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Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma.

The effect of site-specific mutations in the mouse platelet-derived growth factor (PDGF) beta-receptor on activation of the Na+/H+ exchanger was examined in normal murine mammary gland epithelial (NMuMG) and Chinese hamster ovary (CHO) cells. These cells, which do not normally express PDGF receptors, were stably transfected with PDGF beta-receptor cDNA. Intracellular pH and Ca2+ were monitored using fluorescent probes. In both NMuMG and CHO cells expressing wild-type PDGF beta-receptors, PDGF B/B activated the amiloride-sensitive Na+/H+ exchanger. In both cell types, cell alkalinization was reduced by approximately 50% with a receptor mutant Y708F,Y719F which cannot bind phosphatidylinositol (PI) 3'-kinase. An inhibitor of PI 3'-kinase, LY294002, also inhibited alkalinization by 43% in cells with wild-type, but not Y708F,Y719F receptors. PDGF-induced intracellular Ca2+ release was not affected by this mutation. Both alkalinization and Ca2+ release were reduced by nearly 100% with the mutant Y977F,Y989F, which cannot bind phospholipase C gamma (PLC gamma). Y739F, a mutant that fails to bind the GTPase-activating protein did not affect PDGF-induced alkalinization. In protein kinase C (PKC) down-regulated NMuMG cells (wild-type receptor), PDGF no longer activated the Na+/H+ exchanger. In contrast, in PKC down-regulated CHO cells (wild-type receptor), PDGF-induced alkalinization was attenuated by only 37%. This residual activity was unaffected by the Y708F,Y719F mutation, but was completely eliminated by removal of medium Ca2+. These findings indicate that phospholipase C gamma is essential for activation of Na+/H+ exchange. PI 3'-kinase participates in PKC-dependent activation of Na+/H+ exchange by PDGF. In CHO cells, there is a second, Ca(2+)-dependent mechanism for activation of the exchanger.

pathway is believed to follow the activation of phospholipase C-y (PLC-y) which results in production of diacylglycerol.
The mechanism(s) responsible for activation of Na+/H+ exchange by the PKC-independent pathway(s) remain unclear. In particular, it has not yet been determined whether the PKCindependent pathway requires PLC activity or whether it results from alternative signaling molecules. One candidate for an alternative signaling pathway is the phosphatidylinositol (PI) 3"kinase which is activated by the PDGF receptor (1, 7). By producing novel lipid intermediates, PI 3"kinase could participate in activation of membrane transporters. One approach to distinguishing these possibilities is to analyze PDGF receptor mutations that are deficient in one or more of the signaling pathways of interest.
Binding of PDGF to its receptor initiates a u t o p h o s p h o~l ation on multiple tyrosine residues, including tyrosines in the kinase insert (Ki) region, and the C-terminal domain. After phosphorylation, the receptor physically aossociates with signaling molecules, including PLCy-1 (8)(9)(10)(11)(12)(13), PI 3"kinase (1, 71, and GTPase-activating protein (GAP) (14)(15)(16). Binding sites for PI 3"kinase and GAP are located in the Ki region, while PLC-y binds to the C-terminal domain of the receptor (12). Specific TydPhe mutations prevent binding of these signaling molecules to the PDGF receptor (17).
Bovine serum albumin (fraction V, fatty acid-poor) was obtained from Miles (Napenrille, IL). LY294002 was the kind giR of Chris Vlahos, from Lilly Research Laboratories (Indianapolis, IN).
Cell Culture-NMuMG or CHO cells transfected with PDGF receptors were grown in Dulbecco's modified Eagle's medium or Ham's F-12 medium, respectively, in a humidified atmosphere of 5% CO,, 95% air a t 37 "C. Both media contain 10% (v/v) calf serum, penicillin (50 unitdml), and streptomycin (50 units/ml). Medium for stable transfectants was supplemented with 800 pg/ml G418. Culture medium was changed every 2-3 days until cells were confluent. For measurement of intracellular pH (pH,), cells were transferred and grown to confluence in Leighton tubes (Costar, Cambridge, MA). Cells were made quiescent by replacement of serum with bovine serum ablumin (0.5 mg/ml) and transferrin (5 pg/ml). For measurements of Cap+,, cells were grown on glass coverslips in six-well plates for 2-3 days prior to use.
PDGF Receptor Blots--Anti-PDGF receptor Western blot analysis of PDGF receptors expressed in NMuMG or CHO cells was performed as described previously (17). The expression of the PDGF receptor mutants was quantitated using laser densitometry (Zeineh Soft Laser Scanning Densitometer, model SLR-504-XL, Biomed Instruments, Inc., Fullerton, CA).
Measurements of Intracellular Ca2+-Cells grown on coverslips were incubated with 4.2 p~ fura-2/AM and 0.02% pluronic detergent a t room temperature for 1 h. Fura-2 fluorescence was measured using a Nikon epifluorescence inverted microscope as described previously (3,21). After equilibration, cells on coverslips were washed with solution containing 0 Ca" and EGTA (2 mM) to remove extracellular Ca2+. PDGF (5 ng/ml) was then added immediately in the absence of Ca2+.
Statistics-Values presented are means 2 S.E. or (where indicated) S.D. Differences in mean values between groups were examined using analysis of variance followed by individual contrast (22). Student's t test was used when appropriate. p values < 0.05 were considered statistically significant.

RESULTS
Expression of the PDGF P-receptor mutants in NMuMG cells was analyzed by Western blot (Fig. 1). PDGF receptor protein was undetectable in nontransfected cells (lane 1 ). Cells trans- fected with PDGF receptor mutants Y739F, Y989F, and Y977F,Y989F expressed similar amount of receptor protein as cells transfected with the wild-type receptor. Mutants AKi, Y977F, and Y708F,Y719F expressed 2-3-fold greater PDGF receptor protein than cells transfected with wild-type receptor.
Mutation a t the GAP Binding Site-We next studied the pH, effects of PDGF in cells expressing PDGF receptors with mutations in the kinase insert region. A mutation at the GAPbinding site (Y739F) did not alter the PDGF response. PDGF (20 ng/ml) elicited a rise in pHi of 0.13 -c 0.02 (n = 13, Fig. 3A), not different from that in cells expressing wild-type receptor. In CHO cells expressing theY739F mutation, PDGF caused pH, to rise 0.16 -c 0.02 (n = 9) from a basal of 7.04 2 0.02 (not shown).
The Y708F,Y719F mutation could potentially cause PDGF to elicit additional acid generation, thus giving the appearance of diminished alkalinization. This was not the case, as application of PDGF following amiloride (100 PM) inhibition of Na+/H+ exchange resulted in indistinguishable rates of pH change in wild-type and Y708F,Y719F receptors (wild type, 0.02 2 0.01 pH uniUl5 min, n = 4; Y708F, Y719F, 0.0 f 0.1 pH uniUl5 min, n = 5 ) .
Since even point mutations in the PDGF receptor could theoretically confer multiple defects in signaling, the role of PI 3"kinase was further explored using a recently discovered inhibitor of the enzyme LY294002 (23). This compound, when applied at 20 p~ to NMuMG cells with the wild-type receptor, reduced the PDGF-induced pH, change by 43%, nearly identical to that found in the Y708F, Y719F mutant cells without LY294002 (Fig. 4B). When Y708F,Y719F mutant cells were treated with LY294002 there was no reduction in activation of Na+/H+ exchange, confirming the specificity of LY294002 under these conditions. Thus, the inability to activate PI 3'-kinase, whether by mutation or inhibition, confers a specific deficit in activation of Na+/H+ exchange in two distinct cell types.
PDGF Receptor Mutants and Ca2+,-To show that the Y708F,Y719F receptor mutation could elicit other early responses that were normal, the effect of PDGF on release of intracellular Ca2+ (Ca2+-free medium) was examined (Fig. 5 , Table I). In NMuMG cells expressing wild-type PDGF P-receptor (Fig. 5A), PDGF (5 ng/ml) raised Ca", lasting approximately 3 min. In NMuMG cells expressing the Y708F,Y719F mutation ( Fig. 5B 1, PDGF elicited Ca", responses nearly identical to those with wild-type receptor. In contrast, Y977F,Y989F (a mutation that disrupts binding of PLCy (12)) completely blocked PDGF-induced Ca", release (Fig. 5C). One mutation in the PLCy binding site, Y977F, did not affect PDGF-induced Ca2+* release, while another (Y989F) completely blocked the effects of PDGF on Ca2*, (Table I). Effect of Mutations a t PLCy Binding Site-As for the Ca", response the pHi response to PDGF (20 ng/ml) was unaltered in six of nine NMuMG cell preparations expressing Y977F (Fig.  6A); conversely, in mutant Y989F the PDGF response was absent in 7 out of 10 NMuMG preparations (Fig. 6B). In CHO cells, PDGF failed to raise pH, significantly with either point mutation (not shown). The double mutation, Y977F,Y989F, reduced activation of Na+/H+ exchange by PDGF from 0.15 pH unit in wild-type to nearly zero in both NMuMG cells (Fig. 6C) and CHO cells (not shown). As for the Y708F,Y719F mutation, the Y977F,Y989F mutation did not alter the Na+/H+ exchanger per se, as osmotic agents activated the exchanger in cells expressing this mutation (not shown). These results indicate that PDGF activation of Na+/H+ exchange is entirely dependent upon activation of PLCy in both cell types.
Involvement of PKC in PDGF-induced Alkalinization-The results of Fig. 6 clearly implicate PLCy as playing a critical role in activation of the Na+/H+ exchanger. To assess the role of PKC, pH, was measured after acute administration of PMA or after PKC was down-regulated by pre-treatment of the cells with 20 ng/ml PMA for 24 h (6,24). In control preparations, PMA (250 ng/ml) increased pH, by 0.13 f 0.03 pH units ( n = 9) from 7.14 2 0.04 in NMuMG cells expressing the wild-type receptor (Fig.  7A). A similar response was observed in CHO cells expressing wild-type receptors and Y708F,Y719F receptors. After PKC down-regulation (which does not alter basal pH,), PMAfailed t o elicit alkalinization in all three cell types (Fig. 7A).
In contrast, the effect of PDGF in PKC down-regulated cells varied with cell type (Fig. 7B). PMApre-treatment almost completely blocked PDGF-induced alkalinization of NMuMG cells   , center bars, Fig. 7B), suggesting a second mechanism other than PKC in mediating activation of Na+/H+ exchange. In CHO cells expressing the Y708F,Y719F mutant, PKC downregulation did not affect the PDGF-induced alkalinization response at all (right-hand bars, Fig. 7 B ) . Finally, in cells lacking both PI 3"kinase and PKC activity, the residual activation of Na+/H+ exchange was completely eliminated when cells were depleted of Ca2+ by removal of Ca2+ from the extracellular medium for 15 min. Removal of Ca2+, does not affect Na+/H+ exchange activity per se, since osmotic agents can activate the exchanger normally following Ca2+ removal both in wild type cells (20, 25) and in Y708FE719F cells lacking PKC (not shown). These results suggest the existence of two pathways for activation of Na+/H+ exchange by PDGF in CHO cells. The first requires both PI 3'-kinase and PKC for maximal activity; the second is dependent upon changes in Ca2+.  04 ( n = 9). B, 7 in 10 of Y989F did not respond to PDGF. C , 10 of 11 preparations of Y977F,Y989F did not respond to PDGF, increasing pH, only 0.04 0.02 pH unit ( n = 11, p < 0.05 compared with wild type).

DISCUSSION
When CHO or NMuMG cells, which both lack native PDGF receptors, were stably transfected with a cDNA for the wildtype PDGF P-receptor, PDGF was able to activate the Na+/H+ exchanger in these cells. This observation was exploited to study the effect of signaling mutations on the coupling of the PDGF receptor to Na+lH+ exchange.In both cell lines, PDGF BE3 consistently failed to activate Na+lH+ exchange with mutants that cannot bind PLCy (Fig. 6). Tyr 989 appears to be more important in the interaction with PLCy than Tyr-977, since residual Ca2' (Table I) and pH (Fig. 6) signals were present with Y977F, but not with Y989F. This finding is consistent with previous work with the human receptor, in which Tyr-1021, the homolog of mouse Tyr-989, is more important for binding of PLCy than is Tyr-1009 (12).
Activation of PLCy results in production of diacylglycerol which stimulates PKC, and production of inositol trisphosphate which induces Ca2+, release. Direct activation of PKC by PMA also induces Na+/H+ exchange in both cell lines (Fig. 7A), consistent with a vital role for this enzyme in activation of the exchanger. Moreover, in PKC down-regulated NMuMG cells expressing wild-type receptors, activation of Na+/H+ exchange by PDGF was absent (Fig. 7B), suggesting that the PKC pathway is both sufficient and necessary for activation of the exchanger by PDGF in NMuMG epithelial cells. However, in CHO cells this pathway is not the sole mechanism for PDGF induced Na+/H+ exchange, because PKC-down-regulation attenuated the response t o PDGF by only 37% in this cell line (Fig. 7). This result is consistent with findings made with other ligands and cell types (5, 6, 26). In these cell types, there is a pathway for activation of Na+/H' exchange that is not mediated by any isoform of PKC that can be down-regulated by PMA.
Since activation of Na+/H+ exchange appears to require PLCy, the "PKC-independent" pathway for activation of Na+/H+ exchange most likely involves an alternative action of diacylglycerol or an inositol phosphate. Synthetic diacylglycerols do not activate the exchanger in PKC down-regulated cells (261, but native diacylglycerols could be a source for arachidonic acid products, which could theoretically activate the exchanger (27). Alternatively, the exchanger could be activated by an inositol phosphate or the Ca2+ spike induced by inositol trisphosphate. Depletion of cell Ca2+ by removal of Ca2+ from the medium for 15 min was sufficient to completely block PDGF activation of Na+/H+ exchange in cells lacking PI 3'-kinase and PKC activity (Fig. 7B). This critical role for Ca2+ is consistent with earlier work from this lab with vascular smooth muscle cells (20) and the recent finding that NHE-1 is a Caz+/calmodulin-binding protein which requires Ca2+ for normal activation (28) (Fig. 8).
Although PKC and Ca2+ are clearly important in the regulation of Na+/H+ exchange, the data of this paper suggests the additional participation of PI 3'-kinase. The binding site for PI 3"kinase is in the kinase insert (Ki) region of the receptor. Previous studies showed that a large deletion of the Ki region (AKi) did not interfere with activation of Na+/H+ exchange when the mutant receptor was expressed at levels 5-fold higher than wild type (2). In subsequent work with the AKi mutant expressed at levels closer to wild type, activation of Na+/H+ exchange was reduced (data not shown). Examination of point mutations in the Ki region was used to determine whether Na+/H+ exchange is actually signaled by a molecule that binds in that region.
Tyr Phe mutations at residues 708 and 719 prevent binding of PI 3'-kinase to the receptor (17). Receptors carrying this double mutation were clearly defective in activation of Na+/H+ exchange (Figs. 3 and 4). This was not due to abnormally low expression of these receptors (Fig. 1) and was observed in two different cell lines. Moreover, the PI 3'-kinase mutants exhibited a normal response to phorbol esters (Fig. 7) and a normal Ca", response to PDGF (Fig. 5). Thus, the partial blockade in activation of Na+/H+ exchange in the Y708F,Y719F mutant was not part of a global defect in early signaling events.
It has recently been found that certain PDGF receptor residues, including 708 and 719, may bind multiple signaling molecules. For example, Nck (29) and Shc (30) share binding sites with the PI 3'-kinase on the human receptor. Therefore, the unambiguous assignment of functions to specific sites on the PDGF receptor is not always possible. It seems unlikely that our results with Na+/H+ exchange are due to one of these alternative interactions since a new inhibitor of PI 3'-kinase (LY294002) closely mimicked the effect of the Y708F,Y719F mutation. This similarity in the effect of LY294002 and the Y708F,Y719F mutation leads us to conclude that PI 3"kinase (in addition t o PLCy) plays an important role in activation of Na+/H+ exchange. PI 3'-kinase specifically phosphorylates the 3' position of the inositol ring of various phosphatidylinositols to form 3-phosphorylated compounds, including PI 3,4,5-triphosphate (31, 32). PI 3,4,5-triphosphate is a poor substrate for PLCy (33-351, but has recently been found to activate PKCC (361, a ubiquitous isoform of PKC that does not respond to DAG or phorbol esters (37,38). It is conceivable that the "PKC-independent" activation of Na+/H+ exchange is through activation of PKCC by PI 3,4,5-triphosphate. However, activation of the Na+/H+ ex-