Rainer Harhammer

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.

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 Ga isoforms into cells via a patch pipette. 2 In RINm5F cells, the neuropeptide galanin inhibited dihydropyridine (DHP)‐ and ω‐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 Gα01 subunit. Following PTX treatment of GH3 cells, only the Gα01 subunit restored carbachol‐induced inhibition of ICa, whereas only the Gαo2 subunit restored somatostatin‐induced inhibition of ICa. Gl subtypes had no effect on ICa inhibition. 3 Both cell lines expressed two distinct immunoreactive Go proteins. Whereas in RINm5F cell membranes G01 was found to be the predominant isoform, we detected more G02 than G01 in GH3 cell membranes. Nevertheless, all agonists stimulated incorporation of the photoreactive GTP analogue [α‐ 32P]GTP azidoanilide into both Go isoforms. 4 The results indicate that the same Go subtype, i.e. G01, 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 Gβγ dimers with small GTP‐binding proteins of the Rho family

Gβγ dimers of heterotrimeric G proteins have been shown to be important for the translocation of cytosolic proteins to membranes. The involvement of Gβγ 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 Gβγ to immobilized GST‐Rho fusion proteins. In addition, brain Gβγ, but not transducin Gβγ, was able to inhibit GTPγS binding to GST‐Rho in a concentration‐dependent manner. GTPγS binding to GST‐Rac was also decreased by brain Gβγ whereas nucleotide binding to GST‐Cdc42 was not changed. We conclude that Gβγ dimers may participate in the process of membrane attachment and/or other regulations of Rho and Rac.

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.

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 GTPγS-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.