©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Potentiation of G-mediated Phospholipase C Activation by Retinoic Acid in HL-60 Cells
POSSIBLE ROLE OF G(*)

(Received for publication, November 28, 1994; and in revised form, January 11, 1995)

Taroh Iiri (1) Yoshimi Homma (2) Yoshiharu Ohoka (4) Janet D. Robishaw (3) Toshiaki Katada (4) Henry R. Bourne (1)(§)

From the  (1)Departments of Pharmacology and Medicine, Cell Biology Program, and the Cardiovascular Research Institute, University of California, San Francisco, California 94143, the (2)Department of Biosignal Research, Tokyo Metropolitan Institute of Gerontology, Itabashi, Tokyo 173, Japan, the (3)Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, and the (4)Department of Physical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113 Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Differentiated HL-60 cells acquire responsiveness to fMet-Leu-Phe (fMLP), which activates phospholipase C and O(2) generation in a pertussis toxin-sensitive manner. Addition of retinoic acid (RA) for the last 24 h during dimethyl sulfoxide (Me(2)SO)-induced differentiation enhanced fMLP-dependent signals and interaction between fMLP receptor and G(i). RA modifies both the function and subunit composition of G, the predominant G(i) of HL-60 membranes, as shown by comparing purified G from membranes of Me(2)SO-treated cells (D-G) to G from membranes of cells treated with both Me(2)SO and RA (DR-G). As compared to D-G, DR-G induced more fMLP binding when added to membranes of pertussis toxin-treated HL-60 cells and, in the presence of GTPS, stimulated beta-sensitive phospholipase C in extracts of HL-60 cells to a much greater extent and at lower concentrations. Immunoblots revealed that RA induced expression of the (2) subunit, which was otherwise undetectable in G purified from HL-60 cells or in HL-60 membranes. Possibly by inducing expression of (2), RA alters two functions of the G(i) beta subunit, modulation of fMLP receptor-G coupling and activation of the effector, phospholipase C.


INTRODUCTION

Heterotrimeric (alphabeta) G proteins are GTP-dependent molecular switches that relay signals from cell surface receptors to effector enzymes and ion channels(1) . G proteins consist of two functional subunits, alpha and beta. Historically alpha subunits were assumed to transmit primary signals, while beta subunits were thought to regulate or terminate signals and to be interchangeable among G proteins(2) . Recently, however, accumulating evidence has shown that beta subunits can directly regulate activities of many effectors, including adenylyl cyclases(3, 4) , phospholipase Cbeta (PLCbeta)(^1)(5, 6) , certain K channels(7, 8) , and PI3 kinase(9) , and that beta can mediate hormonal stimulation of the mitogen-activated protein kinase pathway (10) . Moreover, discovery of multiple beta (beta) and () subunits (11, 12, 13, 14, 15) suggested that combinations of different beta and gene products might perform different specific functions. Indeed, experiments with antisense oligonucleotides suggest that specific beta and subunits may determine the specificity of interactions between G proteins and receptors(16, 17) .

One of the signaling systems in which beta, rather than alpha, stimulates the effector is the G(i)-mediated activation of PLCbeta by fMet-Leu-Phe (fMLP) in differentiated HL-60 cells(18) . Several agents, including Me(2)SO and RA (19) induce HL-60 cells to differentiate into neutrophil-like cells. One result of this differentiation program in HL-60 cells is that PLCbeta and the machinery for generating O(2) become responsive to stimulation by fMLP; both responses can be blocked by pertussis toxin (PTX) (20, 21) and are mediated by G(i)(22) , possibly by both G and G, although the former predominates in HL-60 cells(23, 24) . beta, rather than alpha(i), is thought to activate PLCbeta, based on the observations that beta can activate PLCbeta isoenzymes (5, 6) and that no known PLCbeta can be stimulated by alpha(i)(25) .

The mechanism by which differentiation factors allow fMLP-dependent activation of PLC is not known. Possible targets of these agents include the fMLP receptor, G(i), and PLCbeta. Differentiation of HL-60 cells induced by Me(2)SO or dibutyryl cAMP (Bt(2)cAMP) is associated with increases in the number of fMLP-binding sites (26, 27, 28, 29, 30) and in the G(i) content of membranes(23, 28, 31) . In surprising contrast, differentiation induced by RA alone is not associated with detectable increases in either fMLP-binding sites, fMLP receptor transcripts(26, 27, 29, 32) , or G(i) content, (^2)although it clearly increases responses to fMLP. Thus, the mode of action of RA differs from that of Me(2)SO.

Here we report studies of the potentiation by RA of the Me(2)SO-induced fMLP-dependent activation of PLCbeta in HL-60 cells. This potentiation occurs, at least in part, via an effect on G. RA enhances both the interaction of G with fMLP receptors and its ability to activate beta-sensitive PLCbeta. RA treatment increases expression of a specific polypeptide, (2), which may be responsible for both changes in the function of G.


EXPERIMENTAL PROCEDURES

Preparation of Membranes and Cytosol from Differentiated HL-60 Cells

HL-60 cells (approximately 5 times 10^5/ml) were differentiated into neutrophil-like cells by treatment with 1.3% Me(2)SO for 5-6 days either with or without 1 µM RA for the final 24 h. The differentiated cells were resuspended at a final concentration of 1 times 10^8 cells/ml in cavitation buffer (20 mM Tris-HCl, pH 7.5, 250 mM sucrose, 1.5 mM MgCl(2), 3 mM benzamidine, 1 µM leupeptin, 1 µM (p-amidinophenyl)-methanesulfonyl fluoride, and 2 µg/ml soybean trypsin inhibitor) and disrupted by N(2) cavitation followed by addition of 1.25 mM EGTA as described(33) . The disrupted cells were centrifuged at 500 times g for 10 min, and the resulting supernatant was further centrifuged at 40,000 times g for 30 min. The supernatant was used as cytosol, and the pelleted membranes were obtained and resuspended at 5-8 mg of protein/ml in buffer A (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM dithiothreitol, and 25 kallikrein inhibitory units/ml aprotinin) and stored at -85 °C(33, 34) .

fMLP-binding Assay

Membranes (15-25 µg of protein) were incubated at 30 °C for 20 min with various concentrations of [^3H]fMLP (1.25-160 nM) in 50 µl of buffer N (20 mM HEPES/NaOH, pH 7.4, 1 mM EGTA, 0.2 mM MgCl(2), 0.1 mM EDTA, 1 mM dithiothreitol, and 50 kallikrein inhibitory units/ml aprotinin). The reaction mixture further contained 10 µM GTPS where indicated. The reactions were terminated and fMLP binding was quantitated by rapid filtration on GF/C glass filters as described (35) . GTPS-sensitive fMLP binding was determined as a difference between values for specific binding assayed in the presence and absence of 10 µM GTPS.

For the reconstitution of GTPS-sensitive fMLP binding in HL-60 cell membranes by addition of purified G, the membranes were prepared from differentiated HL-60 cells which had been cultured in the presence of 50 ng/ml PTX for 24 h. The membranes from PTX-treated cells were incubated on ice for 15 min with indicated amounts of purified G and then assayed for fMLP binding with 80 nM [^3H]fMLP, as described(34) .

GTPase Assay

Membranes (10-12.5 µg of protein) were incubated at 30 °C for 5 min in 25 µl of buffer N containing 0.5 mM App[NH]p, 1 µM [-P]GTP (3-7 times 10^3 counts/min/pmol), an ATP-regenerating system consisting of 0.1 mM ATP, 3 mM phosphoenolpyruvate, 20 µg/ml pyruvate kinase, and various concentrations of fMLP. The reactions were terminated, and phosphate release was quantitated by charcoal absorption, as described (36) .

Purification of G from HL-60 Cell Membranes

Membranes were solubilized with CHAPS and G was purified essentially as described(34) . The membranes were solubilized with 5 ml of buffer A containing 1% CHAPS and 100 mM NaCl. After centrifugation at 200,000 times g for 30 min at 4 °C, the clear supernatant fractions, in buffer B-1 (buffer A containing 0.7% CHAPS and 50 mM NaCl), was applied to a column of DEAE-Toyopearl 650(S) (1 times 10 cm) which had been equilibrated with 50 ml of buffer B-1. The column was washed with 10 ml of buffer B-1 and then eluted at a flow rate of 1.0 ml/min with a 40-ml linear gradient of NaCl (50-250 mM) in buffer B-1. G and G were recovered at 100 and 130 mM NaCl, respectively. The second major peak fractions containing G were concentrated to approximately 1 ml (using Centricon-10 filters, Amicon) and then fractionated on a column (1.6 times 50 cm) of Sephacryl S-300(HR) which had been equilibrated with buffer B-2 (buffer A containing 0.5% sodium cholate, 1 µM GDP, and 100 mM NaCl). G fractions were further applied to a column of Mono Q HR5/5 which had been equilibrated with 20 ml of buffer B-1. The column was washed with 2 ml of buffer B-1 and then eluted at a flow rate of 0.75 ml/min with a 20-ml linear gradient of NaCl (100-300 mM) in buffer B-1. Fractions containing G, in buffer B-3 (buffer A containing 10% glycerol, 0.5% sodium cholate, and 1 µM GDP), were applied to a column of Hi-Trap Heparin (Pharmacia LKB Biotechnol; 1-ml bed volume) which had been equilibrated with 5 ml of buffer B-3. G was recovered in flow-through fractions. The fractions containing G were concentrated and stored at -85 °C.

Fractionation and Assay of PLC Activity from HL-60 Cell Cytosol

Cytosol of HL-60 cells (1-2 times 10^9 cells) was diluted with equal volume of buffer C (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 10% glycerol, and 0.5% sodium cholate) and applied to a column of Hi-Trap Heparin (1-ml bed volume) which had been equilibrated with 5 ml of buffer C. The column was washed with 3 ml of buffer C and then eluted at a flow rate of 0.5 ml/min with a 25-ml linear gradient of NaCl (0-500 mM) in buffer C.

Phospholipase C activity was measured using sonicated micelles of 50 µM [inositol-2-^3H]phosphatidylinositol-4,5-bisphosphate (20,000 counts/min/tube) and 450 µM phosphatidylethanolamine in a solution containing 50 mM MES-NaOH, pH 7.0, 1.5 mM MgCl(2), 3 mM EGTA, and 1.2 mM CaCl(2) (to give 0.3 µM free Ca)(37) .The final concentration of sodium cholate was 0.08%. Reactions were terminated and released IP3 was quantitated as described(38) . Before the assay, G was activated by incubation with 10 µM GTPS in a reaction mixture containing 10 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5% cholate, and 10 mM MgCl(2) at 30 °C for 30 min.

Immunoblotting Assay

For immunoblotting of subunits, purified G or the cholate-solubilized particulate fractions of HL-60 cells, prepared as described(14) , were resolved by SDS-polyacrylamide gel electrophoresis and then transferred to nitrocellulose blots, using a high temperature transfer procedure(39) . The blots were incubated with either the -specific antibody (B-17), the (5)-specific antibody (D-9), or the (7)-specific antibody (A-67) at a serum dilution of 1:200. The specificities of these antibodies against various recombinant subunits have been described previously(15) ; (2) is distinguished from (3) by their differing mobility on SDS-polyacrylamide gel electrophoresis(15) . Antibody binding was detected by incubation of the blots with I-labeled goat anti-rabbit F(ab`)(2) fragment (Du Pont NEN). Immunoblot analysis of alpha and beta subunits was performed as described(40) . Rabbit polyclonal antibody AS/7 and EC/2 were purchased from DuPont NEN. The specificities of rabbit polyclonal antibodies, beta-636, beta-637, and beta-638 to beta(1), beta(2), and beta(3), respectively, were described previously(11) . The specific antibody for beta(4) was purchased from Santa Cruz Biotechnology.

Miscellaneous

Bovine brain G, alpha, and beta subunit were purified as described(41) . ADP-ribosylation of G(i) alpha subunits by PTX, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing was performed as described(28, 34) . fMLP-stimulated O(2) generation was measured by assaying the reduction of cytochrome c, as described (42) . fMLP-dependent IP(3) production in intact cells was measured using an IP(3) assay kit (Amersham Corp.), as described(43) .


RESULTS

RA Potentiates Effects of Me(2)SO on Responses to fMLP

In agreement with the idea that the mode of action of RA differs from that of Me(2)SO, exposure of HL-60 cells to RA for 24 h barely increased fMLP-dependent O(2) generation, but O(2) generation was dramatically increased by addition of RA during the last 24 h of differentiation induced by Me(2)SO (Fig. 1A). Similarly, RA treatment enhanced activation of PLCbeta by fMLP over and above the effect of Me(2)SO alone (Fig. 1B) although RA alone (24 h) did not induce detectable activation of PLCbeta by fMLP (not shown). Differentiation induced by Me(2)SO or RA was followed by termination of cell proliferation (after 1 day for RA or 4-5 days for Me(2)SO). With either agent, HL-60 cells began to die 24-48 h after proliferation ceased (not shown).


Figure 1: Effects of RA or Me(2)SO on fMLP-dependent O(2) generation or PLC activation in intact HL-60 cells. A, cells were exposed to RA alone (1 µM), Me(2)SO alone (1.4%), or Me(2)SO (DMSO) (1.4%) for the indicated number of days plus RA (1 µM) during the last 24 h of treatment. Cells (4 times 10^6) were then incubated with 1 µM fMLP for 10 min at 37 °C, and O(2) generation was measured as described under ``Experimental Procedures.'' Bars in panel A represent the mean ± S.E. of four determinations. B, cells were treated with Me(2)SO alone (1.4%) for 5 days (D) or with Me(2)SO (1.4%) for 5 days plus RA (1 µM) during the last 24 h of treatment (DR). Cells (5 times 10^6) were then incubated with 1 µM fMLP for 1 min at 37 °C, and production of IP(3) was measured as described under ``Experimental Procedures.'' Bars represent the mean ± S.E. of three determinations.



fMLP Receptor-G(i) Interaction Is Altered by RA

fMLP binding and fMLP-dependent GTPase assays in membranes suggested that RA enhances the interaction between G(i) and fMLP receptor. We first investigated fMLP binding in membranes from HL-60 cells treated with Me(2)SO alone (5 days) or with Me(2)SO (5 days) plus RA (during the last day of treatment) (D versus DR). As assessed by PTX-catalyzed ADP-ribosylation of alpha(i), the membrane G(i) content of D and DR membranes were almost equal (25 versus 28 pmol/mg protein, respectively), indicating that co-exposure to RA did not affect the expression of G(i).

Like the binding of many agonists to G(i)-coupled receptors, fMLP binding is diminished in the presence of GTPS(35, 44) ; presumably, this is because receptor-G(i) complexes have a higher binding affinity for agonists, and binding of GTPS to alpha(i) promotes dissociation of G(i) from receptors. D and DR membranes contained apparently equal numbers of fMLP-binding sites, as measured in the presence of GTPS, while the number of sites sensitive to GTPS was greater in DR than in D membranes (Fig. 2, A and B). The fMLP-binding sites that disappear in the presence of GTPS are assumed to represent fMLP receptors that would be coupled to G(i) in the absence of the guanine nucleotide, while the GTPS-insensitive-binding sites are thought to represent receptors uncoupled from G(i). Following this interpretation, the data in Fig. 2, A and B, show that treatment with Me(2)SO plus RA enhanced coupling of fMLP receptors to G(i) more than did treatment with Me(2)SO alone.


Figure 2: fMLP binding (A and B) and fMLP-stimulated GTP hydrolysis (C) in membranes of Me(2)SO (D) or Me(2)SO plus RA (DR)-treated HL-60 cells. A, D (circles) and DR (triangles) membranes (20 µg) were incubated at 25 °C for 20 min in 50 µl of a reaction mixture containing the indicated concentration of [^3H]fMLP. The specific binding was measured in the presence (open symbols) or in the absence (closed symbols) of 10 µM GTPS, as described under ``Experimental Procedures.'' B, GTPS-sensitive fMLP binding, estimated as the difference between the two specific binding curves performed in the presence and absence of GTPS, depicted in panel A. C, D (circles) and DR (triangles) membranes (10 µg) were incubated at 30 °C for 5 min in the presence of the indicated concentration of fMLP, and GTPase activity was measured as described under ``Experimental Procedures.''



Although GTP hydrolysis stimulated by maximally effective concentrations of fMLP was almost equal in D and DR membranes, fMLP stimulated GTPase activity with an EC that was almost 10-fold lower in DR, as opposed to D membranes (Fig. 2C). By this criterion also, RA enhanced the interaction between fMLP receptors and G(i).

Membranes from HL-60 cells treated with RA alone (for 1 or 2 days) showed no detectable fMLP-binding or fMLP-stimulated GTPase activity. In the presence of Me(2)SO, exposure of the cells to RA for more than 24 h inhibited the induction of fMLP binding and the G(i) increase induced by Me(2)SO (data not shown).

RA Enhances Receptor and Effector Interactions of pure G

The principal PTX substrate in HL-60 cells is the alpha subunit, alpha, of G; these cells contain a small amount of alpha, but do not express other potential PTX substrates, such as alpha or alpha(o)(23, 24, 28, 45) . To assess differentiation-induced changes in G, we purified the G heterotrimer from D or DR membranes, without detectable contamination of G(34) .

To assess interactions of pure G with fMLP receptors, we prepared acceptor membranes from PTX-treated cells; in these membranes, fMLP binding was completely unaffected by the presence of GTPS, indicating that PTX treatment had completely uncoupled endogenous G(i) from the receptors. In order to test specifically the additional differentiating effect of RA, the acceptor membranes were prepared from cells differentiated in the presence of Me(2)SO alone. Addition of pure G to these membranes increased the binding of fMLP to its receptors, and this increased binding was blocked by GTPS. G purified from DR cells (DR-G) was much more effective in enhancing GTPS-sensitive fMLP binding than was pure G from D cells (D-G) (Fig. 3A), in agreement with parallel observations (Fig. 2, A and B) of the effects of GTPS on fMLP binding to sites in DR versus D membranes, in experiments in which the receptors were coupled to endogenous G. We compared the abilities of three purified G preparations to enhance fMLP binding in PTX-treated acceptor membranes; at a concentration of 5 nM, these pure G proteins enhanced fMLP binding with a rank order of DR-G > G purified from bovine brain D-G (Fig. 3B).


Figure 3: Effects of D-G or DR-G on GTPS sensitive fMLP binding. A, the indicated concentrations of pure D-G (circles) or pure DR-G (triangles) were added to PTX-treated D membranes, and specific fMLP binding was assayed in the absence (closed symbols) or in the presence (open symbols) of 10 µM GTPS as described under ``Experimental Procedures.'' B, fMLP binding was assayed in the absence (open circle) or in the presence of 5 nM D-G (closed circles), bovine brain G (open squares), or DR-G (closed triangles); symbols represent duplicate determinations.



In addition to increasing the ability of G to interact with fMLP receptors, RA treatment greatly enhanced the ability of GTPS-activated G to stimulate PLC activity partially purified from HL-60 cytosol. Cytosol, obtained from DR HL-60 cells, was fractionated on a Hi-Trap Heparin column in an NaCl gradient (see ``Experimental Procedures''), and PLC activities of individual fractions were assayed without an activator (control) or in the presence of either 30 nM beta purified from bovine brain or 10 nM GTPS-activated DR-G (Fig. 4A). PLC activity was eluted from the column in two peaks; the second peak of PLC activity was similarly activated by both GTPS-activated DR-G and bovine brain beta, in keeping with previous evidence (18, 25, 46, 47) that G in neutrophils (and HL-60 cells) activates PLC via its beta subunit, while activated alpha cannot stimulate PLC in neutrophils or other cells rather than via alpha-GTP.


Figure 4: G and beta dependent activation of PLC activity in HL-60 cytosol extract fractionated on a Hi-Trap Heparin column. A, the cytosol fraction of DR HL-60 cells (see ``Experimental Procedures'') was applied to a Hi-Trap Heparin column, and PLC activities in fractions eluted on an NaCl gradient were assayed in the absence of any stimulus (open circles) or in the presence of GTPS-activated DR-G (10 nM; filled triangles) or 30 nM beta (filled circles), as described under ``Experimental Procedures.'' B, the indicated concentrations of GTPS-activated D-G, DR-G, bovine brain alpha (open squares), or bovine brain beta subunit were added to aliquots of fraction 26 from the Hi-Trap Heparin column, and PLC activities were assayed.



Column fraction 26, which showed maximal fold stimulation by both activated DR-G and beta, was used to test the molar potency of pure beta, DR-G, and D-G as stimulators of beta-sensitive PLC activity. On a molar basis, GTPS-activated DR-G appeared slightly more potent as a stimulator of PLC than did pure beta from bovine brain. Both were much more effective stimulators than was GTPS-activated D-G (Fig. 4B). In accord with work by others (18, 25, 46, 47) , GTPgS-activated alpha from bovine brain did not stimulate this PLC activity.

Retinoic Acid Induces Expression of a Specific Subunit, (2)

Because DR-G interacted with the fMLP receptor and stimulated a beta-sensitive PLC activity more effectively than did D-G, we asked whether the difference might be accounted for by a qualitative difference between the beta subunits of the two proteins. The immunoblots of Fig. 5A show that both the D- and DR-G preparations contained similar relative amounts of beta1, beta2, and beta4 (beta3 was not detectable in either type of preparation; result not shown). Although both G preparations contained (5) and (7), only the DR-G contained detectable (2) (Fig. 5B). Indeed, immunoblots of membrane proteins from undifferentiated or Me(2)SO-treated HL-60 cells revealed no immunoreactive (2), although (2) could be detected in immunoblots of membranes of cells treated with RA alone (for 2 days) and was much more prominent in membranes from DR cells (Fig. 5C). Although the (2) band was detected with an antibody that binds both (2) and (3), the presence of (3) was ruled out by virtue of its different mobility from (2), and also because a (3)-specific antibody failed to detect (3) in extracts of DR cells (results not shown). Thus, Me(2)SO and RA synergistically increased fMLP-dependent activation of PLC (Fig. 1C) in parallel with their synergistic effect on expression of (2).


Figure 5: Immunoblot analysis of beta in D-G, DR-G, and membranes of HL-60 cells. A, D-G (lane 2), DR-G (lane 3), or bovine brain beta (lane 1) were resolved on a 11% SDS-polyacrylamide gel, and then immunoblotted with a beta1 specific antibody (beta-636), a beta2 specific antibody (beta-637), or a beta(4)specific antibody as described under ``Experimental Procedures.'' B, D-G (lane 1) or DR-G (lane 2) were resolved on a 15% SDS-polyacrylamide gel, and then immunoblotted with a -specific antibody (B-17), a (5)-specific antibody (D-9), or a (7)-specific antibody (A-67) as described under ``Experimental Procedures.'' C. cholate extracts of membrane fractions (100 µg) from undifferentiated cells or cells treated with RA (2 days), Me(2)SO (5 days), or Me(2)SO (DMSO) (5 days) plus RA (during the last 24 h of treatment) were resolved on a 15% SDS-polyacrylamide gel and then immunoblotted with a -specific antibody (B-17). The standard represents 4 µg of purified bovine brain G proteins containing a mixture of (2) and (3) subunits.



After ADP-ribosylation by PTX and radioactive NAD, D- and DR-G(i) preparations were subjected to SDS-polyacrylamide gel electrophoresis and isoelectric focusing. Apparent molecular weights and isoelectric points of alpha in the two preparations were identical (results not shown), suggesting that differentiation in the presence of RA did not produce a change in size or charge of alpha.


DISCUSSION

In this report we show that RA potentiates Me(2)SO as an inducer of fMLP-dependent PLC activation in HL-60 cells and that this potentiation results, at least in part, from a qualitative change in the beta subunit of G. RA enhances the abilities of G both to interact with fMLP receptor and also to activate a beta-sensitive PLC in the cytosol of differentiated HL-60 cells. RA induces expression of a specific subunit, 2, which is otherwise not found in either G or membrane fractions of HL-60 cells. This is the first demonstration that a differentia-tion factor regulates both the function and the polypeptide composition of a G protein beta subunit. Moreover, if expression of (2) is responsible for the enhanced signaling ability of G, this is the first demonstration that the specific composition of a beta subunit physiologically accounts for the ability of a G protein to activate a specific effector.

In this section, we shall first discuss the three novel observations in this report. Then we shall return to the question of whether expression of 2 accounts for the qualitative change in G function we observed.

Novel Observations

Our first new observation is the synergism of two differentiation factors, Me(2)SO and RA, in increasing fMLP-dependent signals in HL-60 cells (Fig. 1, A and B). This agrees with earlier inferences that the two factors produce their effects on HL-60 cells in different ways. (^3)Stimulation of fMLP receptors is known to activate multiple effector pathways, involving activities of PLC, PI3K, phospholipase A(2), phospholipase D, and NADPH oxidase(48, 49) . The pathways (or combination of pathways) that lead to increases in most of these activities, including NADPH oxidase, are not fully understood, making it difficult to pinpoint the mechanism(s) by which RA synergistically increases many responses to fMLP. In the case of PLC activation, however, the interactions that link the fMLP receptor, G(i), and PLC are direct; consequently, at least one of these elements must be a differentiation target of RA.

Indeed, our second new observation is that G, more specifically, its beta subunit, is a regulatory target of RA in HL-60 cells. RA induced changes in the ability of G to interact with fMLP receptors, as shown by measurements of fMLP binding and stimulation of GTP hydrolysis in HL-60 membranes (Fig. 2) and also by reconstitution into PTX-treated membranes of pure G preparations from cells treated with Me(2)SO plus RA or Me(2)SO alone (DR-GversusD-G; Fig. 3). In addition, reconstitution of a partially purified PLCbeta with GTPS-activated G indicated that DR-G was a more effective and more potent activator of PLC, on a molar basis, than was D-G (Fig. 4). The latter result clearly implicated the beta subunit of DR-G as the key element that accounts for the difference between DR-G and D-G, because (a) the PLCbeta preparation was quite sensitive to stimulation by beta but not by alpha-GTPS (both purified from bovine brain); (b) PLC preparations from HL-60 and other cells (6, 18, 25) do not respond to activated alpha(i) subunits; (c) although both D-G and DR-G(i) contained equal amounts of alpha subunit, the latter produced a very large stimulation of PLC in response to GTPS, whereas the former had hardly any effect (Fig. 4B). Taken together, the enhanced abilities of DR-G to interact with the fMLP receptor and to activate PLCbeta explain, at least in part, the synergistic effect of RA on Me(2)SO-induced responsiveness of HL-60 cells to fMLP.

The third new finding is that RA induces expression of a specific subunit, (2), which is not present in HL-60 cells unless RA is added. Several reports have shown differentiationrelated changes in the amount of different Galpha subunits in various cell types (23, 31, 50, 51) . To our knowledge, however, differentiation factor-regulated expression of a specific G subunit has not previously been reported, although different cells and tissues of mammals do contain different complements of beta and subunits.

Thus, while (5) and (7) are expressed in a variety of tissues, (2) and (3) are preferentially expressed in the brain(14) . beta subunits are widely distributed in different tissues, with the exception of beta5, which is selectively expressed in brain(13) . Immunocytochemistry techniques have shown more precise and specific localizations. In the retina, beta1 and 1 are found in the outer segments of rod cells, while beta3 and 2 are found in cone cells(52) . (5) has been reported to co-localize with vinculin and actin filaments in cultured cells (53) .

Does RA-induced Expression of (2) Account for the Altered Functions of G?

It is likely that specific expression and localization of different beta and subunits reflect specific roles of the individual subunits in transmitting different signals. In this context, it is reasonable to suspect that RA-induced expression of (2) and the consequent formation of complexes (beta(x)(2)) containing (2) and various beta subunits play a role in the parallel effects of RA on fMLP signaling in HL-60 cells.

Clear precedents indicate that specific beta complexes can influence the specificity of interactions between receptors and alpha subunits, as is likely to be the case for beta(x)(2) in HL-60 cells. Antisense experiments have shown that a somatostatin and an m4-muscarinic receptor in a single cell require beta subunits composed of different, specific beta and polypeptides to trigger G(o)-dependent inhibition of Ca currents(16, 17) . beta complexes of different composition differ in their ability to facilitate rhodopsin-catalyzed binding of GTPS to alpha(t)(54, 55, 56) . Moreover, a farnesylated peptide mimicking the C terminus of (1) can directly stabilize metarhodopsin II, the active form of rhodopsin(57) ; this shows that structurally specific segments of a subunit can interact directly with a receptor and, by implication, help to determine receptor-G protein specificity.

Our data suggest that a specific beta (beta(x)(2)) activates PLC in HL-60 cells with greater efficiency than other beta/ combinations. In other tissues, what precedents indicate the specificity of different beta subunits as regulators of effectors? First, a strong general argument: if all beta complexes were equivalent, all G proteins should regulate beta-sensitive effectors in the same way; for example, stimulation of G(s) by beta-adrenoreceptors should stimulate PLC activity and open K channels just as well as does stimulation of G(i). This is demonstrably not the case, suggesting that different beta complexes are not equivalent.

In addition to this general argument, several studies have used purified beta subunits of different composition to search for specific regulation of effectors, including K channels (7) and PLCbeta(58, 59) . In general these studies indicate that beta(1)(1), the beta of retinal rod outer segments, regulates these effectors less effectively than do other beta complexes, but that the other beta complexes are more or less indistinguishable in potency or efficacy. In a study using PLCbeta3 purified from bovine brain, a recombinant beta containing (2) appeared to be a somewhat more potent stimulator than other beta complexes (see Fig. 5of (58) ), although the authors concluded that all complexes were equivalent.

If beta(x)(2) is indeed a superior stimulator of PLCbeta, how can this fact have escaped previous investigators? One possibility is that the right combination of beta and subunits has not been tested. If beta(x)(2) is the key activator of PLCbeta in RA/Me(2)SO-treated HL-60 cells, beta(x) could be beta1, beta2, or beta4; the last of these has not been tested as a stimulator of PLC in combination with (2). (^4)It is also possible that differentiated HL-60 cells contain a PLCbeta that is specifically more sensitive to beta(x)(2), but which has not yet been tested in experiments with recombinant proteins. HL-60 cells have been reported to contain PLCbeta2 (which is found only in HL-60 cells; Refs. 5, 60) and PLCbeta3, both of which are sensitive to activation by beta(5, 6) ; PLCbeta3 is more sensitive to beta than is PLCbeta2(5, 6) . We have not established the identity of the PLC used in our experiments, which was partially purified from HL-60 cytosol; the active fractions contained material that was detected by anti-PLCbeta2 antibody, but did not react with an antiserum to the carboxyl terminus of PLCbeta3 (results not shown). Thus we cannot rule out the presence of a previously unknown beta-responsive PLC in our experiments. (^5)

In summary, we have shown that RA, a differentiation factor, enhances fMLP signals in HL-60 cells and that the enhanced stimulation of PLC depends on a qualitative change in the beta subunit of G. In parallel with these changes, RA induces expression of a specific polypeptide, (2). From this circumstantial evidence, we infer that RA-induced (2) accounts both for enhanced interactions of G with fMLP receptors and for its more effective stimulation of PLC. Critical tests of this inference will require further experiments. It will be necessary to show that (a) pure beta(x)(2) does activate a PLC in these cells more effectively than do other beta complexes, and (b) removal or specific inactivation of (2) blocks the effect of RA on fMLP-stimulated PLC activity.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants GM27800 and CA54427 (to H. R. B.), the Human Frontier Science Program and Yamanouchi Foundation (to T. I.), NIH Grant GM 39867 (to J. D. R.), Grants 05271215 and 06264217 from the Ministry of Education, Science, and Culture of Japan (to Y. H.), and the Scientific Research Fund of Ministry of Education, Science, and Culture of Japan (to T. K.). 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: Box 0450, S-1210, Dept. of Pharmacology, University of California Medical Center, San Francisco, CA 94143-0450. Tel.: 415-476-8161; Fax: 415-476-5292.

(^1)
The abbreviations used are: PLCbeta, phospholipase Cbeta; fMLP, fMet-Leu-Phe; RA, retinoic acid; PTX, pertussis toxin; GTPS, guanosine 5`-O-(thiotriphosphate); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MES, 4-morpholineethanesulfonic acid;

(^2)
T. Iiri, Y. Homma, Y. Ohoka, J. D. Robishaw, T. Katada, and H. R. Bourne, unpublished observation.

(^3)
RA has been reported not only to increase the number of fMLP-binding sites in HL-60 cells, but also to block the increase in fMLP-binding sites induced by Me(2)SO in the same cells(26) . In contrast, we found that RA did not alter the increased number of fMLP-binding sites induced by Me(2)SO. The discrepancy may be explained by a difference in experimental procedures. In the previous study, HL-60 cells were treated simultaneously with RA and Me(2)SO, whereas in our experiments RA was added only for the last 24 h of a 5-day Me(2)SO treatment.

(^4)
Interestingly, transient expression experiments in COS cells have shown that co-expression of beta(5) and (2) activates PLCbeta2 more strongly than do several other combinations(13) . It is not clear, however, whether the greater effect on PLCbeta2 is due to enhanced potency of the beta(5)(2) dimer or to greater stability (and therefore a higher membrane content) of the dimer.

(^5)
A truncated form of PLCbeta3, lacking part of the COOH terminus, is reportedly (61) activated more than 100-fold by beta.


ACKNOWLEDGEMENTS

We thank colleagues in Henry Bourne's laboratory for advice and useful useful discussions, and Bernard Fung (UCLA School of Medicine) for antisera directed against beta1, beta2, and beta3.


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