MP at activating the GTPase of reconstituted Gi/G(o)-proteins, whereas in HL-60 membranes, MP and MP 7 were similarly effective. MP and MP 7 were similarly effective at activating [3H]GTP formation from [3H]GDP and GTP in HL-60 membranes and by NDPK purified from bovine liver mitochondria. Our data suggest the following: (1) MP activates Gi-proteins in HL-60 cells, but (2) the venom does not simply mimic receptor activation. (3) MP and MP 7 may activate GTP hydrolysis in HL-60 membranes indirectly through interaction with NDPK. (4) MP 7 is a more effective direct activator of PTX-sensitive G-proteins than MP, whereas with regard to NDPK, MP and MP 7 are similarly effective.

Rapid isolation of G alpha 13 from bovine brain membranes: supportive effect of ethylene glycol.

G13 belongs to the G12-subfamily of heterotrimeric regulatory G-proteins. Employing specific antibodies, we isolated G alpha 13 from bovine brain by a four-step purification protocol combining conventional and affinity chromatography. The use of ethylene glycol as a protective agent influenced the elution properties of G alpha 13 markedly. Only in the presence of ethylene glycol (30% v/v) a clear separation of G alpha 13 from other G-proteins was achieved during the initial anion exchange chromatography. This allowed isolation of G alpha 13 by subunit exchange chromatography on beta gamma-agarose. G alpha 13 was only released from immobilized beta gamma-dimers via activation by AMF but not by GTP gamma S, pointing to a poor basal nucleotide exchange of this protein. In contrast, N-terminally truncated G alpha 13 did not bind to immobilized beta gamma-dimers.

Purification of the G-protein G13 from rat brain membranes.

Significant amounts of G13, a member of the recently described G12-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G13 (G alpha 13) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G13 from most of the other G-proteins, including Gq/11. Moreover, G alpha 13-enriched fractions obtained from this chromatographic step were devoid of beta gamma-dimers, despite the absence of G-protein-activating agents. Nevertheless, the purified G alpha 13 retained its ability to interact with beta gamma-dimers under appropriate conditions, i.e. the addition of Lubrol PX instead of cholate as detergent and the omission of ethylene glycol routinely used as a protecting additive. The interaction was demonstrated by (i) the binding of G alpha 13 to immobilized beta gamma-complexes and (ii) the formation of stable heterotrimers during sucrose-density-gradient centrifugation. Furthermore, our studies on G alpha 13 provide evidence for an extremely slow basal GDP/GTP exchange rate. The purified protein showed negligible binding of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]). Accordingly, dissociation of G alpha 13 from immobilized beta gamma-complexes was achieved by AlF4-/Mg2+, but not by GTP[S]. These data indicate that G13 exhibits properties highly distinct from those of other G-proteins.

Purification of a novel G-protein alpha 0-subtype from mammalian brain.

Three distinct G-protein alpha o-subtypes, i.e. alpha o1, alpha o2 and a newly observed 'alpha o3', are present in membranes of mammalian brain, each appearing as two isoforms on SDS/PAGE. Only alpha o1 and alpha o2 appear to be substrates for pertussis toxin (PTX) when membranes or partially purified proteins are examined. In order to elucidate the apparent PTX-resistance of the third alpha o-subtype, we purified alpha o3 from porcine and bovine brain membranes. During the purification procedures, alpha o3 occurred in its dissociated monomeric form and, together with beta gamma-complexes, as a heterotrimer. In a first attempt, we used purified G-protein alpha i/alpha o-mixtures to obtain a final separation of alpha o3. By using f.p.l.c. anion-exchange chromatography on a Mono Q column, complete separation of alpha i1 and alpha o2 was achieved. Partial resolution of alpha o1, alpha i2 and alpha o3 was observed; alpha o3 was eluted between alpha o1 and alpha i2. If alpha o-subunits free from alpha i contaminants were loaded on to the Mono Q column, all three alpha o-subtypes were resolved. The identity of the third subtype as an alpha o-subtype was confirmed by sequence analysis of tryptic fragments. All three alpha o-subtypes bound GTP[S]. Purified alpha o3 was ADP-ribosylated when subjected to PTX treatment in the presence of beta gamma-subunits, and on SDS/PAGE the mobility of alpha o3 was similar to that of ADP-ribosylated alpha o1. On the basis of results obtained with subtype-specific antibodies, the third alpha o-subtype is immunologically more related to alpha o1 than to alpha o2. Purified alpha o3 failed to reconstitute carbachol-mediated inhibition of Ca2+ current in PTX-pretreated SH-SY5Y-cells, whereas alpha o1 and alpha o2 did successfully restore this effect. We conclude that the novel alpha o3 forms differs from alpha o1 and alpha o2 in its primary structure and may be involved in signal-transduction pathways other than those described for alpha o1 and alpha o2.

Lipophilic beta-adrenoceptor antagonists and local anesthetics are effective direct activators of G-proteins.

We studied the effects of various beta-adrenoceptor (beta AR) antagonists and local anesthetics (LAs), i.e. substances possessing one basic and one lipophilic domain each, on activation of regulatory heterotrimeric guanine nucleotide-binding proteins (G-proteins). In membranes of differentiated HL-60 cells, propranolol activated high-affinity GTP hydrolysis with a half-maximal effect at 0.19 mM and a maximum at 1 mM. There was a close correlation between the log Q values (logarithm of the octanol: water partition coefficient) of beta AR antagonists and the logarithm of their effectiveness at activating GTPase (EC 3.6.1.-) in HL-60 membranes. The lipophilic LA, tetracaine, was also an effective activator of GTPase in HL-60 membranes, whereas more hydrophilic LAs were less stimulatory (bupivacaine and lidocaine) or even inhibitory (procaine). Propranolol and tetracaine also stimulated binding of guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) to HL-60 membranes, but their stimulatory effects on GTP[gamma S] binding were smaller than on GTP hydrolysis. The stimulatory effects of propranolol and tetracaine on GTPase and GTP[gamma S] binding were inhibited by pertussis toxin. Propranolol and tetracaine effectively activated GTP hydrolysis of a reconstituted mixture of bovine brain Gi/Go-proteins, but the concentrations of substances needed for GTPase activation were higher than in HL-60 membranes. Procaine showed stimulatory effects on the GTPase of Gi/Go-proteins. Our data show that beta AR antagonists and LAs activate pertussis toxin-sensitive G-proteins, presumably through interaction with the C-terminus of their alpha-subunits. Apparently, the lipophilic domain of beta AR antagonists and LAs is more important for G-protein activation than the basic domain. We discuss the possibility that activation of nucleoside diphosphate kinase by beta AR antagonists and LAs contributes to their stimulatory effects on GTP hydrolysis in HL-60 membranes.

Binding of cholecystokinin-8 (CCK-8) peptide derivatives to CCKA and CCKB receptors.

The structural requirements for the selective binding of cholecystokinin-8 (CCK-8)-related peptides to peripheral (CCKA) receptors are not sufficiently understood. In this study, the interaction of a series of newly shortened analogues of CCK-8 with both receptor subtypes was analyzed by displacement studies using [3H]-CCK-8 and 125I-Bolton-Hunter (BH)-CCK-8 as radioligands. The pentapeptide derivative of CCK-8, succinyl-Tyr (SO3H)-Met-Gly-Trp-Met-phenethylamide, was found to bind selectively with high affinity to the CCKA receptor. The replacement of Met28 and/or Met31 by norleucine and of L-Trp30 by its D-analogue had no significant effect on the binding properties of the peptide. Further C-terminal shortening resulted in a drastic loss of affinity and selectivity of the CCK receptor binding.

G12 and G13 alpha-subunits are immunochemically detectable in most membranes of various mammalian cells and tissues.

The cDNAs of two putatively pertussis toxin-insensitive G-protein alpha-subunits, alpha 12 and alpha 13, were recently cloned. mRNA analyses based on the reverse transcriptase polymerase chain reaction indicated a widespread distribution of both mRNAs [Strathmann, M. P., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. USA 88, 5582-5586]. Generating specific antibodies directed against internal and C-terminal peptide sequences, we identified alpha 12 protein in all and alpha 13 protein in most tissues and cell lines tested. No species differences were observed, indicating a high degree of identity between mammalian species. Strong immunoreactive signals of both proteins were obtained in neuronal cell membranes of various species. Our results support the hypothesis that G12 and G13 are involved in pertussis toxin-insensitive pathways of signal transduction common to most tissues.

Studies with new ergoline derivatives on the effects of central and peripheral 5-hydroxytryptamine receptors.

The antiserotonin properties of a series of new ergoline derivatives were investigated in several pharmacological test systems which have been proposed for the characterization of putative antagonists at central and peripheral 5-HT2 receptors. In radioligand binding studies with [3H]ketanserin among the new ergolines only 1-methyl-2-brom-9,10-dihydrolysergic acid-bis(beta-acetoxyethyl)-amide (AWD 52-336) showed high affinity at cortical 5-HT2 receptors (Ki-5.4 nmol/l). In 5-HT-amplified ADP-induced aggregation of human platelets 6-nor-6-propyl-9,10-dihydro ergometrine (AWD 52-227) and 9,10-dihydrolysergic acid-di-ethanol-amide (AWD 52-302) were potent inhibitors of 5-HT response. Comparison of this two in vitro tests demonstrated a significant correlation (r = 0.636; p < 0.05) between the ability of the ergolines to block the 5-HT aggregation mediated by platelet 5-HT2 receptors and their affinity to [3H]ketanserin-labelled binding sites in rat cortical membranes. In the used in vivo tests (tryptamine tremor, 5-HTP-induced head twitches) 1-methyl-9,10-dihydrolysergic acid-bis(beta-acetoxyethyl)-amide (AWD 52-83) and AWD 52-336 were found to antagonize the behavioural responses with comparatively moderate potency. The results suggest, therefore, that AWD 52-83 and AWD 52-336 may be both central and peripheral acting 5-HT2 antagonists, whereas AWD 52-227 and AWD 52-302 seem to be potent blockers at peripheral 5-HT2 receptors. Furthermore, the obtained results allow to reveal structure-activity relationships of ergolines. Substitution in position 1 in the tetracyclic ergoline ring system may be important with respect to the efficacy at central 5-HT2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

CCK-8-related C-terminal tetrapeptides: affinities for central CCKB and peripheral CCKA receptors.

We investigated the binding affinity of new tetrapeptides derived from the C-terminal sequence of CCK8 to central CCKB and peripheral CCKA receptors. Compound 1 (Boc-Trp-Met-Asp-Phe-NH2) showed high affinity for central CCKB receptors (Ki 4.2 x 10(-8) M, pancreas/cortex ratio = 283). Compounds 2 (Suc-Trp-Met-Asp-Phe-NH2) and 3 (Suc-Trp-Leu-Asp-Phe-NH2) also exhibited high affinity (Ki 2.7 x 10(-8) M and 5.6 x 10(-8) M, respectively) but their CCKB selectivity was nearly 50 times higher (Ki ratio greater than 14,000). Replacement of Met or Leu by other amino acids resulted in less effective tetrapeptides.

[A preclinical study of the organ distribution and radiation dosage of radioiodinated iodolisuride]. / Vorklinische Untersuchung zur Organverteilung und Strahlenbelastung von Radiojod-Jodlisurid.

The distribution in rats of 125I-iodo-lisuride was studied. Three rats each were sacrificed at fixed intervals between 5 min and 24 h p.i., and the radioactivity was measured in isolated organs and parts of the body. The organ distribution and biexponential blood disappearance were similar to values for unlabeled lisuride. The radiation dose was estimated for man assuming a 123I label. The resulting doses were comparable to those from other radiopharmaceuticals in clinical use.