©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rapid and Persistent Desensitization of m3 Muscarinic Acetylcholine Receptor-stimulated Phospholipase D
CONCOMITANT SENSITIZATION OF PHOSPHOLIPASE C (*)

(Received for publication, February 2, 1995; and in revised form, May 17, 1995)

Martina Schmidt Birgit Fasselt Ulrich Rümenapp Christine Bienek Thomas Wieland Chris J. van Koppen Karl H. Jakobs (§)

From the Institut für Pharmakologie, Universität GH Essen, Hufelandstrasse 55, D-45122 Essen, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 G(i) 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.


INTRODUCTION

A large number of signal transduction systems utilizes the PLC(^1)-catalyzed breakdown of phosphoinositides, generating the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. The former stimulates the release of calcium from the endoplasmic reticulum, while the latter activates protein kinase C(1, 2) . In recent years, activation of PLD has been demonstrated for a variety of hormones, neurotransmitters, and growth factors, including those that act on receptors that possess intrinsic tyrosine kinase activity as well as those acting on receptors coupled to heterotrimeric G proteins(3, 4, 5) . PLD preferentially hydrolyzes the phospholipid phosphatidylcholine, resulting in the formation of phosphatidic acid and choline. Phosphatidic acid may act as an intracellular signaling agent by itself(6, 7) . Upon deacylation and dephosphorylation of phosphatidic acid, respectively, the extra- and/or intracellular signaling molecules, lysophophatidic acid, and diacylglycerol are formed. Lysophosphatidic acid apparently stimulates cells via a distinct cell surface receptor coupled to G proteins(8, 9) . Phosphatidylcholine-derived diacylglycerol species may activate specific isoforms of protein kinase C(10, 11) .

Receptor coupling mechanisms to PLC enzymes have been recently elucidated in great detail. While tyrosine kinase-linked receptors apparently phosphorylate and thereby activate distinct PLC isozymes (PLC), receptors coupled to heterotrimeric G proteins activate PLCbeta isozymes by either GTP-activated alpha subunits (alpha) or free beta dimers (for reviews, see Refs. 12 and 13). Much less clear are the mechanisms that couple receptors to PLD. For the G protein-coupled receptors, it can be hypothesized that G proteins are involved, although direct interaction of PLD with heterotrimeric G proteins or G protein subunits has as yet not been demonstrated. Recently, evidence has been provided that small molecular weight G proteins, ADP-ribosylation factors, and Rho proteins, can stimulate PLD in cell-free preparations(14, 15, 16, 17) .

The human m3 mAChR, stably expressed in HEK cells, interacts with both PTX-insensitive G and PTX-sensitive G(i) proteins in these cells(18) . Receptor activation causes efficient stimulation of both PLC and PLD in a PTX-insensitive manner(18, 19) . We recently reported that PLD can be stimulated in HEK cells by various mechanisms, apparently involving protein kinase C, calcium-dependent events as well as tyrosine phosphorylation, and that the coupling of m3 mAChR to PLD is largely independent of concomitant PLC activation but rather involves a tyrosine kinase-dependent mechanism(20) . During these studies, we noted that, in contrast to PLC activation, mAChR-stimulated PLD apparently rapidly decays(20) . In the present study, we demonstrate that short term agonist treatment persistently desensitizes m3 mAChR-stimulated PLD, whereas the receptor-induced stimulation of PLC is sensitized. The long lasting loss of the PLD response is not due to receptor internalization or loss of PLD activity, but it seems to be due to a loss of an as yet undefined essential transducing component.


EXPERIMENTAL PROCEDURES

Materials

ATP, GTPS, staurosporine, and phenylmethylsulfonyl fluoride were obtained from Boehringer Mannheim. DMEM/F-12 growth medium, fetal calf serum, and G418 were from Life Technologies, Inc. Streptomycin, poly D-lysine, and penicillin G were from Seromed. Digitonin (99% pure) was from E. Merck. Carbachol, pilocarpine, PMA, atropine, benzamidine, leupeptin, cycloheximide, and soybean trypsin inhibitor were from Sigma. BAPTA/AM and genistein were from Calbiochem. PTX was from List Biological Laboratories. [9,10-^3H]Oleic acid (10 Ci/mmol), myo-[2-^3H]inositol (24.4 Ci/mmol), [^3H]NMS (79.5 Ci/mmol), and [S]GTPS (1344 Ci/mmol) were obtained from DuPont NEN. Nitrocellulose filters were from Amersham Corp.

Cell Culture

HEK cells stably expressing the human m3 mAChR at a density of about 200,000 receptors/cell (21) were cultured in DMEM/F-12 growth medium containing 10% fetal calf serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin. The cells were grown in culture dishes precoated with poly D-lysine (>300 kDa; 0.05-0.1 mg/dish). Stocks of the transfected cells were maintained in the presence of G418 (0.5 mg/ml). For experiments, cells were subcultured in DMEM/F-12 medium lacking G418 and grown to near confluence (175-cm^2 culture flasks or 35-mm culture dishes).

Assay of PLD and PLC Activities in Intact Cells

For measurement of intact cell PLD and PLC activities, cellular phospholipids were labeled by incubating nearly confluent monolayers of cells for 20-24 h with [^3H]oleic acid (2-2.5 µCi/ml) and myo-[^3H]inositol (1.25 µCi/ml), respectively, in growth medium. In [^3H]oleic acid-prelabeled cells, the radioactivity associated with the phospholipids was between 7.5 and 10 10^5 cpm/dish. For PTX treatment, the cells were incubated during the last 16 h of the labeling period with 100 ng/ml PTX. After the labeling period, the cells were equilibrated twice for 10 min at 37 °C in HBSS, containing 118 mM NaCl, 5 mM KCl, 1 mM CaCl(2), 1 mM MgCl(2), and 5 mMD-glucose, buffered at pH 7.4 with 15 mM HEPES. For desensitization experiments, prelabeled cells were treated for 2 min with agonist in HBSS and then washed twice with HBSS. Then, carbachol or other stimulatory agents were added in the presence of ethanol (400 mM) for the indicated periods of time to measure [^3H]PtdEtOH accumulation. LiCl (10 mM) was added 10 min before the stimulatory agents when myo-[^3H]inositol phosphate formation was monitored. When effects of protein kinase inhibitors or the calcium chelator BAPTA/AM were studied, they were added 30 min before carbachol. The reactions performed at 37 °C were stopped by adding 1 ml of ice-cold methanol to the dishes. The cells were scraped off from the dishes, and the phospholipids and inositol phosphates were extracted and analyzed as described previously(20) . Radioactivity was measured by liquid scintillation spectrometry, with a counting efficiency of about 40%.

Protein levels were measured by the method of Bradford (22) in separate culture dishes. All experiments were performed in triplicate culture dishes and repeated as indicated. Formation of [^3H]PtdEtOH is expressed as percentage of total phospholipids. [^3H]Inositol phosphate formation is normalized for protein content and is given as cpm/mg of protein.

Assay of PLD Activity in Permeabilized Cells

Prior to permeabilization, [^3H]oleic acid-prelabeled (2 µCi/ml) cells were treated for 2 min with or without 1 mM carbachol followed by two washing steps with HBSS. Thereafter, the cells were detached from the culture flasks, resuspended in DMEM/F-12 medium without serum, pelleted by centrifugation, and resuspended in phosphate-buffered saline (150 mM NaCl, 2.7 mM KCl, 6.5 mM Na(2)HPO(4), and 1.5 mM KH(2)PO(4), pH 7.4). After recentrifugation and resuspension in buffer A, containing 135 mM KCl, 5 mM NaHCO(3), 5 mM EGTA, 4 mM MgCl(2), 2 mM ATP, 1.5 mM CaCl(2) (corresponding to 40 nM free Ca), 5.6 mMD-glucose, and 20 mM HEPES, pH 7.2, at a cell concentration of about 1 10^7 cells/ml, the assay of PLD activity was carried out for 30 min at 37 °C in a total volume of 200 µl, containing 100 µl of the cell suspension (0.5-1 10^6 cells), 8 µM digitonin, and 300 mM ethanol as well as the test agents(20) . The reaction was stopped by the addition of 2 ml of chloroform/methanol (1:1) and 1 ml of H(2)O. [^3H]PtdEtOH formed was analyzed as described above.

mAChR Binding Assay

HEK cells on 35-mm tissue culture dishes were pretreated for 2 min with or without 1 mM carbachol, followed by two washing steps with ice-cold HBSS. Thereafter, cell surface mAChRs were measured by binding of the membrane-impermeant mAChR antagonist, [^3H]NMS (2 nM), in HBSS in a total volume of 1 ml for 4 h at 4 °C, by which time binding equilibrium was reached(23) . Bound and free ligand were separated by rapidly washing the cells in ice-cold HBSS. The cells were then solubilized in 0.5 ml of 1% (w/v) Triton X-100 and scraped into scintillation vials to measure radioactivity. Nonspecific binding was determined in the presence of 1 µM atropine and amounted to 5% of total binding. mAChR binding is expressed as pmol of [^3H]NMS bound per culture dish.

Membrane Preparation

HEK cells pretreated or not with carbachol were detached from the culture flasks, pelleted, washed once in phosphate-buffered saline, recentrifuged, and resuspended in homogenization buffer containing 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 250 mM sucrose, 3 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 1 mg/ml soybean trypsin inhibitor, and 20 mM Tris-HCl, pH 7.5. The cells were homogenized by nitrogen cavitation and pelleted by centrifugation for 10 min at 1000 g. Crude membranes were prepared from the resulting supernatant by centrifugation for 60 min at 50,000 g. The membranes were suspended in buffer B, containing 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, and 20 mM Tris-HCl, pH 7.5, and stored at -80 °C.

GTPS Binding in HEK Membranes

Binding of GTPS was performed in a total volume of 100 µl containing 0.2 nM [S]GTPS, 1 mM dithiothreitol, 1 mM EDTA, 5 mM MgCl(2), 150 mM NaCl, 50 mM triethanolamine hydrochloride, pH 7.4, and the indicated additions at 30 °C as described before(18) . The reaction was started by the addition of membranes (5 µg of protein/tube) to the temperature-equilibrated reaction mixture. The incubation was terminated after 60 min by the addition of 2.5 ml of ice-cold buffer C containing 5 mM MgCl(2) and 50 mM Tris-HCl, pH 7.5. Separation of membrane-bound and free radioactivity was performed by rapid filtration through glass fiber filters (Whatman GF/C). The filters were washed 4 times with 2.5 ml of buffer C and counted in a liquid scintillation spectrometer. Nonspecific binding was defined as the binding not competed for by 10 µM unlabeled GTPS. The data were normalized for protein content and are presented as pmol of GTPS bound per mg of protein.

GTPS Binding in Permeabilized Adherent HEK Cells

Culture dishes with a confluent cell layer (about 750,000 cells/dish) were kept for 10 min at 37 °C in DMEM/F-12 growth medium for equilibration. Then, the cells were incubated in medium for 2 min with or without 1 mM carbachol, followed by two washing steps with medium. The medium was then removed and replaced by 1 ml of buffer D containing 5 mM MgCl(2), 1 mM EDTA, 150 mM NaCl, 10 µM digitonin, and 50 mM triethanolamine hydrochloride, pH 7.4. After 15 min, binding of GTPS to permeabilized adherent HEK cells was initiated by replacing buffer D with fresh buffer D, containing in addition 10 nM [S]GTPS (0.1 µCi) with or without 1 mM carbachol, and continued for 10 min at 37 °C as described previously(24) . Then, the buffer was removed and replaced by 0.5 ml of ice-cold buffer C. Cells were scraped off, and the suspension was rapidly filtered through nitrocellulose filters. The dish was additionally washed with 0.5 ml of buffer C, also passed through the filters. The filters were washed 4 times with 2.5 ml of ice-cold buffer C and counted in a liquid spectrometer. Nonspecific binding was defined as the binding not competed for by 10 µM unlabeled GTPS. The data are presented as fmol of GTPS bound per mg of protein.


RESULTS

In HEK cells stably expressing the human m3 mAChR, the mAChR agonist carbachol causes a marked activation of PLD measured as accumulation of its transphosphatidylation product, PtdEtOH(18, 19, 20) . Interestingly, maximal levels of labeled PtdEtOH were obtained already after 2 min of incubation with carbachol (1 mM), followed by a stable plateau up to at least 30 min of incubation (Fig. 1). The very rapid cessation of carbachol-induced PtdEtOH formation was not due to the activation with a supramaximally effective agonist concentration (1 mM). An identical time course of [^3H]PtdEtOH formation, although at a lower level, was obtained when the cells were treated with carbachol at a half-maximally effective concentration (3 µM) (Fig. 1). To study whether the observed PLD desensitization is the result of a negative feedback by carbachol-induced increase in cytoplasmic calcium, activation of protein kinase C, or tyrosine phosphorylation, the cells were pretreated with 20 µM BAPTA/AM, a condition completely preventing carbachol-induced increase in cytosolic calcium, 0.1 µM staurosporine, inhibiting PMA (0.1 µM) induced PLD activation by more than 90%, or with the tyrosine kinase inhibitor, genistein (10 µM), causing about half-maximal inhibition of carbachol-induced protein tyrosine phosphorylation in HEK cells(20) . As illustrated in Fig. 2, none of these pretreatments had any effect on the shape of the time course of carbachol-induced PtdEtOH formation, although under all of these conditions mAChR-stimulated PLD was reduced by 50-60%. The BAPTA/AM treatment was performed in calcium-free medium, suggesting that the observed reduction in PLD stimulation is due to the removal of both extra- and intracellular calcium. The staurosporine-induced inhibition of mAChR-stimulated PLD is apparently not due to inhibition of protein kinase C, since it is not mimicked by specific protein kinase C inhibitors(20) , but it may be due to inhibition of tyrosine kinases by this rather unselective inhibitor(4) . The use of the tyrosine kinase inhibitor, genistein, at a maximally effective concentration was not possible in this type of experiments, since under this condition genistein completely prevented the carbachol-induced PLD activation(20) .


Figure 1: Time course of mAChR-induced PtdEtOH formation. Formation of [^3H]PtdEtOH was measured in m3 mAChR-expressing HEK cells prelabeled with [^3H]oleic acid in the absence () and presence of either 3 µM (black square) or 1 mM carbachol (bullet) for the indicated periods of time. Data are from one representative experiment (mean ± S.D., triplicate culture dishes), characteristic of six similar ones.




Figure 2: Influence of BAPTA/AM, staurosporine and genistein on the time course of mAChR-induced PtdEtOH formation. [^3H]Oleic acid-prelabeled HEK cells were pretreated for 30 min without (Control) and with BAPTA/AM (20 µM), staurosporine (0.1 µM), or genistein (10 µM) as indicated. Thereafter, carbachol (1 mM) induced [^3H]PtdEtOH formation was measured in the presence of ethanol (400 mM) for the indicated periods of time. For the BAPTA/AM experiment, extracellular Ca was additionally removed from the incubation medium. Data are from one representative experiment (mean ± S.D., triplicate culture dishes), characteristic of three separate ones.



In order to exclude ethanol and PtdEtOH, the PLD product, as causative agents for the rapid desensitization, HEK cells were pretreated for 2 min with carbachol (1 mM) in the absence of ethanol and LiCl. After wash-out of carbachol, the cells were immediately restimulated with carbachol in the presence of ethanol and LiCl. As shown in Fig. 3, mAChR-mediated stimulation of PLD was abrogated by prior cell treatment with carbachol. In complete contrast, mAChR-stimulated PLC activity, measured as accumulation of [^3H]inositol phosphates, was not impaired but was significantly (p < 0.01, n = 12) enhanced (by 70-100%) upon pretreatment of the cells with carbachol.


Figure 3: Desensitization of mAChR-stimulated PLD and sensitization of PLC stimulation. m3 mAChR-expressing HEK cells prelabeled with [^3H]oleic acid and myo-[^3H]inositol were pretreated for 2 min in the absence of ethanol and LiCl without (Control) and with 1 mM carbachol (Carbacholpretreated), followed by wash-out of carbachol, as described under ``Experimental Procedures.'' Thereafter, basal and carbachol (1 mM) stimulated formation of [^3H]PtdEtOH (leftpanel) and myo-[^3H]inositol phosphates (rightpanel) were measured for 10 min in the presence of ethanol (400 mM) and LiCl (10 mM) as indicated. Data are from one experiment (mean ± S.D., triplicate culture dishes), representative of six separate experiments.



Activation of purinergic receptors endogenously expressed in HEK cells (25) by the agonist, ATP (1 mM), stimulated PLD activity to about 30% of the level observed with 1 mM carbachol (Fig. 4); however, with identical time kinetics (data not shown), suggesting that the ATP-stimulated PLD also rapidly desensitizes. Indeed, 2 min of ATP treatment followed by wash-out of the agonist completely prevented subsequent purinergic receptor-mediated PLD stimulation (Fig. 4B). Agonist-induced desensitization can be either homologous or heterologous. Therefore, mutual interactions between activation of mAChRs and purinergic receptors were studied. Carbachol pretreatment (1 mM for 2 min) abolished mAChR-stimulated PLD, while ATP-induced PLD stimulation was still present but significantly (p < 0.01, n = 9) reduced by about 50% (Fig. 4A). Vice versa, after ATP treatment (1 mM for 2 min), carbachol still effectively stimulated PLD, but the response was significantly (p < 0.01, n = 9) reduced by about 25% (Fig. 4B).


Figure 4: Desensitization of mAChR- and purinergic receptor-stimulated PLD. m3 mAChR-expressing HEK cells prelabeled with [^3H]oleic acid were pretreated for 2 min in the absence of ethanol without (Control) and with 1 mM carbachol (Carbacholpretreated) (A) or without and with 1 mM ATP (ATPpretreated) (B), followed by wash-out of carbachol and ATP. Thereafter, formation of [^3H]PtdEtOH was measured for 10 min in the presence of ethanol (400 mM) without (Basal) and with 1 mM carbachol or 1 mM ATP as indicated. Data are from one representative experiment (mean ± S.D., triplicate culture dishes), repeated three times.



To study at which level the PLD response to mAChR activation was blunted, several possibilities were examined. First, we investigated whether activation of PLD itself by direct activation of protein kinase C with the phorbol ester PMA and by direct activation of heterotrimeric G proteins with AlF(4) was altered in carbachol-pretreated intact cells. In contrast to the abolished mAChR stimulation, pretreatment of the cells for 2 min with carbachol (1 mM) had no effect on the PMA- and AlF(4)-induced activation of PLD (Fig. 5A). Furthermore, pretreatment of intact HEK cells with carbachol had no effect on stimulation of PLD by the stable GTP analog GTPS measured in permeabilized cells (Fig. 5B). Collectively, these data indicated that the rapid desensitization of the mAChR-induced PLD stimulation was not due to an inactivation of the PLD enzyme itself and its activation by protein kinase C and G proteins.


Figure 5: Influence of carbachol pretreatment on PLD activation by PMA, AlF(4), and GTPS. [^3H]Oleic acid-prelabeled HEK cells were pretreated for 2 min in the absence of ethanol without (Control) and with 1 mM carbachol (Carbacholpretreated), followed by wash-out of carbachol. Thereafter, [^3H]PtdEtOH formation was measured in intact cells (A) in the absence (Basal) and presence of carbachol (1 mM, 10 min), AlF(4) (10 mM NaF plus 10 µM AlCl(3), 60 min) or PMA (0.1 µM, 60 min) as indicated. In addition, following digitonin permeabilization (B), basal and GTPS (100 µM) stimulated formation of [^3H]PtdEtOH was determined as described under ``Experimental Procedures.'' Data are from one representative experiment (mean ± S.D., triplicate culture dishes), characteristic of three similar ones.



Second, the rapid desensitization of the mAChR-stimulated PLD could be due to a loss of cell surface receptors. However, preexposure of the cells to 1 mM carbachol for 2 min had no effect on binding of the hydrophilic mAChR antagonist ligand [^3H]NMS, used at a receptor-saturating concentration of 2 nM(26) , to intact HEK cells (Fig. 6A). These data indicated that the desensitization of the PLD response was not due to m3 mAChR internalization.


Figure 6: Influence of carbachol pretreatment on mAChR number and mAChR-induced GTPS binding. m3 mAChR-expressing HEK cells were pretreated for 2 min with (filledcolumns) and without (opencolumns) 1 mM carbachol. After wash-out of carbachol, binding of [^3H]NMS to intact cells was determined as described under ``Experimental Procedures'' (A). Data are from two experiments (mean ± S.D., quadruplicate culture dishes). In addition, in membranes of control and carbachol-pretreated cells, agonist-induced binding of [S]GTPS was determined (B). Data are from three separate experiments (mean ± S.D.) each performed in triplicate. Basal [S]GTPS binding was 0.90 ± 0.05 pmol and 0.77 ± 0.10 pmol/mg protein in membranes of control and carbachol-pretreated cells, respectively.



Third, desensitization of mAChR-induced PLD activation might be due to a receptor-G protein uncoupling. Therefore, agonist-induced binding of GTPS to membranes of cells pretreated or not for 2 min with carbachol (1 mM) was studied. Pretreatment of intact cells with carbachol caused an almost complete abrogation of agonist-induced GTPS binding to membranes of these cells (Fig. 6B). Similar data were obtained when we measured GTPS binding to permeabilized adherent cells pretreated with carbachol (Fig. 7A). These data suggested that the rapid mAChR desensitization of PLD activation may be due to uncoupling of the receptor from the G protein(s) mediating the mAChR action. This hypothesis was further examined by measuring the time courses of recovery of agonist-stimulated GTPS binding and PLD activation following pretreatment of intact cells with carbachol (1 mM for 2 min). After 4 h of culturing of the cells in agonist-free medium, the carbachol-induced binding of GTPS measured in permeabilized adherent HEK cells was fully restored (Fig. 7A). However, mAChR-stimulated PLD activity remained completely abolished during this time scale (Fig. 7B). Stimulation of PLD by PMA and AlF(4) was not altered (data not shown). After 24 h of culturing of the cells in agonist-free medium, PLD stimulation by carbachol was restored by about 50%, with the carbachol-induced PtdEtOH formation being 0.13 ± 0.02 and 0.23 ± 0.02% of total phospholipids in carbachol-pretreated and untreated control cells, respectively (mean ± S.D., n = 3, from one experiment, representative for two independent ones). mAChR-stimulated PLC was sensitized by prior short term carbachol treatment, up to about 2-fold (Fig. 7C). This supersensitivity to carbachol disappeared after 2 h of cell culturing in agonist-free medium.


Figure 7: Time courses of recovery of mAChR-induced GTPS binding and stimulation of PLD and PLC. A, HEK cells were pretreated for 2 min with 1 mM carbachol. After wash-out of carbachol, the carbachol (1 mM) induced GTPS binding was determined at the indicated periods of time in permeabilized adherent cells as described under ``Experimental Procedures.'' The opensymbol at 0 h indicates the agonist-induced GTPS binding to permeabilized cells not pretreated with carbachol. Basal GTPS binding was 46 ± 3.6 and 31.5 ± 2.2 fmol/mg of protein in untreated and carbachol-pretreated cells, respectively. B and C, HEK cells prelabeled with [^3H]oleic acid and myo-[^3H]inositol were pretreated for 2 min with 1 mM carbachol in the absence of ethanol and LiCl. After wash-out of carbachol, the carbachol (1 mM) induced formation of [^3H]PtdEtOH (B) and myo-[^3H]inositol phosphates (C) was measured at the indicated periods of time for 10 min in the presence of ethanol (400 mM) and LiCl (10 mM). Opensymbols at 0 h indicate the agonist-induced stimulation of PLD and PLC in cells not pretreated with carbachol. Basal accumulation of [^3H]PtdEtOH was unchanged. Basal accumulation of myo-[^3H]inositol phosphates was 5.8 ± 1.7 10^3 and 9.7 ± 1.5 10^3 cpm/mg of protein in untreated and carbachol-pretreated cells, respectively. Data are from one experiment (mean ± S.D., triplicate culture dishes). Identical results were obtained in two separate experiments.



Fourth, we assessed whether PLD stimulation during short term agonist treatment was a prerequisite for the long lasting loss of the mAChR response. Therefore, HEK cells were pretreated with genistein (100 µM for 30 min) before a 2-min challenge with carbachol, followed by a 4-h recovery in the absence of either agent. The genistein pretreatment completely inhibited acute carbachol and AlF(4) stimulation of PLD as reported before(20) , but it did not prevent the desensitization of mAChR-stimulated PLD (Fig. 8). Independent of whether short term carbachol treatment was performed in the absence or presence of genistein, receptor-mediated stimulation of PLD, measured after a 4-h recovery period, was reduced by more than 80%. On the other hand, AlF(4) stimulation of PLD was not affected by prior carbachol alone treatment and reduced by about 40% in cells pretreated with genistein and carbachol.


Figure 8: Influence of genistein on PLD stimulation and desensitization. HEK cells prelabeled with [^3H]oleic acid were pretreated without (Control) and with genistein (100 µM) for 30 min followed immediately by measurement of [^3H]PtdEtOH accumulation in the absence (Basal) and presence of carbachol (1 mM, 10 min) or AlF(4) (10 mM NaF plus 10 µM AlCl(3), 60 min) as indicated. In addition, control (Carbachol) and genistein-pretreated cells (Genistein + Carbachol) were treated for 2 min with 1 mM carbachol in the absence of ethanol followed by wash-out of carbachol and genistein. After a 4-h recovery, basal and carbachol- or AlF(4)-stimulated [^3H]PtdEtOH accumulation was determined as described above. Data are from one experiment (mean ± S.D., triplicate culture dishes).



Fifth, the desensitization of mAChR-stimulated PLD may be due to activation of G proteins not directly involved in PLD stimulation. We have previously reported that the m3 mAChR can couple to both PTX-insensitive G proteins and PTX-sensitive G(i) proteins in HEK membranes(18) . Although PLD stimulation is PTX-insensitive (18, 20) (Fig. 9A), the concomitant activation of G(i) by the m3 mAChR could induce a signal resulting in the long lasting PLD desensitization. However, as demonstrated in Fig. 9B, PTX pretreatment (100 ng/ml for 16 h) did not affect desensitization of mAChR-stimulated PLD. As in nonintoxicated cells, following 2 min of treatment with 1 mM carbachol, subsequent carbachol-induced PLD stimulation was completely suppressed in PTX-pretreated cells, even after 4 h of culturing of the cells in agonist-free medium.


Figure 9: Influence of PTX on desensitization of mAChR-stimulated PLD. m3 mAChR-expressing HEK cells were treated for 16 h without (Control) and with 100 ng/ml PTX as described under ``Experimental Procedures.'' A, [^3H]PtdEtOH formation was measured in the absence (Basal) and presence of 1 mM carbachol for 10 min in control and PTX-pretreated cells. B, control and PTX-pretreated HEK cells were treated for 2 min with 1 mM carbachol in the absence of ethanol followed by wash-out of carbachol. After a 4-h recovery, basal and carbachol-stimulated [^3H]PtdEtOH formation was determined as described above. Data are from one representative experiment (mean ± S.D., triplicate culture dishes). Similar results were obtained in two separate experiments.



Finally, to study whether synthesis of a protein causing a long lasting inhibition of PLD stimulation is induced by carbachol, the effect of the protein synthesis inhibitor, cycloheximide, on mAChR-stimulated PLD and its desensitization was studied. For this, cells were pretreated for 1 h with 350 µM cycloheximide, which inhibited [^3H]leucine incorporation in HEK cells by 95% (data not shown). In these cells, a similar time course of [^3H]PtdEtOH formation, although at a lower level (about 50%), was obtained as in untreated controls (Fig. 10A). Furthermore, treatment of HEK cells immediately after a 2-min carbachol challenge with cycloheximide (350 µM) for 4 h did not result in recovery of mAChR-stimulated PLD. In the short term carbachol-pretreated cells, agonist-induced [^3H]PtdEtOH formation was reduced to about 6 and 9% of initial response after 4 h of treatment with and without cycloheximide, respectively (Fig. 10B).


Figure 10: Influence of protein synthesis inhibition on desensitization of mAChR-stimulated PLD. A, formation of [^3H]PtdEtOH in response to 1 mM carbachol was measured for the indicated periods of time in [^3H]oleic acid-prelabeled HEK cells pretreated for 1 h without (bullet) and with (black square) 350 µM cycloheximide. Data are from one experiment (mean ± S.D., triplicate culture dishes). B, carbachol (1 mM) stimulated formation of [^3H]PtdEtOH was measured for 10 min in the presence of ethanol in untreated HEK cells (Con) and in cells pretreated for 2 min with 1 mM carbachol in the absence of ethanol, followed by wash-out of carbachol and an additional 4 h of treatment without (Carbpretreated) and with 350 µM cycloheximide (Carb + CHXpretreated). Data are from one representative experiment (mean ± S.D., triplicate culture dishes), characteristic of three similar ones.



The discrepancy between the largely reduced GTPS binding and the even enhanced PLC response in the carbachol-pretreated cells suggested that there exists a large signal reserve in the stimulatory PLC pathway. Therefore, we studied the effects of the partial mAChR agonist, pilocarpine(27) , on GTPS binding and on PLC and PLD activities. Pilocarpine, at a receptor-saturating concentration of 1 mM, increased GTPS binding to permeabilized HEK cells by about 75% of that induced by 1 mM carbachol (61 ± 4.0 versus 79 ± 4.1 fmol/mg protein, n = 8). With regard to activation of PLD, pilocarpine exhibited about 50% maximal efficiency compared with carbachol (Fig. 11A). However, similar to the full agonist, carbachol, pretreatment of HEK cells for 2 min with pilocarpine (1 mM) completely abrogated subsequent pilocarpine- or carbachol-stimulated PLD. On the other hand, pilocarpine was a full agonist in activating PLC, both in control cells and in carbachol-pretreated cells. Similar to carbachol, inositol phosphate accumulation induced by pilocarpine was almost 2-fold higher in carbachol-pretreated than in control cells (Fig. 11B).


Figure 11: Comparison of carbachol- and pilocarpine-induced stimulation of PLD and PLC. Formation of [^3H]PtdEtOH (A) and myo-[^3H]inositol phosphates (B) was measured in the presence of LiCl (10 mM) and ethanol (400 mM) in control HEK cells prelabeled with [^3H]oleic acid and myo-[^3H]inositol for 10 min in the absence (Basal) and presence of 1 mM carbachol or 1 mM pilocarpine as indicated. In addition, after treatment of [^3H]oleic acid-prelabeled HEK cells for 2 min with 1 mM pilocarpine in the absence of ethanol (Pilocarpinepretreated) and of myo-[^3H]inositol-prelabeled cells for 2 min with 1 mM carbachol in the absence of LiCl (Carbacholpretreated), followed by wash-out of the agonists, basal and carbachol- or pilocarpine-stimulated formation of [^3H]PtdEtOH (A) and myo-[^3H]inositol phosphates (B) was measured in the presence of ethanol (400 mM) and LiCl (10 mM), respectively. Data are from one experiment (mean ± S.D., triplicate culture dishes), representative of three separate experiments.




DISCUSSION

Activation of m3 mAChR stably expressed in HEK cells stimulates both PLC and PLD(18, 19, 20) . In addition, receptor activation leads to a rapid increase in cytosolic calcium concentration and protein tyrosine phosphorylation(20) . We recently reported that PLD activity in HEK cells can be stimulated by various mechanisms, apparently involving GTP-binding proteins, calcium, and protein kinase C as well as tyrosine phosphorylation(20) . On the other hand, mAChR-mediated PLD stimulation is apparently independent of concomitant PLC stimulation, including activation of protein kinase C and increase in cytosolic calcium, but it most likely involves PTX-insensitive G proteins and a tyrosine kinase-dependent mechanism(20) . Data presented in this report corroborate this apparent PLC independence of receptor-stimulated PLD.

Recently, rapid desensitization of receptor-stimulated PLD has been reported for various receptors, including m1 and m3 mAChR, in various cellular systems(28, 29, 30, 31, 32) . However, in contrast to m3 mAChR-expressing HEK cells, receptor stimulation of PLD in these cells was protein kinase C-dependent, as demonstrated by the use of inhibitors and/or protein kinase C down-regulation(28, 29, 30, 31, 32) . Since mAChR-stimulated PLC and PLD in HEK cells exhibited distinct kinetics, with rapid cessation of PtdEtOH accumulation in contrast to prolonged accumulation of inositol phosphates(20) , we studied the apparent desensitization of PLD stimulation and possible mechanisms underlying this refractoriness. As demonstrated here, mAChR-stimulated PLD in HEK cells is rapidly and completely desensitized upon receptor activation, whereas PLC stimulation is not impaired but sensitized. The mechanisms possibly underlying the desensitization of the PLD response were analyzed at various levels.

First, we considered that cellular responses and factors elicited by mAChR activation, in addition to PLD stimulation, may act as negative feedback inhibitors. However, neither prevention of cytosolic calcium increase by BAPTA/AM pretreatment, nor inhibition of protein kinase C by staurosporine, nor inhibition of mAChR-induced tyrosine phosphorylation by genistein affected the time kinetics of carbachol-stimulated PtdEtOH formation. Furthermore, down-regulation of protein kinase C by prolonged PMA treatment and inhibition of protein kinase C with the selective inhibitor, calphostin C, did not substantially reduce nor resulted in increased agonist-induced PtdEtOH accumulation(20) . Finally, although m3 mAChR stimulation of PLD is PTX-insensitive in HEK cells, agonist-activated receptor has been shown to activate G(i) proteins in membranes of these cells(18) , raising the possibility that G(i) and G(i)-derived signals are involved in desensitization of PLD stimulation. However, PTX treatment affected neither direct PLD stimulation nor desensitization of this response by m3 mAChR. Collectively, these data suggest that desensitization of mAChR-stimulated PLD is not due to cellular responses and factors independent of receptor-induced and G protein-mediated PLD stimulation.

Second, the desensitization was not due to mAChR down-regulation or internalization. Identical kinetics of PtdEtOH accumulation were obtained with a supramaximally and a half-maximally effective concentration of carbachol. Moreover, under conditions where PLD stimulation was completely abrogated, mAChR-stimulated PLC was not impaired. Most important, as observed in Chinese hamster ovary cells transfected with the human m3 mAChR(26) , binding of the hydrophilic mAChR antagonist ligand NMS to intact HEK cells preexposed for 2 min to carbachol, a condition causing complete desensitization of the PLD response, was not affected. In the other cellular systems exhibiting rapid desensitization of receptor-stimulated PLD, participation of receptor internalization or down-regulation was either not studied (28, 29, 30, 32) or was even made likely, e.g. for thrombin and bombesin receptors in CCL39 and Swiss 3T3 fibroblasts, respectively (28, 31) .

Third, the desensitization might be due to PLD products or may occur at the PLD level. The PLD product, PtdEtOH, and its co-substrate, ethanol, could be excluded as causative agents, as full desensitization was obtained in the absence of ethanol and concomitant PtdEtOH formation. The natural PLD product, phosphatidic acid, as causative compound was also made unlikely by the observation that in cells pretreated with genistein, which completely inhibited acute agonist-induced PLD stimulation(20) , mAChR-stimulated PLD was desensitized by a 2-min carbachol treatment. A PLD substrate depletion was made improbable by experiments using a low carbachol concentration that exhibited the same decay of PtdEtOH formation as observed with a maximally effective concentration. Furthermore, treatment of cells with the partial m3 mAChR agonist, pilocarpine, caused the same long lasting desensitization of PLD stimulation as the full receptor agonist, carbachol. Finally, in cells completely insensitive to mAChR stimulation, PLD was fully sensitive to activation by various other stimuli. PMA-stimulated protein kinase C, apparently not involved in m3 mAChR-stimulated PLD in HEK cells(20) , caused an identical increase in PLD activity in desensitized cells as in naive control cells. A similarly unaltered phorbol ester responsiveness despite full desensitization of receptor-stimulated PLD was also reported in other cells(28, 30, 32) . However, since the G protein-linked m3 mAChR and PMA-activated protein kinase C are independent activators of PLD activity in HEK cells, it is feasible that different PLD isozymes (33) or different pools of PLD substrate (34) are utilized.

Since activated receptors and G proteins apparently stimulate PLD via a common pathway(20) , PLD stimulation by directly activated GTP-binding proteins was examined in mAChR refractory HEK cells. Stimulation of PLD activity in intact and permeabilized HEK cells, respectively, by AlF(4), an activator of heterotrimeric G proteins, and the stable GTP analog GTPS, which can activate heterotrimeric and small molecular weight G proteins, was not affected by prior carbachol challenge. In contrast, in CCL39 fibroblasts expressing the human m1 mAChR, desensitization of PLD stimulation was heterologous, i.e. both mAChR and thrombin receptor as well as AlF(4) stimulation of PLD was blunted upon preexposure to carbachol(28) , suggesting that the desensitization is due to a modification of more distal components in the PLD signal transduction cascade. In 1231N1 astrocytoma cells, treatment with carbachol resulted as well in heterologous desensitization of receptor-stimulated PLD (m3 mAChR and thrombin receptor)(32) . Since stimulation of PLD by GTPS in cell sonicates was not reduced by prior carbachol treatment (no data on AlF(4) stimulation), it was concluded that G proteins are not uncoupled from PLD(32) . However, recent data suggest that GTPS stimulation of PLD activity in cell-free preparations is mainly due to small molecular weight G proteins (ADP-ribosylation factors and Rho proteins)(14, 15, 16, 17) . Thus, it remains to be seen whether or not PLD stimulation by heterotrimeric G proteins is unaltered concomitant with the abrogated receptor-stimulated PLD in 1231N1 astrocytoma cells. In HEK cells, rapid and complete desensitization of PLD stimulation was not only observed with the transfected m3 mAChR but also upon activation of endogenously expressed purinergic receptors. Short term (2 min) exposure of the cells to either mAChR or purinergic receptor agonist, followed by agonist wash-out and immediate restimulation with the other receptor agonist, demonstrated that desensitization of receptor-mediated PLD stimulation is, at least in part, also heterologous in this cell type, although no complete cross-desensitization was observed as in other cellular systems(28, 32) . This partial heterologous desensitization may be due to receptor-G protein uncoupling induced, for example, by activated protein kinase C or G protein-coupled receptor kinase(s)(35) .

Since both heterotrimeric and small molecular weight G proteins are apparently involved in mAChR stimulation of PLD in HEK cells (20) , (^2)and since stimulation of PLD by directly activated G proteins was not desensitized, we considered that receptor activation of G proteins may be defective in the mAChR refractory cells. Indeed, short term carbachol treatment almost completely abolished agonist-induced binding of GTPS to G proteins, as determined in membrane preparations and permeabilized adherent cells. However, in measuring the recovery after removal of carbachol, a complete dissociation of these two events was observed. Whereas agonist-stimulated GTPS binding was fully restored after 4 h of culturing in agonist-free medium, mAChR-stimulated PLD remained completely suppressed and only partially (by about 50%) recovered after 24 h. It has to be noted here that, although m3 mAChR stimulation of PLD and PLC in HEK cells is PTX-insensitive, receptor-induced binding of GTPS is to a large extent to PTX-sensitive G(i)-type G proteins(18) . Thus, although overall receptor-stimulated GTPS binding was fully restored, the data cannot exclude that receptor coupling to the PTX-insensitive G protein(s) specifically mediating the PLD response is defective even up to 24 h after removal of the desensitizing agonist.

In CCL39 fibroblasts, m1 mAChR-stimulated PLD was fully restored 30 min after removal of the agonist(28) , suggesting that rapid processes, e.g. receptor internalization-externalization and/or phosphorylation-dephosphorylation, are involved in desensitization of receptor-stimulated PLD. On the other hand, the very long lasting desensitization, geq24 h, of m3 mAChR-stimulated PLD in HEK cells suggested that either an inhibitory factor is formed upon receptor activation or that the activated receptor induces the loss or permanent inactivation of an essential coupling component. To study whether the synthesis of an inhibitory protein responsible for the long lasting inhibition of PLD stimulation is induced by carbachol, cells were treated with the protein synthesis inhibitor, cycloheximide, either before or immediately after carbachol challenge. However, neither treatment prevented the desensitization of mAChR-stimulated PLD. Thus, we would rather like to suggest that an essential transducing component is degraded or persistently redistributed and that new synthesis of this component apparently involved in m3 mAChR coupling to the PLD-specific G protein(s) is required for resensitization of the receptor response.

In contrast to the rapid and long lasting desensitization of receptor-stimulated PLD, mAChR stimulation of PLC was not desensitized following short term agonist treatment of HEK cells. On the contrary, 2 min of preexposure to carbachol led to an about 2-fold increase in receptor-mediated inositol phosphate formation. This supersensitivity disappeared 2 h after agonist removal. The mAChR agonist, pilocarpine, which acted as a partial agonist with regard to PLD stimulation and G protein activation(27) , behaved as a full agonist in activation of PLC. Both in naive and in carbachol-pretreated cells, with a largely reduced agonist-induced GTPS binding but a 2-fold enhanced PLC response, pilocarpine stimulated inositol phosphate formation to the same extent as the full receptor agonist, carbachol. These data suggest that the G proteins mediating m3 mAChR stimulation of PLC, most likely G(18) , are very efficiently activated by the receptor and/or that only a rather small fraction of these G proteins needs to be activated to cause a full receptor-stimulated PLC. The apparent PLC supersensitivity observed in HEK cells preexposed to agonist contrasts with data reported in Chinese hamster ovary cells transfected with and stably expressing the same human m3 mAChR(26) . In these cells, 5 min of treatment with carbachol led to an about 50% reduction in initial (first 10 s) inositol trisphosphate accumulation, while at later time points, no effect of agonist preexposure was observed. Thus, it remains to be studied whether in HEK cells the very initial phase of inositol trisphosphate accumulation is reduced by agonist pretreatment as observed in Chinese hamster ovary cells. Nevertheless, the 2-fold enhanced accumulation of inositol phosphates, being mainly inositol monophosphate (data not shown), observed in agonist-pretreated HEK cells indicates that at least one of the phases, initial or late, of receptor-stimulated PLC is supersensitive. Finally, the very distinct time kinetics of loss of PLC supersensitivity and recovery of PLD stimulation suggest that these two processes involve distinct molecular mechanisms.

In conclusion, the data presented here indicate that stimulation of PLD in HEK cells by the transfected m3 mAChR and the endogenously expressed purinergic receptor is very rapidly and fully desensitized. As extensively studied on mAChR-stimulated PLD, this rapid desensitization is not due to a loss of cell surface receptors or PLD activation by G proteins, but it may involve an initial receptor uncoupling from the responsive G proteins. On the other hand, the long lasting desensitization of receptor-stimulated PLD is rather due to a loss of an as yet undefined essential transducing component. In contrast to the fully desensitized PLD response, m3 mAChR stimulation of PLC was sensitized by agonist preexposure, indicating that regulation of these two phospholipases by agonist-activated m3 mAChR involves very distinct molecular mechanisms.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 354). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-201-723-3460; Fax: 49-201-723-5968.

(^1)
The abbreviations used are: PLC, phospholipase C; PLD, phospholipase D; PTX, pertussis toxin; mAChR, muscarinic acetylcholine receptor; DMEM, Dulbecco's modified Eagle's medium; G protein, guanine nucleotide-binding protein; GTPS, guanosine 5`-O(3-thiotriphosphate); PMA, phorbol 12-myristate 13-acetate; BAPTA/AM, bis-(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid tetra(acetoxymethylester); NMS, N-methylscopolamine; HBSS, Hank's balanced salt solution; HEK, human embryonic kidney; PtdEtOH, phosphatidylethanol.

(^2)
U. Rümenapp, M. Geiszt, F. Wahn, M. Schmidt, and K. H. Jakobs, submitted for publication.


ACKNOWLEDGEMENTS

We thank M. Hagedorn and K. Rehder for expert technical assistance and Dr. E. G. Peralta for providing the m3 mAChR-expressing HEK cells.


REFERENCES

  1. Cockcroft, S., and Thomas, G. M. H. (1992) Biochem. J. 288,1-14 [Medline] [Order article via Infotrieve]
  2. Hug, H., and Sarre, T. F. (1993) Biochem. J. 291,329-343 [Medline] [Order article via Infotrieve]
  3. Billah, M. M. (1993) Curr. Opin. Immunol. 5,114-123 [Medline] [Order article via Infotrieve]
  4. Thompson, N. T., Garland, L. G., and Bonser, R. W. (1993) Adv. Pharmacol. 21,199-238
  5. Boarder, M. R. (1994) Trends Pharmacol. Sci. 15,57-62 [CrossRef][Medline] [Order article via Infotrieve]
  6. Cockcroft, S. (1992) Biochim. Biophys. Acta 1113,135-160 [Medline] [Order article via Infotrieve]
  7. Fukami, K., and Takenawa, T. (1992) J. Biol. Chem. 267,10988-10993 [Abstract/Free Full Text]
  8. Van Corven, E. J., Van Rijswijk, A., Jalink, K., Van Der Bend, R. L., Blitterswijk, W. J., and Moolenaar, W. H. (1992) Biochem. J. 281,163-169 [Medline] [Order article via Infotrieve]
  9. Howe, L. R., and Marshall, C. J. (1993) J. Biol. Chem. 268,20717-20720 [Abstract/Free Full Text]
  10. Thompson, N. T., Bonser, R. W., and Garland, L. G. (1991) Trends Pharmacol. Sci. 12,404-407 [CrossRef][Medline] [Order article via Infotrieve]
  11. Ha, K.-S., and Exton, J. H. (1993) J. Biol. Chem. 268,10534-10539 [Abstract/Free Full Text]
  12. Rhee, S. G., and Choi, K. D. (1992) J. Biol. Chem. 267,12393-12396 [Free Full Text]
  13. Sternweis, P. C. (1994) Curr. Opin. Cell Biol. 6,198-203 [Medline] [Order article via Infotrieve]
  14. Bowman, E. P., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268,21509-21512 [Abstract/Free Full Text]
  15. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C. (1993) Cell 75,1137-1144 [Medline] [Order article via Infotrieve]
  16. Cockcroft, S., Thomas, G. M. H., Fensome, A. M., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, O., and Hsuan, J. J. (1994) Science 263,523-526 [Medline] [Order article via Infotrieve]
  17. Malcolm, K. C., Ross, A. H., Qiu, R.-G., Symons, M., and Exton, J. H. (1994) J. Biol. Chem. 269,25951-25954 [Abstract/Free Full Text]
  18. Offermanns, S., Wieland, T., Homann, D., Sandmann, J., Bombien, E., Spicher, K., Schultz, G., and Jakobs, K. H. (1994) Mol. Pharmacol. 45,890-898 [Abstract]
  19. Sandmann, J., Peralta, E. G., and Wurtman, R. J. (1991) J. Biol. Chem. 266,6031-6034 [Abstract/Free Full Text]
  20. Schmidt, M., Hüwe, S. M., Fasselt, B., Homann, D., Rümenapp, U., Sandmann, J., and Jakobs, K. H. (1994) Eur. J. Biochem. 225,667-675 [Abstract]
  21. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Ramachandran, J., and Capon, D. J. (1988) Nature 334,434-437 [CrossRef][Medline] [Order article via Infotrieve]
  22. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  23. Van Koppen, C. J., Sell, A., Lenz, W., and Jakobs, K. H. (1994) Eur. J. Biochem. 222,525-531 [Abstract]
  24. Wieland, T., Liedel, K., Kaldenberg-Stasch, S., Meyer zu Heringdorf, D., Schmidt, M., and Jakobs, K. H. (1995) Naunyn-Schmiedeberg's Arch. Pharmacol. 351,329-336
  25. Watson, P. A., Krupinski, J., Kempinski, A. M., and Frankenfield, C. D. (1994) J. Biol. Chem. 269,28893-28898 [Abstract/Free Full Text]
  26. Tobin, A. B., Lambert, D. G., and Nahorski, S. R. (1992) Mol. Pharmacol. 42,1042-1048 [Abstract]
  27. Lazareno, S., Farries, T., and Birdsall, N. J. M. (1993) Life Sci. 52,449-456 [CrossRef][Medline] [Order article via Infotrieve]
  28. McKenzie, F. R., Seuwen, K., and Pouysségur, J. (1992) J. Biol. Chem. 267,22759-22769 [Abstract/Free Full Text]
  29. Ben-Av, P., Eli, Y., Schmidt, U.-S., Tobias, K. E., and Liscovitch, M. (1993) Eur. J. Biochem. 215,455-463 [Abstract]
  30. Moehren, G., Gustavsson, L., and Hoek, J. B. (1994) J. Biol. Chem. 269,838-848 [Abstract/Free Full Text]
  31. Briscoe, C. P., Plevin, R., and Wakelam, M. J. O. (1994) Biochem. J. 298,61-67 [Medline] [Order article via Infotrieve]
  32. Nieto, M., Kennedy, E., Goldstein, D., and Brown, J. H. (1994) Mol. Pharmacol. 46,406-413 [Abstract]
  33. Massenburg, D., Han, J.-S., Liyanage, M., Patton, W. A., Rhee, S. G., Moss, J., and Vaughan, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,11718-11722 [Abstract/Free Full Text]
  34. Jiang, H., Alexandropoulos, K., Song, J., and Foster, D. A. (1994) Mol. Cell. Biol. 14,3676-3682 [Abstract]
  35. Premont, R. T., Inglese, J., and Lefkowitz, R. J. (1995) FASEB J. 9,175-182 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.