©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
G Protein Control of Drosophila Photoreceptor Phospholipase C (*)

Jennifer L. Running Deer (§) , James B. Hurley (¶) , Stuart L. Yarfitz

From the (1) Howard Hughes Medical Institute and Department of Biochemistry, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Light stimulates phosphatidylinositol bisphosphate phospholipase C (PLC) activity in Drosophila photoreceptors. We have investigated the mechanism of this reaction by assaying PLC activity in Drosophila head membranes using exogenous phospholipid substrates. PLC activation depends on the photoconversion of rhodopsin to metarhodopsin and is reduced in norpA PLC and ninaE rhodopsin mutants. NorpA PLC is stimulated by light at free Ca concentrations between 10 nM and 1 µM. This finding is consistent with a Ca-mediated positive feedback mechanism that contributes to the rapid temporal response of invertebrate photoreceptor cells. The guanyl nucleotide dependence of light-stimulated PLC activity indicates that a G protein regulates NorpA. This was confirmed by the observation that light stimulation of PLC activity is deficient in mutants that lack the eye-specific G protein subunit Ge. These results indicate that Ge functions as the subunit of the G protein coupling rhodopsin to NorpA PLC.


INTRODUCTION

Excitation of invertebrate photoreceptors is initiated by photoconversion of rhodopsin into metarhodopsin. This stimulates a cascade of events in photoreceptor cell rhabdomeres which leads to opening of plasma membrane cation channels. It has been demonstrated that photoactivated rhodopsin catalyzes nucleotide exchange on a G protein and that light stimulates hydrolysis of PIP() into IP and diacylglycerol (1) . Light causes an increase in cytoplasmic Ca, opening of plasma membrane cation channels, and influx of Na and Ca.

The Drosophila norpA gene encodes a photoreceptor PLC that is obligatory for photoexcitation. Strong norpA alleles are blind and exhibit a loss of light-induced electrical responses (2, 3) . About 90% of the PLC activity in Drosophila head homogenates is associated with membranes from the compound eyes. This activity is strongly reduced in norpA mutants (4) . The major PLC from Drosophila heads which is absent in norpA mutants has been purified (5) . This protein, the product of the norpA gene, is a homolog of mammalian PLC- (6). Immunological analysis indicates that NorpA is present in all adult photoreceptor cell types and is specifically localized to rhabdomeres (7) .

Several invertebrate G protein subunits that may participate in phototransduction have recently been identified. , , and subunits of light-stimulated G protein have been isolated from squid photoreceptors (8, 9, 10, 11) . Two photoreceptor-specific G protein isoforms, DGq1 and DGq2 (12) , and a photoreceptor-specific G, Ge (13), have been cloned from Drosophila. Recent studies suggest that DGq1 is the subunit of a G protein that couples light-stimulated rhodopsin to PLC (14) and that Ge is the subunit of this G protein. Immunoreactive Ge is present in photoreceptor cell rhabdomeres (15) , and Ge mutants are deficient in light-stimulated GTPS binding in compound eyes (15, 16) . Ge mutants also exhibit reduced light sensitivity and slowed excitation and deactivation kinetics (16) .

These findings suggest that NorpA PLC is coupled to light activated rhodopsin (metarhodopsin) via a G protein with Ge as its subunit. To confirm this, we determined the nucleotide dependence of PLC activity in Drosophila head membranes and compared light stimulation of PLC activity in Ge and Ge mutant flies. We found that light-sensitive NorpA PLC activity in Drosophila head membranes is guanyl nucleotide-sensitive and dependent on normal Ge function.


EXPERIMENTAL PROCEDURES

Materials

Bovine brain PIP and PE were purchased from Sigma, PS from Avanti, and [H]PIP (74-370 GBq/mmol) from DuPont NEN. PS and PE were stored in CHCl, PIP, and [H]PIP in CHCl:MeOH:HCl (75:25:1) under argon at -20 °C. Nucleotides, creatine phosphate, creatine phosphokinase, and protease inhibitors were purchased from Sigma. Nucleotides were made up as described (17) and stored at -70 °C in Tris buffer. The buffer for the guanine nucleotides also contained 1 mM dithiothreitol.

All fly stocks were white-eyed to eliminate the light blocking effects of colored pigments. w, w;Ge, w;Ge;P[w,Rh1-Ge], and w;ninaE flies were maintained as described previously (15) . w,norpA were raised in the dark to prevent light-dependent retinal degeneration (18) . Heads were collected from ninaE flies that were less than 3 days old to avoid time-dependent retinal degeneration (18) .

Head Membrane Preparation

Flies were dark-adapted for 2-24 h, frozen in liquid nitrogen, and stored at -70 °C. Heads were collected as described (13) and crude membranes prepared by a procedure similar to that of Devary et al.(19) . Heads were homogenized in 250 mM sucrose, 120 mM KCl, 5 mM MgCl, 1 mM EGTA, 10 mM MOPSO (pH 7.0), 1 mM dithiothreitol, 10 µg/ml leupeptin, 1 µg/ml pepstatin A) using a glass/Teflon homogenizer. The suspension was allowed to settle for 5 min to remove large debris, decanted, and crude membranes prepared by centrifugation at 14,000 g, followed by resuspension in 0.5 volume of the same buffer. Membrane preparations were aliquoted and stored in foil-wrapped tubes in liquid nitrogen. Protein concentration was assayed by Bradford (Bio-Rad) using bovine serum albumin standards. All procedures were performed in a darkroom under infrared illumination using night vision goggles.

Substrate Preparation

Total head membrane PLC activity was assayed using OGM (100 mM NaCl, 50 mM HEPES (pH 7.1), 2% octyl--glycopyranoside, 100 µM PIP, 35,000 dpm [H]PIP/reaction). Light-inducible PLC activity was measured on PLV (100 mM NaCl, 50 mM HEPES (pH 7.1), 500 µM mixed phospholipids (1:2:2 molar ratio of PIP:PE:PS), 35,000 dpm [H]PIP/reaction) (20) . Phospholipids were mixed, dried under a stream of argon in a siliconized polypropylene tube, suspended in argon-saturated substrate buffer (100 mM NaCl, 50 mM HEPES (pH 7.1), plus 2% octyl--glycopyranoside for OGM), vortexed 5 min (PLV only), then sonicated at room temperature for 2 min in a Branson water bath sonicator. The substrates were stable for several hours at room temperature and were used within 1 h of preparation.

PLC Assay

Membrane preparations were thawed and illuminated at room temperature using a 100-watt tungsten quartz halogen light source with red (Schott RG-610) or blue (Schott BG-28) filters. In the standard procedure, assays were initiated by addition of 10 µl of illuminated membranes (8-10 µg of protein) to 40 µl of reaction mixture composed of 20 µl of phospholipid substrate, 10 µl of reaction buffer (150 mM HEPES, pH 7.1, 300 mM NaCl, 2.5 mg/ml bovine serum albumin, 12.5 mM MgCl, 9 mM EGTA), 5 µl of ATP regenerating system (10 mM ATP, 50 mM creatine phosphate, 500 units/ml creatine phosphokinase), 2.5 µl of 200 µM GTPS, and 2.5 µl of either 6.6 mM (for PLV substrate) or 38.2 mM CaCl (for OGM substrate) and incubated for 4 min at 30 °C. The reaction was terminated by addition of 50 µl of 10% (w/v) trichloroacetic acid and 25 µl of 10 mg/ml bovine serum albumin. After 15 min on ice, samples were centrifuged at 4 °C for 4 min at 9000 g, and 100-µl supernatants containing soluble inositol phosphates scintillation counted for [H]IP. Substrate blanks obtained by adding trichloroacetic acid to the reaction mix before the membranes were subtracted from each data point. Ca concentrations were varied by adding equal volumes of CaCl stocks of differing concentrations. Free Ca concentration was calculated using MaxC (Shareware from C. Patton, Hopkins Marine Station, Stanford University), and the estimated concentrations of free Ca produced by the CaCl, EGTA, MgCl, and HEPES stocks used in these experiments verified using Rhod-2 (21) .

Final reaction mixtures contained 50 mM HEPES (pH 7.1), 100 mM NaCl, 50 mM sucrose, 24 mM KCl, 2 mM MOPSO, 0.5 mg/ml bovine serum albumin, 3.5 mM MgCl, 2 mM EGTA, 1 mM ATP, 5 mM creatine phosphate, 50 units/ml phosphocreatine kinase, 10 µM GTPS, 0.2 mM dithiothreitol, 2 µg/ml leupeptin, 0.2 µg/ml pepstatin A, 35,000 dpm [H]PIP], plus 20 µM PIP, 40 µM PE, 40 µM PS, 50 nM free Ca (PLV) or 0.4% octyl--glycopyranoside, 20 µM PIP, 5 µM free Ca (OGM).


RESULTS

Assay of Light-stimulated NorpA PLC Activity in Drosophila Head Membranes

Blue and red illuminations were used to regulate PLC activity in Drosophila head membrane preparations. Rh1 rhodopsin, the predominant photopigment in Drosophila compound eyes, absorbs maximally at 480 nm and is converted to its active metarhodopsin form by blue light. Rh1 metarhodopsin absorbs maximally at 560 nm and is photoconverted back to inactive rhodopsin by red light.

PLC activity in Drosophila head membranes was assayed in the presence of 10 µM GTPS using exogenous [H]PIP incorporated into either PLV or OGM. With the PLV substrate, blue illumination of head membranes from dark-adapted control (w ) flies stimulated PLC activity 2-fold above dark levels (Fig. 1). There was no stimulation with red light. High light-independent levels of PLC activity were found using the OGM substrate (Fig. 1), suggesting that the detergent uncouples PLC from receptor activation.


Figure 1: Blue light stimulation of phospholipase C activity in Drosophila head membranes. Phospholipase C activity of w head membranes on [H]PIP-labeled phospholipid vesicles (PLV) or octyl--glycopyranoside micelles (OGM). The flash times were 1 min for red (Schott RG-610) and 10 s for blue (Schott BG-28) illumination. IP is mixed inositol phosphates. Values are means ± sem from multiple experiments (PLV, n = 18) (OGM, n = 4).



The relationship between metarhodopsin formation and PLC stimulation with PLV substrate was examined by titrating Drosophila head membranes with blue light (Fig. 2). With the light intensity attenuated by a 3 OD neutral density filter the level of PLC activity increased with exposure time. Maximal PLC activity was stimulated by a 10-s flash. A 1000-fold increase in intensity of the 10-s flash had little additional effect on PLC activity. However, illumination times of 30 s or 1 min reduced activity (data not shown). A possible explanation for this decrease in activity might be decay caused by extended incubation of the membranes at room temperature. To test for this decay, membranes were flashed for 10 s at maximal intensity and held for varying lengths of time before addition to the substrate mix. We found that PLC activity remained at maximal levels for at least 2 min following the flash of blue light. The activity then slowly decreased to background levels by 10 min (data not shown). Since the membranes maintained full potential for PLC stimulation for 2-min post-flash, the reduced PLC activity at 30-s and 1-min flash times is likely to be the result of specific inactivation mechanisms rather than nonspecific decay.


Figure 2: Changes in light-stimulated phospholipase C activity with intensity and time of illumination. w head membranes were flashed with blue light for different times and assayed for PLC activity on PLV substrate. Neutral density filters of optical density (OD) 1, 2, and 3 were employed to vary light intensity. Values are means ± S.E. (n = 3 or 4) from multiple experiments.



Light stimulation of PLC depends on the state of the phototransduction apparatus prior to preparation of the membranes. Exposure to red illumination did not reduce PLC activity in membranes prepared from dark-adapted flies. However, when membranes were prepared from non-dark-adapted flies, blue light did not stimulate PLC, and red illumination reduced PLC activity to the level found in dark-adapted flies (data not shown). Dark adaptation of previously frozen heads was ineffective in lowering basal activity. We unexpectedly found that when membranes from dark-adapted flies were first flashed with red light, they became refractory to subsequent stimulation by blue light. This suggests that red light induces an insufficiency of some factor required for PLC activation. Byk et al.(22) reported that illumination of Musca eye membranes with orange light, which photoconverts metarhodopsin to rhodopsin, caused release of arrestin from the membranes. It is possible that other components of the phototransduction cascade are also released from rhabdomeric membranes under these conditions.

PLC Activity in norpA and ninaE Mutants

It has been reported that most of the PLC activity in Drosophila head membranes corresponds to NorpA PLC (4, 5, 23) . Consistent with these reports, we found that PLC activity in norpA mutants was only 5% of blue light-stimulated activity of control white-eyed flies when assayed with PLV substrate (Fig. 3). norpA is a strong allele which exhibits severe deficiencies in electroretinogram light sensitivity (24) and NorpA protein levels (23) . When assayed with the OGM substrate, PLC activity in norpA head membranes was approximately 25% of wild type activity (Fig. 3). The levels of Rh1, DGq, and Ge proteins in dark raised norpA head membranes were normal when analyzed by immunoblotting (data not shown).


Figure 3: Phospholipase C activity in norpA and ninaE mutants. Head membranes from control w and white-eyed norpA and ninaE flies were flashed with red (1 min) or blue (10 s) light and assayed on either PLV or OGM substrates. Values are means ± S.D. (n = 3) from a representative experiment.



To confirm that light stimulation of PLC activity requires rhodopsin, ninaE head membranes were assayed. ninaE encodes the opsin component of Rh1 rhodopsin (25, 26) . ninaE mutants exhibit electroretinogram abnormalities and express less than 2% of wild type Rh1 rhodopsin levels (24) . ninaE PLC activity assayed with PLV substrate was insensitive to blue light and was only 25% of unstimulated wild type levels (Fig. 3). The low basal activity of ninaE suggests that most of the basal activity of wild type membranes on PLV substrate is metarhodopsin-dependent. Nearly wild type levels of PLC activity were observed with OGM substrate, indicating that normal levels of norpA PLC are present in the ninaE membranes. Immunoblot analyses demonstrated that the levels of DGq and Ge in ninaE head membranes were normal (data not shown).

Effect of Free Caon PLC Activity

Ca is an important mediator of both excitation and inactivation processes in invertebrate photoreceptors (1). Cytoplasmic Ca increases as a consequence of IP-induced mobilization from internal Ca stores and uptake of extracellular Ca through light-sensitive plasma membrane cation channels.

We examined the Ca dependence of PLC activation. With PLV substrate, PLC activity increased when free Ca was raised from 10 nM to 1 µM (Fig. 4, upper panel). Blue light stimulated PLC activity over this range of free Ca concentration. The largest blue light effect was between 10 and 200 nM free Ca. PLC activity was maximal at 1 µM free Ca, but was nearly insensitive to light at this Ca level. PLC activity assayed with OGM substrate was also dependent on Ca. Maximal activity occurred at 5 µM and was independent of light over the entire range of Ca concentration (Fig. 4, lower panel). This is similar to the previously reported Ca sensitivity of PIP hydrolysis using crude and partially purified norpA PLC (5) .


Figure 4: Ca titration of phospholipase C. w head membranes were maintained in the dark or flashed with red (1 min) or blue light (10 s) and assayed for PLC activity at different free Ca concentrations. PLV substrate (upper panel). Values are means ± S.E. (n = 3 or 4) from multiple experiments. OGM substrate (lower panel). Values are means ± S.D. (n = 3) from a representative experiment.



G Protein Requirement for Light-stimulated PLC Activity

Stimulatory or inhibitory effects of guanine nucleotide analogs on enzymatic activities are classic indicators of G protein regulation. Guanine nucleotide analogs affect PLC activity in various vertebrate cells (20) as well as in the photoreceptors of Musca(19) , Limulus(27) , and squid (28) .

To examine the role of G protein in NorpA PLC activation, we measured the effects of GTP, GTPS, and GDPS on PLC activity in Drosophila head membranes using the PLV substrate. Our standard PLC assay included 10 µM GTPS, a nonhydrolyzable GTP analog which irreversibly activates G protein subunits following nucleotide exchange. Varying GTPS concentration from 1 µM to 1 mM did not affect either basal or light-stimulated PLC activity levels (data not shown). When assayed in the absence of added nucleotide, or in the presence of 10 µM to 1 mM GTP, PLC activity was insensitive to light and about 50% lower than basal activity in the presence of GTPS (Fig. 5). The reduced basal activity and loss of light sensitivity in the absence of GTPS is consistent with G protein regulation of norpA PLC. GDPS, an analog of GDP resistant to phosphorylation by nucleoside diphosphate kinase (29) , inhibits G protein-mediated signaling pathways by maintaining G in the inactive, GDP bound state. The presence of 250 µM GDPS in our assays inhibited PLC activity on PLV substrate and eliminated blue light stimulation. This provides further evidence that NorpA PLC is regulated by a G protein (Fig. 5).


Figure 5: Effects of guanine nucleotides on light stimulation of phospholipase C. w head membranes were maintained in the dark or flashed with red (1 min) or blue (10 s) light and assayed for PLC activity on PLV substrate in the presence or absence of different guanine nucleotides. Black filled columns, no guanine nucleotide; black with white diagonal stripe columns, 10 µM GTPS; gray filled columns, 1 mM GTP; white with black diagonal stripe columns, 250 µM GDPS. Values are means ± S.E. (n = 3 to 9) from multiple experiments.



We used another Drosophila mutant to determine whether light activation of NorpA PLC requires Ge, a photoreceptor-specific G protein subunit. Ge flies have less than 1% of wild type levels of immunoreactive Ge and are defective in light-stimulated GTPS binding (15, 16) . The photoreceptors of Ge flies also exhibit abnormal excitation and recovery as measured in whole cell patch clamp recordings (16) . We found that the basal PLC activity in Ge head membranes using the PLV substrate was about 50% of control levels, whereas the activity following blue illumination was 38% of control. No significant (p = 0.19 by analysis of variance) blue light stimulation in the Ge membranes was evident (Fig. 6, left panel). Analysis of a P element-transformed rescue strain Ge;P[w,Ge] (16) confirmed that the Ge mutation was responsible for the elimination of metarhodopsin-dependent PLC activation. These transgenic flies have about 10% of normal levels of Ge, but exhibit 75% of wild type light induced GTPS binding activity (15) and nearly normal activation and deactivation kinetics (16) . The rescue gene restored blue light-stimulated PLC activity on mixed PLV substrate to 73% of control (Fig. 6, left panel). This blue light stimulation was highly significant (p = 0.00025 by analysis of variance). The PLC activities of the Ge and Ge;P[w,Ge] head membranes on the OGM substrate were equivalent to wild type, indicating normal levels of functional NorpA PLC (Fig. 6, right panel).


Figure 6: Phospholipase C activity in ge mutants. Head membranes from w, Ge, and GeP[ge] flies were maintained in the dark or flashed with red (1 min) or blue (10 s) light and assayed for PLC activity. PLV substrate (left panel). Values are means ± S.E. (n = 5 or 7) from multiple experiments. OGM substrate (right panel). Values are means ± S.E. (n = 3) from multiple experiments.




DISCUSSION

G proteins couple photoexcitation of rhodopsin to activation of PLC in invertebrate photoreceptors (1, 30) . Although there have been no previously published measurements of light and G protein-dependent PLC activity in Drosophila heads, Devary et al.(19) have reported evidence that light and a G protein stimulates inositol phospholipid hydrolysis in Musca eye membranes. Our findings with Drosophila are similar to that report, but there are some notable differences. Whereas Devary et al. (19) reported that blue light stimulates inositol phosphate production in the absence of added nucleotide and in the presence of 100 µM GTP, we found no stimulation under these conditions. In their study, GTPS stimulated PLC under both blue and red illumination, but in our study GTPS was only effective under blue illumination. The differences in guanine nucleotide effects probably reflect differences in the substrates and membranes used in the PLC assays. Devary et al. (19) used endogenous H-labeled inositol phospholipid substrate, whereas we used exogenous [H]PIP in either phospholipid vesicles or detergent micelles. We did not assay PLC using the Devary et al.(19) method, because we were unable to label endogenous PIP with [H]myo-inositol using their conditions. It is clear from our results using the two different [H]PIP substrates, PLV and OGM, that PLC regulation by G protein depends on the substrate environment. With the PLV substrate, we observed metarhodopsin-dependent, G protein-mediated PLC stimulation. However, with OGM, we observed high levels of light and G protein-independent PLC activity. Other differences in our preparations may also account for these discrepencies. Devary et al.(19) prepared membranes from Musca eyes, whereas we used whole Drosophila heads, which contain additional G proteins (31, 32) . The eye and brain G proteins in our preparations may hydrolyze the added GTP to GDP, and the resulting high levels of GDP may be inhibitory. The absence of a GTP effect on other G protein-coupled signaling systems has been reported by others (20) .

Although both we and Devary et al.(19) have interpreted the guanyl nucleotide effects to indicate involvement of a G protein, these results do not preclude the possibility that GTPS or GDPS may also have direct effects on NorpA PLC. Potential GTP binding motifs have been identified in the NorpA protein sequence (33) , but the ability of NorpA PLC to bind or hydrolyze GTP has not been demonstrated.

We have demonstrated that a G protein is absolutely required for blue light stimulation by analyzing PLC activation in the G protein-deficient mutant Ge. The loss of light-stimulated PLC activity in the Ge mutant and its restoration in the Ge;P[w,Ge] rescue flies suggests that Ge functions as the subunit of the G protein that couples NorpA to metarhodopsin. Ge is required for light-stimulated G protein function (15, 16) and it is necessary both for photoexcitation and for termination of the phototransduction cascade (16) . Additionally, immunoprecipitation of Ge is enhanced by GTPS, suggesting interaction between Ge and a G subunit (15) .

Recent evidence suggests that the phototransduction G protein subunit is DGq1 (14) . DGq is present in rhabdomeres, and affinity-purified DGq antibody blocks light-stimulated GTP hydrolysis (14). Dominantly active DGq1 mutants exhibit light-independent GTPase activity and abnormal electrophysiological light responses. Furthermore, interaction between dominant DGq1 and light-dependent degeneration mutants indicate that the DGq1 mutations cause constitutive activation of PLC (14) . Interaction of DGq with PLC is consistent with its structure. DGq is most similar in deduced amino acid sequence to the vertebrate Gq class (12, 34) , which activate PLC (35, 36, 37) .

The mechanism by which the photoreceptor G protein activates NorpA PLC is not known. NorpA is most similar in amino acid sequence to vertebrate PLC-4 (33, 38) . Reconstitution studies have shown that different PLC- isoforms can be directly activated by either or both Gq or G subunits (39, 40, 41) . Vertebrate PLC-4 was stimulated by Gq and not by G in transiently transfected cells (42) . However, in common with other PLC isoforms (43) , NorpA PLC has a pleckstrin homology domain in its amino-terminal region. This motif may be a site of G interaction in the -adrenergic receptor kinase (44, 45). A recent study reports binding between G and pleckstrin homology domains from nine different proteins (46) . Our results do not resolve the issue of whether NorpA PLC is directly regulated by DGq or by a complex containing Ge. This will require the identification of a Drosophila phototransduction G subunit and expression and reconstitution studies.

The Ca dependence of PLC activity in Drosophila heads is consistent with a positive feedback model of PLC activation in which light-induced Ca mobilization accelerates the response to light. PLC enzymes require Ca for catalytic activity, and activation of PLC by micromolar Ca has been reported for many tissues and cell types (47, 48) . Payne and Fein (49) reported a nonlinear decrease in time to peak of the light response of Limulus ventral photoreceptors with increasing flash intensity. They suggested that a messenger produced as part of the signaling cascade accelerates the production of new messenger. They proposed that this messenger is Ca, since injection of the Ca buffer EGTA suppressed nonlinearity and slowed the response to bright flashes.

The predominant source of light-mobilized Ca in Drosophila photoreceptors reportedly is from entry of extracellular Ca through light-activated channels (50, 51) . The light-dependent Ca influx generates highly localized transients adjacent to rhabdomeres (51) . Experiments with Ca chelators of various affinities indicate that local Ca concentrations extend into the micromolar Ca range (51) . Positive feedback of NorpA PLC during the Ca transients may contribute to the rapid temporal responses of photoreceptor cells.

The decrease in PLC activity that we observed at 5 µM free Ca indicates that Ca-regulated inactivation mechanisms act at or before NorpA in the phototransduction cascade. This may reflect Ca and diacylglycerol-dependent stimulation of inaC protein kinase C (52, 53) or arrestin phosphorylation and activation by calmodulin kinase (54) .


FOOTNOTES

*
This research was supported by the Howard Hughes Medical Institute and by National Eye Institute Grant EY06641 (to J. B. H.). 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.

§
Current address: ICOS Corp., Bothell, Washington 98021.

To whom correspondence and reprint requests should be addressed: Howard Hughes Medical Institute, University of Washington, Box 357370, Seattle, WA 98195. Tel.: 206-543-2871; Fax: 206-685-2320.

The abbreviations used are: PIP, phosphatidylinositol 4,5-bisphosphate; IP, inositol trisphosphate; PLC, phosphatidylinositol-specific phospholipase C; IP, mixed inositol phosphates; PLV, mixed phospholipid vesicles; PE, phosphatidylethanolamine; PS, phosphatidylserine; OGM, octyl--glycopyranoside micelles; MOPSO, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; GTPS, guanosine 5`-3-O-(thio)triphosphate; GDPS, guanyl-5`-yl thiophosphate.


ACKNOWLEDGEMENTS

We thank Drs. Patrick Dolph and Charles Zuker for Ge and Ge;P[w,Ge] and Dr. William Pak for norpA and w;ninaE flies. We thank Julietta Schoo for maintaining fly stocks, Dr. David Teng for comments on the manuscript, and Dr. David Hyde for communicating results prior to publication.


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