0163-769X/92/1304-0707$03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society

Vol. 13, No. 4 Printed in U.S.A.

Subcellular Organization of Receptor-Mediated Phosphoinositide Turnover* MARIE E. MONACO AND MARVIN C. GERSHENGORN Department of Physiology and Biophysics, New York University Medical Center and the Research Program, Manhattan Veterans' Administration Hospital, New York, New York 10010 (M.E.M.); and the Division of Molecular Medicine, Department of Medicine, Cornell University Medical College and The New York Hospital, New York, New York 10021 (M.C.G.)

I. Introduction II. Evidence for Coupling of Resynthesis of Ptdlns to Hydrolysis of PtdIns-4,5-P2 A. General B. The phosphoinositide cycle III. Agonist-Responsive and -Unresponsive Pools of Phosphoinositides IV. Relationship of Hormone-Responsive Pools to Receptor Activation V. Model of Genesis of an Agonist-Responsive Phosphoinositide Pool VI. Conclusion

I. Introduction

M

UCH of the biochemistry of agonist-induced phosphoinositide metabolism has been elucidated during the past decade; however, less is known concerning the subcellular organization of these biochemical events. Questions remain regarding: 1) the anatomical location of the biochemical reactions involved in the signal transduction events, 2) the relationship between PtdIns-4,5-P2 hydrolysis and resynthesis of phosphatidylinositol (Ptdlns) and 3) the nature of the agonistresponsive phosphoinositide pool and its relationship to the activated receptor complex. In this review, we will outline the information presently available that is relevant to these questions and propose a model that incorporates these data. Studies of a number of biochemical pathways, including those for steroidogenesis, glycolysis, nucleotide biosynthesis, and the urea cycle, suggest a functional association or compartmentation within cells of the enzymes

involved (for review, see Ref. 1). Existing data support the thesis that such organization may extend to signal transduction pathways as well. Several lines of evidence suggest that stimulation of the adenylyl cyclase-cAMP signaling pathway leads to cAMP accumulation that occurs in discrete areas of the cell. These include findings that: 1) equivalent increases in the levels of cAMP stimulated by two different agonists do not induce similar effects in either Leydig cells (2) or rat heart (3); 2) there is differential activation of particulate and soluble protein kinases by different agonists (4); and 3) there is differential subcellular localization of cAMP that is dependent on the agonist employed (5). Phosphoinositide cycling (Fig. 1), which is stimulated during signal transduction by agonists that use inositol-l,4,5-trisphosphate and 1,2-diacylglycerol (DAG) as second messenger molecules (for review, see Ref. 6), also displays some properties usually associated with compartmentation. In this manuscript, we will review the evidence that supports functional organization of the agonist-stimulated phosphoinositide cycle, including coupling of resynthesis of phosphatidylinositol (Ptdlns) to hydrolysis of Ptdlns4,5-bisphosphate (PtdIns-4,5-P2) and the compartmentation of the phosphoinositides into agonist-responsive and -unresponsive pools. A third piece of compelling evidence supporting the compartmentation hypothesis is the spatial aspect of the calcium mobilization response to different agonists. These data have been reviewed elsewhere (7) and will not be discussed here. Ptdlns

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Address requests for reprints to: Marie E. Monaco, Ph.D., Research Program (151A), Veterans Administration Hospital, 423 East 23rd Street, New York, New York 10010. "This work was supported in part by a grant from the National Science Foundation, DCB-8901476, and a Merit Review Award from the Veterans' Administration (to M.E.M.) and USPHS Grant DK43036 (to M.C.G.).

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FIG. 5. Effect of preincubation with vasopressin and lithium chloride on subsequent incorporation of 32Pi into Ptdlns in WRK-1 cells. Cells were preincubated for 90 min as shown above. After incubation, the cells were washed and further incubated for an additional 60 min with (shaded) or without (hatched) 0.2 /xM vasopressin. Values shown represent means ± 1 SD. [Reproduced with permission from M. E. Monaco and J. R. Adelson: Biochem J 279:337-341,1991 (45).]

synthesis (Fig. 5). In addition, when excess exogenous Ins was added during stimulation by vasopressin in the presence of lithium, there was no increase in Ptdlns synthesis during the period after factors were removed (Table 2). Therefore, accumulation of Ins phosphates under conditions that prevented a decrease in Ptdlns levels was not sufficient to stimulate resynthesis upon removal of vasopressin and lithium. Since WRK-1 cells do not accumulate CDP-DAG when stimulated by vaso-

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TABLE 2. Effect of exogenous inositol on the ability of pretreatment with vasopressin and lithium chloride to stimulate subsequent 32Pj incorporation into Ptdlns

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FIG. 4. Effects of lithium chloride and unlabeled Ins on 32P labeling during prolonged stimulation of GH3 cells by TRH. Cells prelabeled with [3H]Ins were incubated in buffer without (O, A) or with (•, A) 1 MM TRH and with 32Pi (time zero) for an additional 3 h. (a) No further additions; (b) plus 10 mM lithium chloride; (c) plus 10 mM lithium chloride and 100 mM unlabeled Ins. At the times indicated, the incubations were terminated and 32P and 3H radioactivities in Ptdlns (O, • ) and 32P radioactivity in PtdA (A, • ) were measured (3H radioactivity shown in insets). [Reproduced with permission from A. B. Cubitt et al.: Biochem J 271:331-336,1990 (77).]

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WRK-1 cells were preincubated with the above compounds for 90 min. The cells were then washed and incubated for an additional 30 min with radioactive phosphate. Values shown are the means of triplicate determinations ± 1 SD. [Reproduced with permission from M. E. Monaco and J. R. Adelson: Biochem J 279:337-341, 1991 (45).]

pressin in the presence of lithium, we concluded that it was the decrease in the level of Ptdlns that was responsible for the increased rate of Ptdlns synthesis and not an effect stimulated by the second messenger molecules or by a direct receptor-mediated process. Previous studies in WRK-1 cells with the calcium ionophore, A23187, support this conclusion (46). Treatment of cells with A23187 caused a decrease in Ptdlns and an increase in Ins phosphates. The Ins phosphates generated under these conditions were found extracellularly (due to permeabilization of the cells, as demonstrated by trypin blue uptake) so there could be no increase in cellular Ins, and yet the overall rate of Ptdlns synthesis increased.

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712

MONACO AND GERSHENGORN

In conclusion, during signaling by agonists, stimulation of resynthesis of Ptdlns is coupled to hydrolysis of PtdInsP 2 . It appears that stimulation of Ptdlns synthesis is independent of direct agonist-receptor action and is not mediated by second messenger molecules or a rise in precursor concentrations. It appears, therefore, that stimulation of Ptdlns resynthesis occurs via activation of PtdlnsS, which is released from inhibition by its product as the level of Ptdlns is transiently lowered.

III. Agonist-Responsive and -Unresponsive Pools of Phosphoinositides Another indicator of cellular compartmentation is the existence of distinct metabolic or functional pools of a substrate within the cell. A number of studies indicate that not all of the Ptdlns in a cell serves as precursor for PtdIns-4,5-P2 during cell signaling in response to a specific agonist. Hokin and Hokin (47) were the first to postulate two separate metabolic pools of Ptdlns in avian salt glands, one involved in phosphoinositide cycling and the other not, based on kinetics of incorporation of radioactive isotopes in response to agonist. In 1979, Fain and Berridge (48) observed that short-term labeling of blow-fly salivary glands with radioactive phosphoinositide precursors resulted in the preferential labeling of an agonist-responsive pool of Ptdlns. They showed also that signaling ceased when only 5% of Ptdlns was depleted under conditions in which resynthesis was inhibited and that signaling resumed when resynthesis of that 5% of the Ptdlns was restarted. Data from experiments designed to assess the existence of agonist-responsive and -unresponsive pools of phosphoinositides in mammalian tissues have also been reported. A number of studies have produced compelling data that are consistent with the presence of distinct agonist-responsive and -unresponsive pools in several mammalian tissues. These include studies using pancreas (49, 50), brain (51-53), adrenal medulla (54), neutrophils (55), erythrocytes (56, 57), platelets (58-60), canine trachealis muscle (61), C3H fibroblasts (62), WRK-1 (6368) cells, and GH3 cells (69). Interpretation of pool or compartmentation data can be complicated by heterogeneity of the cell population being studied. Observations of what appear to be distinct metabolic or functional pools could result from multiple cell types or differences in the stage of the cell cycle in a population of cells. For this reason, the most convincing data are derived from cloned cell lines that consist of a single cell type and that can be synchronized with respect to cell cycle. The most extensive characterization of agonist-responsive and -unresponsive lipid pools has been carried out in clonal WRK-1 rat mammary tumor cells. It was demonstrated in these vasopressin-respon-

Vol. 13, No. 4

sive cells that a lag of approximately 2 h occurred in labeling of agonist-responsive Ptdlns with radioactive phosphate under nonstimulatory conditions. In contrast, in the presence of agonist, the responsive pool was labeled rapidly (Fig. 6). In addition to demonstrating the existence of agonist-responsive and -unresponsive pools of phosphoinositides, the data also suggested that hydrolysis and resynthesis of phosphoinositides occurred in a "closed cycle" in that the lipid synthesized in response to agonist was subsequently preferentially sensitive to agonist-induced hydrolysis. For example, although only 15% of Ptdlns in equilibrium-labeled cells was responsive to agonist as measured by disappearance of label in the presence of unlabeled phosphate, all of the lipid synthesized as a result of cycling during a 2 h labeling period was responsive to agonist (Fig. 6). These studies in WRK-1 cells were confirmed (67), and the same results were obtained using recloned and synchronized cells (64). The results from recloned cells showed also that the relative size of the responsive pool could vary, most probably as a function of receptor number (see below). As predicted, the polyphosphoinositides were subsequently shown to exist in agonist-responsive and -unresponsive pools in WRK-1 cells as well (65). Two other clonal cell lines have also been shown to possess distinct metabolic phosphoinositide pools. In the pituitary GH3 cell line, we demonstrated that incubating cells with radioactive Ins under conditions that promote the exchange reaction rather than de nouo synthesis of Ptdlns results in labeling of a pool of lipid that is not sensitive to TRH-induced hydrolysis (Table 3) (69). These results are consistent with those previously reported for manganese stimulation of Ins incorporation into an agonist-unresponsive phosphoinositide in brain (51-53), since manganese is known to stimulate base exchange of Ins rather than de nouo synthesis. In C3H/

1

2 3 Time (Hours)

4

FIG. 6. Turnover of Ptdlns in WRK-1 cells. Cells were preincubated with 32Pi for 1 h in the presence (A) or absence (O) of vasopressin. After the preincubation period, cells were washed free of radioactivity and hormone, and the incubation continued in the presence ( ) or absence ( ) of vasopressin. [Reproduced with permission from M. E. Monaco and D. Woods: J Biol Chem 258:15125-15129,1983 (64).]

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ORGANIZATION OF PI CYCLE

November, 1992

TABLE 3. Comparison of the effects of TRH to stimulate formation of [aH]IPs in intact GH cells, in permeabilized cells that were labeled as intact cells in situ ("prelabeled"), and in permeabilized cells that were labeled by base exchange in vitro ("postlabeled") 3

H Radioactivity (per 105 dpm in lipids)

Intact cells Prelabeled cells" Postlabeled cells*

PIP2 (dpm)

IPs (dpm/20 min)

3,100 ± 320 2,480 ± 330 1,500 ± 234

29,895 ± 2,451 10,095 ± 1,806 106 ± 39

[Reproduced with permission from A. B. Cubitt et al: J Biol Chem 265:9707-9714,1990 (69).] PIP2, PtdIns-4,5-P2. " Prelabeled cells were stimulated immediately after permeabilization. b Postlabeled cells were stimulated 20 min after permeabilization and incubation with 0.1 mM [3H]Ins (48 MCi

10T1/2 fibroblasts stimulated by platelet-derived-growth factor, the evidence suggests compartmentation of the polyphosphoinositides involved in cycling (62). In other studies, there was a failure to observe distinct metabolic pools of phosphoinositides (70-75). Interestingly, conflicting results were sometimes observed in the same tissue, and this seems to have been a result of the experimental design employed. In studies with WRK-1 cells, in which phosphoinositide compartmentation was clearly shown using radioactive phosphate as the phosphoinositide precursor (64, 68), it was not possible to show separate metabolic pools when radioactive Ins was the precursor (66, 74, 75). Neither variations in time nor hormonal treatment altered the pattern of incorporation of labeled Ins into phospholipids. Even more sensitive double label studies failed to differentiate between pools of phosphoinositides (76). These data in WRK-1 cells make it clear that failure to observe evidence for multiple metabolic pools does not exclude their existence. As discussed above, alternate pathways of incorporation for inositol, such as exchange, might explain this observation. Furthermore, a distinction must be made between metabolic and functional pools. For example, although labeling with radioactive Ins provided no evidence for distinct metabolic lipid pools, the inability of hormone to induce breakdown of all the labeled phosphoinositide in the presence of excess unlabeled inositol in both WRK-1 and GH3 cells is consistent with the existence of a hormone-unresponsive lipid pool (45, 77).

IV. Relationship of Hormone-Responsive Pools to Receptor Activation More recent data have expanded the idea of agonistresponsive and -unresponsive pools of phosphoinositides and provided additional support for the hypothesis of functional compartmentation of this cycle. For example, while it is clear that there is considerable overlap in the

713

phosphoinositides responsive to different agonists in the same cell (78, 79), there is also evidence in WRK-1 cells that bradykinin can access Ptdlns that is unresponsive to vasopressin (67). Thus "responsivity" is defined only with regard to a specific agonist, and it is possible that all of the cellular Ptdlns has the potential for cycling when stimulation is provoked by more than one agonist. Taking this idea one step further, we have demonstrated that a submaximal dose of TRH cannot stimulate turnover of the same total amount of prelabeled Ptdlns in GH3 cells as a maximally effective dose of TRH, even when the incubation is carried out for long periods of time (77) (Fig. 7). That is, in the presence of lower concentrations of agonist, disappearance of Ptdlns, which had been labeled with radioactive Ins to steady state, reached a new steady state level that was higher (less radioactive Ptdlns metabolized) than that seen with maximally stimulatory doses of TRH. These experiments were performed in the presence of 100 mM unlabeled Ins, which prevented reincorporation of released, radiolabeled Ins. Similar results have been obtained in WRK-1 cells stimulated by vasopressin (M. E. Monaco, unpublished data). Thus, it appears that the concentration of agonist not only determines the rate at which Ptdlns is metabolized, but also delimits which lipid molecules are recruited into the cycle. Most of the PtdInsP 2 in the cell appears to be localized to the plasma membrane (80, 81), although it exists in other subcellular organelles also (28, 82, 83). Ptdlns, on the other hand, appears generally distributed among subcellular membranes (8), and agonist stimulation appears to result in metabolism of Ptdlns in several intracellular membrane compartments (84-87). In some cell systems, 90% of Ptdlns can be metabolized as a result of receptor activation (67), and a value of 50% is quite common (69), so Ptdlns in membranes other than the plasma membrane must be involved in the response. It

1

2

3

4

5

Time (h)

FIG. 7. Time course of the effect of two doses of TRH on 3H-labeled Ins lipids in GH3 cells prelabeled with [3H]Ins and incubated in buffer with 10 mM lithium chloride and 100 mM unlabeled Ins. Closed circles, control; closed triangles, 1 juM TRH; closed squares, 0.3 nM TRH; open squares, 1 MM TRH added to cells previously incubated with 0.3 nM TRH for 3 h. [Reproduced with permission from A. B. Cubitt et al.: Biochem J 271:331-336,1990 (77).]

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714

MONACO AND GERSHENGORN

has been suggested that turnover of the bulk of the Ptdlns constitutes a second phase of cellular response and is occurring directly, i.e. without prior conversion to PtdInsP 2 (88, 89); however, in WRK-1 cells, hormonestimulated increases in radiolabeled phosphate incorporation into Ptdlns are accompanied by similar increases in incorporation of radioactivity into the 1-phosphate of PtdInsP 2 for at least 6 h (65); and the kinetics of inositol phosphate generation and disappearance are consistent with a model in which Ptdlns is metabolized via PtdInsP 2 (90, 91). Is Ptdlns from other membranes transported to the plasma membrane via a transfer protein as plasma membrane Ptdlns is utilized for resynthesis of PtdInsP2? Or is there cycling PtdInsP 2 that is not located at the plasma membrane? Does resynthesis take place at the plasma membrane or in the endoplasmic reticulum, or both? The observation that a submaximal concentration of agonist cannot lead to turnover of all agonist-responsive Ptdlns suggests a close relationship between the specific receptor occupied and the specific Ptdlns metabolized (77). However, it is also clear that different agonists that bind different receptors can cause turnover of the same Ptdlns (78, 79). In any case, it appears that Ptdlns not present within the plasma membrane before stimulation is recruited into the cycle, either by being metabolized within the membranes in which it was synthesized or after transfer to the plasma membrane. The specific Ptdlns that is recruited, moreover, appears to depend, to some degree, on which agonist is acting and on the concentration of that agonist. Attempts to understand the organization of the cycle by determining its anatomical location within the cell have been unsuccessful. As mentioned above, Ptdlns metabolized in response to agonists is found throughout subcellular membranes, as are the various enzyme activities involved in cycling (92-95). It is possible, in fact likely, that the cycle spans more than one subcellular compartment, as has been shown to be the case for the urea cycle that contains both mitochondrial and soluble components that are tightly linked (96, 97). A model for compartmentalized phosphoinositide cycling is described below that attempts to explain involvement of multiple subcellular sites.

V. Model of Genesis of an Agonist-Responsive Phosphoinositide Pool In this section, we will describe a model that attempts to explain the functional compartmentation of phosphoinositides into agonist-responsive and -unresponsive pools. The central hypothesis is that the agonist-responive pool is delimited by a subcellular domain in which agonist-bound, activated receptors signal. In this model, phosphoinositides, primarily but not exclusively Ptdlns-

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4,5-P2, may serve as substrates for the generation of second messenger molecules only if they are in functional proximity to receptor-activated phospholipase C. Agonist-unresponsive phosphoinositides, in contrast, are those phosphoinositides that are located in the cell within domains that are not functionally accessible to receptor-activated phospholipase C. It is important to note that these functionally distinct domains may not be anatomically separate. In fact, in our model the sites of second messenger generation are in domains within the cell surface membrane, and phosphoinositides that were present in intracellular membranes before agonist stimulation are recruited to the plasma membrane where they can be hydrolyzed. In our model, the specific anatomical site of resynthesis of Ptdlns is not important, but the mechanism of activating PtdlnsS is a central component of delimiting the agonist-responsive pool. Ptdlns resynthesis may occur in membranes that are at the cell surface or within vesicles, the Golgi apparatus or the endoplasmic reticulum. The anatomical site is not important because these membranes are in constant flux cycling among these subcellular compartments, and the rate of cycling is increased during agonist action. By contrast, the mechanism of activation of PtdlnsS is important because the synthesis of only phosphoinositides involved in cycling should be increased during agonist stimulation. We propose that activation of PtdlnsS is mediated by a decrease in the level of Ptdlns, that is, release of PtdlnsS from product inhibition by Ptdlns. This mechanism causes Ptdlns resynthesis only in those membranes in which activated phospholipases C have lowered the phosphoinositides as they are used to generate second messenger molecules. Hence, only phosphoinositides that are to be used for signaling are synthesized at an increased rate. Our model is based on information obtained in several cell sytems, in particular, from studies of TRH action in GH3 cells and vasopressin action in WRK-1 cells and in liver. Therefore, not all data on which this model is based have been obtained in a single system. We suggest that the mechanism may be common to all agonists that use the phosphoinositide signaling pathway. There are two central tenets in our hypothesis regarding the receptors that activate phosphoinositide signaling. One of these ideas is different from principles that have been developed from studies of stimulatory receptors that interact with Gstimuiatory protein and couple to adenylyl cyclase (98, 99); namely, that upon binding agonist, receptors change from a form that is of lower affinity to one of higher affinity and, therefore, not all receptors for a given agonist are equivalent once cells have been exposed to an agonist. Thus, previously occupied receptors, which have a higher affinity, may bind agonist preferentially over receptors that were not previously bound. Changes

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November, 1992

ORGANIZATION OF PI CYCLE

in receptor affinity have been well documented for TRH receptors and Via vasopressin receptors (Refs. 100 and 101 and Nussenzveig, D. R. and M. C. Gershengorn, unpublished data). And second, receptors that activate phospholipase C, like those that couple to adenylate cyclase, cycle from the cell-surface membrane through intracellular vesicular compartments and then back to the plasma membrane (102, 103). Our hypothesis is as follows (Fig. 8). Agonist binds to receptor and rapidly activates, via a G protein (104), phospholipase C-mediated hydrolysis of phosphoinositides, primarily PtdIns-4,5-P2, at the cell surface. Upon G protein activation, agonist-receptor binary complex may dissociate from the quaternary complex. The agonist-receptor complex is then internalized ("sequestered") within an endocytic vesicle. G protein and phospholipase C remain in the same region of the plasma membrane, perhaps limited in their mobility by interactions with the cytoskeleton (105). An alternate possibility is that the agonist-receptor-G protein-phospholipase C complex may be relatively stable (106) and the quaternary complex may be internalized. The endocytic vesicle then fuses with an endosome that contains an acidic intravesicular core in which agonist dissociates from receptor. (The endocytic vesicle may have an acidic intravesicular space also.) The endosome contains Ptdlns, which was originally within intracellular mem-

Secretory vesicle

FIG. 8. Model of the genesis of an agonist-responsive phosphoinositide pool. In this hypothesis, the receptor transforms to a higher affinity state upon binding agonists and remains in this conformation even after agonist dissociates in the acidic environment of the endosome. The receptor-containing endocytic vesicle fuses with an endosome and then recycles to the plasma membrane as shown. PI is replenished in the plasma membrane, which is the primary site of phospholipase Cmediated hydrolysis of PtdInsP2, by recruitment of Ptdlns from intracellular membranes—fusion of receptor-containing endosomes and, for example, of secretory vesicles—and by local synthesis (not shown). PI in this figure represents PtdIns-4,5-P2 and PtdIns-4-P as well as Ptdlns.

715

branes, that diffuses throughout the receptor-containing endosomal membrane and thereby replenishes the metabolized Ptdlns in the membrane domain in proximity to the activated receptor. The endosomal vesicle then recycles to the cell-surface membrane. Plasma membrane Ptdlns may also be replenished by the fusion of other intracellular vesicles, such as secretory vesicles, and by local synthesis (see above). At the cell surface, agonist within the extracellular space preferentially binds to these recycled receptors because they are in a higher affinity conformation, and the cycle starts over again. In this way, the same subset of receptors is used repeatedly and Ptdlns is recruited to the cell surface from other intracellular membranes for conversion to PtdInsP 2 . Only those Ptdlns molecules that can be incorporated into the plasma membrane in the domain delimited by activated receptors are part of the agonist-responsive pool. The unresponsive pool is comprised of phosphoinositides that are not present within cell surface domains in proximity to activated receptors. VI. Conclusion The evidence presented here is consistent with the existence of a functional organization of phosphoinositides within cells. We suggest that the key element to the separation into agonist-responsive and -unresponsive pools is the receptor that initiates the signal transduction cascade. The receptor, like other membrane proteins and lipids, is sorted into certain plasma membrane domains and therein can become associated with G proteins and phospholipases C. It is only that portion of cellular phosphoinositides that are cycled into these domains that can be hydrolyzed by the activated phospholipase C ("agonist-responsive pool"). A corollary to our hypothesis is that signal transduction by receptors that couple via a G protein to phospholipase C use a cellular process that is different from that used by receptors that couple via Ggtimuiatory to adenylyl cyclase. This, perhaps, should not be surprising since the chemical nature and intracellular locations of the two substrates, PtdIns-4,5-P2 and ATP, are so different. We present our working model with the full knowledge that it is unproven and that its proof or refutation awaits further experimentation. We would like its presentation to provoke additional studies of the phosphoinositide signal transduction pathway. References 1. Srere PA 1987 Complexes of sequential metabolic enzymes. Annu Rev Biochem 56:89-124 2. Dufau ML, Homer KA, Hayashi K, Tsuruhara T, Conn PM, Katt KJ 1978 Actions of choleragen and gonadotropin in isolated Leydig cells. Functional compartmentalization of the hormoneactivated cyclic AMP response. J Biol Chem 253:3721-3729 3. Hayes JS, Brunton LL, Brown JH, Reese JB, Mayer SE 1979

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Hormonally specific expression of cardiac protein kinase activity. Proc Natl Acad Sci USA 76:1570-1574 4. Buxton ILO, Brunton LL 1983 Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. J Biol Chem 258:10233-10239 5. Barsony J, Marx SJ 1990 Immunocytology on microwave-fixed cells reveal rapid and agonist-specific changes in subcellular accumulation patterns for cAMP and cGMP. Proc Natl Acad Sci USA 87:1188-1192 6. Berridge MJ 1987 Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem 56:159-193 7. Berridge MJ, Cobbold PH, Kuthbertson KSR 1988 Spatial and temporal aspects of cell signalling. Phil Trans R Soc Lond B 320:325-343 8. Hawthorne JN 1982 Inositol phospholipids. In: Hawthorne JN, Ansell GB (eds) Phospholipids. Elsevier Biomedical Press, Amsterdam, New York, Oxford, pp 263-278 9. Agranoff BW, Bradley RM, Brady RO 1958 The enzymatic synthesis of inositol phosphatide. J Biol Chem 233:1077-1083 10. Paulus H, Kennedy EP 1960 The enzymatic synthesis of inositol monophosphatide. J Biol Chem 235:1303-1311 11. Fischl AS, Carman GM 1983 Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase. J Bacteriol 154:304-311 12. Rao RH, Strickland KP 1974 On the solubility, stability and partial purification of CDPdiacyl-sn-glycerol: inositol transferase from rat brain. Biochim Biophys Acta 348:306-314 13. Ghalayini A, Eichberg J 1985 Purification of phosphatidylinositol synthase from rat brain by CDP-diacylglycerol affinity chromatography and properties of the purified enzyme. J Neurochem 44:175-182 14. Parries GS, Hokin-Neaverson M 1984 Phosphatidylinsotol synthase from canine pancreas: solubilization by n-octyl glucopyranoside and stabilization by manganese. Biochemistry 23:47854791 15. Takenawa T, Egawa K 1977 CDP-diglyceride: inositol transferase from rat liver. Purification and properties. J Biol Chem 252:54195423 16. Takenawa T, Saito M, Nagai Y, Egawa K 1977 Solubilization of the enzyme catalyzing CDP-diglyceride-independent incorporation of myo-inositol into phosphatidyl inositol and its comparison to CDP-diglyceride: inositol transferase. Arch Biochem Biophys 182:244-250 17. Berry G, Yandrasitz JR, Segal S 1983 CMP-dependent phosphatidylinositokmyo-inositol exchange activity in isolated nerve-endings. Biochem Biophys Res Commun 112:817-821 18. Fischl AS, Homann MJ, Poole MA, Carman GM 1986 Phosphatidylinositol synthase from Saccharomyces cerevisiae. J Biol Chem 261:3178-3183 19. Cubitt A, Gershengorn M 1990 CMP activates reversal of phosphatidylinositol synthase and base exchange by distinct mechanisms in rat pituitary GH3 cells. Biochem J 272:813-816 20. Nikawa J, Kodaki T, Yamashita S 1987 Primary structure and disruption of the phosphatidylinositol synthase gene of Saccharomyces cerevisiae. J Biol Chem 262:4876-4881 21. Nikawa J, Kodaki T, Yamashita S 1988 Expression of the Saccharomyces cerevisiae PIS gene and synthesis of phosphatidylinositol in Escherichia coll J Bacteriol 170:4727-4731 22. Kelly MJ, Bailis AM, Henry SA, Carman GM 1988 Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. J Biol Chem 263:18078-18085 23. Thompson W, MacDonald G 1975 Isolation and characterization of cytidine diphosphate diglyceride from beef liver. J Biol Chem 250:6779-6785 24. Van Golde LMG, Raben J, Batenburg JJ, Fleischer B, Zambrano F, Fleischer S 1974 Biosynthesis of lipids in Golgi complex and other subcellular fractions from rat liver. Biochim Biophys Acta 360:179-192 25. Williamson FA, Morre DJ 1976 Distribution of phosphatidylinositol biosynthetic activities among cell fractions from rat liver. Biochem Biophys Res Commun 68:1201-1205

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Subcellular organization of receptor-mediated phosphoinositide turnover.

0163-769X/92/1304-0707$03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society Vol. 13, No. 4 Printed in U.S.A. Subcellular Organization...
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