Biomedical subjects

K H Jakobs

Publications and source records attributed to K H Jakobs.

At least 73 records · Page 4Linked to original sources

PubMed1996

Receptor internalization delays m4 muscarinic acetylcholine receptor resensitization at the plasma membrane.

We analyzed the role of receptor internalization and recycling in muscarinic acetylcholine receptor (mAChR) desensitization and resensitization. Incubation of Chinese hamster ovary cells stably expressing the m4 mAChR with 1 mM carbachol for 1 hr reduced cell surface receptor number by 50-60% with no change in total receptor number. Pretreatment of the cells with 450 mM sucrose, which did not affect the ability of m4 receptors to inhibit forskolin-stimulated cAMP accumulation, completely blocked receptor internalization. On the other hand, the carbachol treatment reduced the ability of m4 receptors to inhibit cAMP accumulation in both sucrose-treated and untreated cells, with a similar onset and to a similar extent. The EC50 value for carbachol was increased approximately 10-fold, and maximal inhibition determined at 100 microM carbachol was reduced approximately 50%. In contrast, thrombin-induced inhibition of cAMP accumulation was not affected. Recycled receptors in cells not treated with sucrose remained refractory to carbachol stimulation for > or = 2 hr after agonist removal, even though cell surface receptor number had recovered completely within 1 hr. In contrast, resensitization of receptor function was very rapid in cells treated with sucrose. Ten minutes on removal of agonist, mAChRs in the plasma membrane of sucrose-treated cells were fully resensitized. Also, an internalization-defective m4 mAChR mutant, T399A, that was found to desensitize similar to the wild-type receptor, resensitized more rapidly than the wild-type receptor. We conclude that desensitization and resensitization of m4 mAChRs in Chinese hamster ovary cells can occur at the plasma membrane and that receptor internalization strongly delays the process of resensitization of desensitized receptors.

Animals↗

PubMed1996

Inhibition by toxin B of inositol phosphate formation induced by G protein-coupled and tyrosine kinase receptors in N1E-115 neuroblastoma cells: involvement of Rho proteins.

G protein-coupled receptors activate phospholipase C (PLC)-beta isoforms by the alpha or beta gamma subunits of G proteins, whereas growth-factor receptors activate PLC-gamma isoforms by phosphorylating tyrosine residues of the enzyme. As a common substrate for PLC enzymes, phosphatidylinositol 4,5-bisphosphate [Ptdins(4,5)P2] may play a pivotal role in the regulation of cellular PLC activity. Because small-molecular-weight G proteins have been implicated in the synthesis of Ptdins(4,5)P2, we studied the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates small G proteins of the Rho family, on receptor-stimulated PLC activity. We report here that in N1E-115 neuroblastoma cells, stimulation of inositol phosphate formation by the G protein-coupled receptor agonists bradykinin and lysophosphatidic acid and by the tyrosine kinase receptor agonist platelet-derived growth factor is largely attenuated by toxin B treatment. Furthermore, inositol phosphate production stimulated by the stable GTP analog guanosine 5'-O-(3-thio)-triphosphate in permeabilized N1E-115 cells was inhibited by C3 exoenzyme, which specifically inactivates Rho proteins. The inhibition by toxin B was apparently not caused by its effect on the cytoskeleton. In addition, the level of platelet-derived growth factor receptors, which was studied with immunoblotting, was unaffected by toxin B. Using exogenous Ptdlns(4,5)P2 as PLC substrate, it was found that the intrinsic enzymatic activity of PLC activated either by Ca2+ or by guanosine 5'-O-(3-thio)triphosphate was not altered by toxin B. However, toxin B decreased strongly, by up to 80%, the cellular level of Ptdins(4,5)P2 in a concentration-dependent manner, without changing those of phosphatidylinositol and phosphatidylinositol 4-phosphate. These results, together with the recent finding that Rho family proteins can regulate phosphatidylinositol 4-phosphate 5-kinase activity, demonstrate that Rho proteins are presumably important regulators of Ptdins(4,5)P2 synthesis and, thereby, play an integral role in the regulation of cellular signaling by PLC enzymes.

ADP Ribose Transferases↗

PubMed1996

m3 Muscarinic receptor-induced and Gi-mediated heterologous potentiation of phospholipase C stimulation: role of phosphoinositide synthesis.

Agonist activation of thrombin and purinergic receptors endogenously expressed in human embryonic kidney (HEK) cells and of the stably expressed m3 muscarinic acetylcholine receptor (mAChR) induces phospholipase C (PLC) stimulation, with the most pronounced PLC stimulation observed on mAChR activation. These receptor responses were pertussis toxin (PTX) insensitive and nonadditive, suggesting that the receptors share common signaling pathways. Short term (2 min) pretreatment of HEK cells with carbachol (1 mM), but not ATP, followed by agonist washout, caused a long-lasting (> or = 90 min) sensitization of PLC responses. At 30 min after carbachol treatment and washout, mAChR-stimulated PLC activity, measured as formation of either total inositol phosphates or of inositol-1,4,5-trisphosphate, was enhanced by 1.5-2-fold. PLC stimulation by thrombin and purinergic receptors was increased by approximately 3-fold. Furthermore, carbachol pretreatment also enhanced, by approximately 2.5-fold, stimulation of PLC activity on direct activation of G proteins by AIF4- and guanosine-5'-O-(3-thio)-triphosphate in intact and permeabilized cells, respectively. In contrast, PLC activities, measured with exogenous phosphatidylinositol-4,5-bisphosphate [Ptdns(4,5)P2] in HEK cell lysates, were not altered, suggesting that carbachol pretreatment may enhance the cellular level of Ptdlns(4,5)P2. Indeed, the level of Ptdlns(4,5)P2 was found to be increased by approximately 50% in HEK cells 30 min after short term carbachol treatment, whereas the level of phosphatidylinositol was not altered and that of phosphatidylinositol-4-phosphate decreased (by 40-50%). Pretreatment of HEK cells with PTX prevented the m3 mAChR-induced PLC potentiation and reduced the elevation in Ptdlns(4,5)P2 level by approximately 50%. In conclusion, short term agonist activation of m3 mAChRs stably expressed in HEK cells can lead to a longlasting heterologous potentiation of PLC signaling, which processes apparently involve PTX-sensitive G proteins and an enhanced PLC substrate supply.

Carbachol↗

PubMed1995

Activation of muscarinic K+ current in guinea-pig atrial myocytes by sphingosine-1-phosphate.

1. Activation of muscarinic K+ current (IK(ACh)) by sphingosine-1-phosphate (Sph-1-P) was studied in isolated cultured guinea-pig atrial myocytes using whole-cell voltage clamp. 2. Sph-1-P caused activation of IK(ACh) with an EC50 of 1.2 nM. The maximal current that could be activated by Sph-1-P amounted to about 90% of the IK(ACh) caused by a saturating concentration of acetylcholine (ACh, 10 microM). Sphingosine (1 microM), which can mimic the signalling effects of Sph-1-P in other cells, failed to cause measurable activation of IK(ACh). 3. IK(ACh) activation by Sph-1-P was completely suppressed in cells treated with pertussis toxin. 4. Desensitization of muscarinic receptors by pre-incubation of the cells with carbachol did not affect the response to Sph-1-P; likewise, pre-incubation of the cells with Sph-1-P resulted in a reduced sensitivity to the phospholipid but not to ACh. In contrast, pre-incubation with either Sph-1-P or a serum phospholipid previously described as activating atrial IK(ACh) resulted in reduced sensitivity to both phospholipids. 5. It is concluded that activation of IK(ACh) by Sph-1-P in atrial myocytes is induced by binding to a novel G protein-coupled phospholipid receptor.

Acetylcholine↗

PubMed1995

The role of membrane proximal threonine residues conserved among guanine-nucleotide-binding-protein-coupled receptors in internalization of the m4 muscarinic acetylcholine receptor.

Many guanine-nucleotide-binding-protein-coupled receptors contain consensus sequences for phosphorylation by cAMP-dependent protein kinase (PKA), often located in the membrane proximal regions critically important for receptor signalling. In the present study, we have evaluated by site-directed mutagenesis the role of the putative PKA phosphorylation sites in the m4 muscarinic acetylcholine receptor (mAChR), i.e. Thr145 in the second cytoplasmic loop and Thr399 in the third cytoplasmic loop, and the influence of PKA on m4 mAChR function and internalization. Antagonist binding was unaltered by any of the mutations studied, while the agonist-binding affinity was either not affected (Thr145 alanine), increased (Thr399 alanine) or decreased (Thr399 serine or aspartic acid). m4 mAChR-mediated inhibition of adenylyl cyclase was unaltered by the mutations, except for an approximately tenfold reduced agonist potency of the Thr399 aspartic acid mutated receptor. Agonist-induced receptor internalization was unaltered with Thr399 serine or aspartic acid mutations of the receptors, but was strongly decreased in its rate and extent upon replacement of Thr399, Thr145 or both of these residues with alanine. These mutational effects could not be reproduced by treatment of wild-type receptor-expressing cells with the PKA inhibitor H-8. Furthermore, maximal stimulation of cellular PKA neither affected receptor internalization nor signalling measured as receptor-mediated Ca2+ mobilization. We conclude that the membrane proximal threonine residues of the m4 mAChR are not required for receptor signalling, but replacement by alanine residues can significantly affect receptor internalization, independently of PKA phosphorylation. Sequence comparisons suggest that threonine residues at corresponding positions may be relevant to internalization of other guanine-nucleotide-binding-protein-coupled receptors.

Amino Acid Sequence↗

PubMed1995

Evidence for ADP-ribosylation-factor-mediated activation of phospholipase D by m3 muscarinic acetylcholine receptor.

Activation of phospholipase D (PLD) is a cellular response to a wide variety of extracellular ligands. However, the exact mechanisms that link cell surface receptors to PLD remain unclear. In this study, we report the involvement of the small-molecular-mass guanine-nucleotide-binding protein, ADP-ribosylation factor (ARF), in the activation of PLD by the muscarinic acetylcholine receptor (mAChR) in human embryonic kidney cells stably expressing the human m3 subtype. PLD stimulation in permeabilized cells by guanosine 5'-O-[gamma-thio]triphosphate (GTP[S]) was dependent on a cytosolic factor and reconstituted by purified recombinant ARF 1. Brefeldin A, a known inhibitor of the ARF guanine-nucleotide-exchange-factor activity in Golgi membranes, inhibited mAChR-stimulated PLD, whereas basal PLD activity and stimulation by GTP[S] were not affected. Upon cell permeabilization without the addition of stimulus, ARF proteins were released. However, the addition of GTP[S] during permeabilization and mAChR activation before permeabilization caused an almost complete and partial (about 60%) inhibition, respectively, of ARF release, indicating that ARF proteins are activated and thereby translocated to membranes. The results indicate that ARF proteins and their nucleotide-exchange factor are apparently involved in the signalling pathway leading from mAChR activation to PLD stimulation in human embryonic kidney cells.

ADP-Ribosylation Factors↗

PubMed1995

Rapid and persistent desensitization of m3 muscarinic acetylcholine receptor-stimulated phospholipase D. Concomitant sensitization of phospholipase C.

Activation of muscarinic acetylcholine receptors (mAChR) in human embryonic kidney (HEK) cells stably expressing the human m3 subtype leads to stimulation of both phospholipase C (PLC) and D (PLD). mAChR-stimulated PLD was turned off after 2 min of receptor activation with either the full (carbachol) or partial agonist (pilocarpine) and remained completely suppressed for at least 4 h. Partial recovery was observed 24 h after agonist removal. This rapid arrest of PLD response was not due to a loss of cell surface receptors and was also not caused by negative feedback due to concomitant activation of protein kinase C, tyrosine phosphorylation, increase in cytosolic calcium, or activation of Gi proteins. Furthermore, PLD stimulation by directly activated protein kinase C and GTP-binding proteins was unaltered in carbachol-pretreated cells. Finally, neither prevention of PLD stimulation during carbachol pretreatment by genistein nor inhibition of protein synthesis by cycloheximide, added before or after carbachol challenge, resulted in recovery of mAChR-stimulated PLD. The short term carbachol pretreatment nearly completely abolished agonist-induced binding of guanosine 5'-O-(3-thiotriphosphate) to membranes or permeabilized adherent cells. Full recovery of this response was achieved after 4 h. Similar to transfected m3 mAChR, PLD stimulation by endogenously expressed purinergic receptors was also fully blunted after 2 min of agonist (ATP) treatment. Preexposure of HEK cells to either receptor agonist partially, but not completely, reduced PLD stimulation by the other agonist. In contrast to desensitization of PLD stimulation, 2 min of carbachol treatment led to a sensitization, by up to 2-fold, of mAChR-stimulated inositol phosphate formation. This supersensitivity was also observed with pilocarpine, which acted as a full agonist on PLC. On the basis of these results, we conclude that the m3 mAChR stimulates PLD and PLC in HEK cells with distinct efficiencies and with very distinct durations of each response. The rapid and long lasting desensitization of the PLD response is apparently not due to a loss of cell surface receptors or PLD activation by GTP-binding proteins, but it may involve, at least initially, an uncoupling of receptors from GTP-binding proteins and most likely a loss of an as yet undefined essential transducing component.

Carbachol↗

PubMed1995

Enhanced cAMP accumulation by the human thyrotropin receptor variant with the Pro52Thr substitution in the extracellular domain.

Recently, a naturally occurring variant of the human thyrotropin receptor with a Pro52Thr substitution in the N-terminal extracellular domain of the receptor has been identified. To determine the functional significance of this substitution, cDNAs of wild-type and variant thyrotropin receptors were stably expressed in Chinese hamster ovary cells. The Pro52Thr substitution did not affect synthesis and membrane localization of the receptor, as evidenced by 125I-thyrotropin binding analysis to intact cells. The variant receptor and the wild-type receptor were expressed in equivalent numbers and displayed identical binding affinity for thyrotropin. Strikingly, thyrotropin increased cAMP accumulation to a much greater extent in cells expressing the variant receptor as compared to the wild-type receptor-expressing cells. Basal and cholera toxin-stimulated or forskolin-stimulated cAMP levels were not different. It is concluded that the Pro52Thr substitution in the N-terminal region of the human thyrotropin receptor produces a receptor protein with enhanced coupling to cAMP production. This naturally occurring hyperactive thyrotropin receptor may participate in hyperthyroidism of patients with Graves' disease.

Animals↗

PubMed1995

G protein regulation of the Na+/H+ antiporter in Xenopus laevis oocytes. Involvement of protein kinases A and C.

We have characterized the regulation of the endogenous Na+/H+ exchanger in Xenopus laevis oocytes by G proteins and protein kinases by measuring the ethylisopropylamiloride-sensitive Li+ uptake. Injection of oocytes with the stable GTP analog GTP gamma S stimulated Li+ uptake up to almost 4-fold, an effect blocked by coinjection with the GDP analog, guanyl-5'-yl thiophosphate. Injection into oocytes of beta gamma subunits of the heterotrimeric G protein transducin enhanced Li+ uptake by about 3-fold. This stimulation was blocked by transducin alpha subunits, which by themselves did not influence Li+ uptake. Using various activators and inhibitors of protein kinases, it is demonstrated that the X. laevis oocyte Na+/H+ antiporter can be stimulated by activation of both protein kinase A and C. Stimulation of Na+/H+ exchanger activity by GTP gamma S but not that induced by transducin beta gamma subunits was blocked by the protein kinase A inhibitor H-89. On the other hand, transducin beta gamma subunit-stimulated activity was prevented by the protein kinase C inhibitor, calphostin C. The non-selective protein kinase inhibitor H-7 blocked both GTP gamma S- and transducin beta gamma subunit-stimulated Na+/H+ exchanger activity. The results suggest that the Na+/H+ exchanger of X. laevis oocytes can be activated by G proteins and that this activation is not direct but mediated by protein kinase A- and/or protein kinase C-dependent pathways.

Animals↗

PubMed1995

Analysis of receptor-G protein interactions in permeabilized cells.

Receptor-induced binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP [gamma S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus alpha-toxin, binding of GTP[gamma S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP[gamma S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP[gamma S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[gamma S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP[gamma S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP[gamma S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Cells, Cultured↗

PubMed1995

Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type.

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca2+ concentration [Ca2+]i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca2+]i with EC50 values of about 2 microM and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP3) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca2+]i were 300-350 nM and mainly, almost 90%, due to influx of extracellular Ca2+. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca2+]i with EC50 values of about 20 microM and 7 microM, respectively. Maximal InsP formation was only 10-15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca2+]i were similar in both cell types. Formation of InsP3 was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca2+]i increases induced by m2 mAChR activation were exclusively due to Ca2+ mobilization from intracellular stores. The efficacy of pilocarpine for stimulating InsP and Ca2+ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca2+]i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca2+ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.

Calcium↗

PubMed1995

Enhanced G protein activation in immortalized lymphoblasts from patients with essential hypertension.

Epstein-Barr virus-immortalized B lymphoblasts obtained from hypertensive patients with enhanced Na+/H+ exchanger activity (HT cells) proliferate distinctly faster upon serum stimulation than those from normotensive controls with low exchanger activity (NT cells) (Rosskopf, D., E. Frömter, and W. Siffert. 1993. J. Clin. Invest. 92:2553-2559). Stimulation with platelet-activating factor (PAF) as well caused an enhanced proliferation of HT cells. In analyzing possible differences in signal transduction between the immortalized NT and HT lymphoblasts, we observed that cell stimulation with PAF and somatostatin caused a twofold higher increase in [Ca2+]i in HT than in NT cell lines. This difference was completely abrogated by pertussis toxin (PTX) treatment. Furthermore, PAF-stimulated formation of inositol 1,4,5-trisphosphate (IP3) was twofold enhanced in HT cell lines. On the other hand, PAF receptor density and affinity, total cellular phospholipase C activity, expression of PTX-sensitive G proteins, and control binding of the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S), to membrane G proteins were not different in NT and HT cell lines. However, PAF- and mastoparan-stimulated binding of GTP gamma S to G proteins, which was fully PTX-sensitive, was 2.5-fold higher in HT than NT cell lines. These data suggest an enhanced receptor-mediated activation of PTX-sensitive G proteins despite unchanged receptor and G protein expression. Thus, this study not only suggests that enhanced signal transduction and cell proliferation are abnormalities in a certain group of patients with essential hypertension but also explains these findings as a result of an enhanced G protein activation in this common disorder.

Animals↗

PubMed1994

Mechanisms of phospholipase D stimulation by m3 muscarinic acetylcholine receptors. Evidence for involvement of tyrosine phosphorylation.

In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTP-binding proteins by A1F4- and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F4(-)-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and tyrosine phosphorylation. The coupling of m3 mAChR to phospholipase D appears to be largely independent of concomitant phospholipase C activation with subsequent increase in cytoplasmic calcium concentration and protein kinase C activity. The data instead suggest the involvement of an essential protein tyrosine phosphorylation mechanism in phopsholipase D activation by the m3 mAChR and heterotrimeric GTP-binding proteins.

Acetylcholine↗

PubMed1994

Unoccupied beta-adrenoceptor-induced adenylyl cyclase stimulation in turkey erythrocyte membranes.

The influence of beta-adrenoceptor ligands and a beta-adrenoceptor-derived peptide was studied on stimulation of adenylyl cyclase in membrane preparations of turkey erythrocytes. In the absence of receptor ligands, the hydrolysis-resistant GTP analogs, guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta,gamma-imino]triphosphate, caused a slow, time-dependent increase in adenylyl cyclase activity, which was accelerated and potentiated by the additional presence of the agonist, isoproterenol. In contrast, the beta-adrenoceptor antagonists, propranolol and pindolol, almost completely prevented stimulation of adenylyl cyclase by the GTP analogs alone, i.e. in the absence of an agonist, in a concentration-dependent and stereo-selective manner. This antagonist action was mimicked by a peptide corresponding to the second cytoplasmic loop of the turkey erythrocyte beta-adrenoceptor. On the other hand, GTP analog-preactivated cyclase activity and enzyme stimulation by fluoride and forskolin were not or only slightly reduced by beta-adrenoceptor antagonists. The data presented suggest that in turkey erythrocyte membranes agonist-free beta-adrenoceptors can cause significant Gs protein and subsequent adenylyl cyclase activation, and that this unoccupied receptor action can be blocked by beta-adrenoceptor antagonists, thus exhibiting by themselves a negative intrinsic activity.

Adenylyl Cyclases↗

PubMed1994

Deletion analysis of the m4 muscarinic acetylcholine receptor. Molecular determinants for activation of but not coupling to the Gi guanine-nucleotide-binding regulatory protein regulate receptor internalization.

In order to investigate whether coupling to and/or activation of guanine-nucleotide-binding proteins (G proteins) is involved in agonist-induced internalization of m4 muscarinic acetylcholine receptors (mAChRs), a deletion mutant [des-(264-394)mAChR] was constructed that lacks a substantial portion of the putative third intracellular loop. The wild-type receptor and des-(264-394)mAChR stably expressed in Chinese hamster ovary cells in essentially comparable amounts, exhibited identical antagonist-binding affinities. Consistent with the reported importance of the third cytoplasmic loop for Gi protein activation, the des-(264-394)mAChR showed a drastically reduced potential to mediate agonist-induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to Gi proteins was not impaired, as demonstrated by a similar guanine-nucleotide-sensitive and pertussis-toxin-sensitive high-affinity agonist-receptor binding for both mAChRs. In contrast, des-(264-394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of Gi proteins rather than ineffective coupling to Gi proteins. Internalization of wild-type receptor and des-(264-394)mAChR was observed with similar agonist concentrations and showed similar maximal values. However, des-(264-394)mAChR displayed a significantly reduced rate of receptor internalization. A similar attenuation of wild-type mAChR internalization was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR involved in Gi-protein coupling and activation are not identical and that activation of, but not coupling to, Gi proteins regulates m4 mAChR internalization.

Adenylyl Cyclases↗

PubMed1994

G protein specificity in receptor-effector coupling. Analysis of the roles of G0 and Gi2 in GH4C1 pituitary cells.

In rat pituitary GH4C1 cells, activation of transfected dopamine D2 receptors (long, D2L, or short, D2S, form) and endogenously expressed somatostatin and muscarinic M4 receptors induced inhibition of cAMP synthesis and of Bay K 8644-induced calcium entry via pertussis toxin-sensitive G proteins. To analyze the role of alpha 0 and alpha i2 in relaying of these signals, alpha 0 or alpha i2 antisense constructs were separately and stably transfected into GH4C1 cells. Reverse transcription-polymerase chain reaction and Western blot analyses indicated specific ablation of alpha 0 or alpha i2 in the antisense transfectant clones. Elimination of alpha 0 selectively abolished receptor-mediated inhibition of calcium entry. Notably, the action of dopamine D2L receptor was partially (about 30%) retained. By contrast, depletion of alpha i2 selectively impaired receptor-mediated inhibition of cAMP accumulation. Inhibition of basal cAMP synthesis by any of the four receptors studied was blocked in alpha i2-depleted clones. Additionally, dopamine D2L, somatostatin and muscarinic M4 receptor-mediated inhibition of vasoactive intestinal peptide-stimulated cAMP formation was also abolished. Remarkably, somatostatin even potentiated (by 30%) the action of vasoactive intestinal peptide in alpha i2-antisense clones. In contrast, the action of dopamine D2S receptor on stimulated cAMP synthesis remained largely unaltered. The results demonstrate that alpha 0 specifically triggers receptor-induced closure of calcium channels, whereas alpha i2 specifically mediates inhibition of adenylyl cyclase in GH4C1 cells. Furthermore, the data suggest that G1 protein specificity in receptor coupling to inhibition of adenylyl cyclase depends critically on the activity state of the enzyme. Moreover, the results indicate an essential difference in coupling of dopamine D2L and D2S receptors to G proteins.

Animals↗

PubMed1994

Receptor-stimulated guanine-nucleotide-triphosphate binding to guanine-nucleotide-binding regulatory proteins. Nucleotide exchange and beta-subunit-mediated phosphotransfer reactions.

In order to study whether phosphate transfer reactions are involved in the binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins, binding of the GTP analogues, guanosine 5'-[gamma-thio]triphosphate, GTP[S], and guanosine 5'-[beta, gamma-imino]triphosphate, p[NH]ppG, and the regulation of binding by the formyl-peptide-receptor agonist, fMet-Leu-Phe, were studied in membranes of differentiated HL-60 cells. For fMet-Leu-Phe-stimulated binding of either GTP analogue, a competing nucleotide was required. With GDP as the competing nucleotide, initial rates of fMet-Leu-Phe-stimulated binding of GTP[S] and p[NH]ppG were similar for up to approximately 30 s. Thereafter, receptor-stimulated binding of p[NH]ppG rapidly reached equilibrium, whereas the binding of GTP[S] proceeded further. At equipotent concentrations of p[NH]ppG and GTP[S], maximal fMet-Leu-Phe-stimulated binding of GTP[S] was approximately twofold higher than that of p[NH]ppG. Finally, for half-maximal receptor-stimulated binding of GTP[S], approximately fivefold higher concentrations of both Mg2+ and GDP were required than for p[NH]ppG binding. With p[NH]ppG as the competing nucleotide, the extent of receptor-stimulated binding of GTP[S] as well as its Mg2+ requirement and time course were similar to the receptor-stimulated p[NH]ppG binding observed in the presence of GDP. However, with GTP[S] as the competing nucleotide, fMet-Leu-Phe reduced the binding of p[NH]ppG, a reaction further enhanced when GDP was additionally present. Under similar conditions as used in the binding studies, GTP[S] thiophosphorylated a 35-kDa protein, which is most likely a guanine-nucleotide-binding regulatory protein beta subunit [Wieland, T., Nürnberg, B., Ulibarri, I., Kaldenberg-Stasch, S., Schultz, G. & Jakobs, K. H. (1993) J. Biol. Chem. 268, 18111-18118]. The thiophosphorylation state of this protein was regulated by guanine nucleotides, Mg2+ and, most importantly, by activated formyl-peptide receptors. The data thus provide evidence for an essential difference between GTP[S] and p[NH]ppG binding to guanine-nucleotide-binding regulatory proteins and suggest that, in addition to the nucleotide-exchange reaction, a (thio)phosphate-group-transfer process via guanine-nucleotide-binding regulatory protein beta subunits is involved in the receptor-stimulated binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins.

Binding, Competitive↗

PubMed1994

Agonist-induced down-regulation of the m4 muscarinic acetylcholine receptor occurs without changes in receptor mRNA steady-state levels.

The regulation of m4 muscarinic acetylcholine receptor mRNA expression by receptor activation was studied in N1E-115 neuroblastoma and AtT-20 pituitary cells that endogeneously express the m4 muscarinic receptor. Receptor concentration was measured by binding of the muscarinic receptor radioligand [3H]quinuclidinyl benzilate, and RNA-RNA solution hybridization/RNase protection assay with a m4 receptor-specific [32P]-cRNA probe was used to evaluate the levels of receptor mRNA. Treatment of both cell lines with a receptor-saturating concentration of the agonist carbachol decreased receptor number. However, there was no change in steady-state levels of m4 mAChR mRNA in both cell lines. Determination of mRNA stability in the presence of the transcription blocker actinomycin D revealed that carbachol treatment increased half-life of receptor mRNA in N1E-115 cells, but not in AtT-20 cells, suggesting that receptor activation can regulate m4 receptor mRNA stability dependently on cell type. Analysis of receptor degradation kinetics in the presence of the protein synthesis inhibitor cycloheximide showed that receptor down-regulation in N1E-115 and AtT-20 cells is sufficiently accounted for by increased receptor degradation. These results indicate than m4 muscarinic receptor down-regulation is substantially different from that of the muscarinic receptor subtypes m2 and m3 which is reported to be associated with agonist-induced reduction in receptor mRNA.

Animals↗