Journal of NeurocheinistrpLippincott-Raven Publishers, Philadelphia

1995 International Society for Neurochemistry

Multiple Levels for Regulation of TrkA in PC 12 Cellsby Nerve Growth Factor

*Jie Zhou, *Janice S. Valletta, tMark L. Grimes, and *$§William C. Mobley

Departments of *Neurology and ::Pediatrics and §The Neuroscience Program, University of California, SanSan Francisco, California, U.S .A . ; and fiDepartment of Cheinistrv and Biochernistrv,

Massey University, Palmerston North, New Zealand

Abstract : TrkA is a receptor tyrosine kinase for nervegrowth factor (NGF) . Recent studies indicate that NGFregulates not only activation of trkA kinase but also ex-pression of the trkA gene . To further define NGF actionson trkA, we examined binding and signaling through trkAafter both short and long intervals of NGF treatment. In-duction of tyrosine phosphorylation on gpl40trkA was rap-idly followed by down-regulation of cell surface and totalcellular gp140trkA . At later intervals, increased expressionof trkA was evident in increased mRNA and protein levels .At 7 days, there was increased binding to gp140trkA andincreased signaling through this receptor . NGF appearsto regulate trkA at several levels . In neurons persis-tently exposed to NGF, maintenance of NGF signalingmay require increased trkA gene expression . KeyWords:Receptor-Signaling-Tyrosine kinase-Phosphoryla-tion-Phospholipase C-y1-Extracellular signal-regu-lated kinase-1 .J. Neurochem. 65, 1146-1156 (1995) .

Nerve growth factor (NGF) regulates the survival,development, and differentiation of the sympatheticand sensory neurons in the PNS and the differentiationof certain cholinergic neurons in the CNS (Levi-Mon-talcini, 1987 ; Thoenen, 1991 ; Longo et al ., 1993 ;Crowley et al ., 1994) . NGF also promotes neuronaldifferentiation of PC 12 cells, a line used extensivelyto investigate ligand-receptor interactions and cellulardifferentiation in response to NGF (Greene andTischler, 1976 ; Halegoua et al ., 1991 ; Longo et al .,1993) . NGF exerts its effects via specific low-affinityreceptors (Kp = 10 -`' M) and high-affinity receptors(Kt , = 10 - '' M) (Sutter et al ., 1979 ; Bernd and Greene,1984 ; Meakin and Shooter, 1992) . Dissociation fromhigh-affinity receptors is considerably slower thanfrom low-affinity receptors (Sutter et al ., 1979 ; Lan-dreth and Shooter, 1980 ; Sehechter and Bothwell,1981 ; Woodruff and Neet, 1986) ; therefore, high-af-finity receptors are also referred to as slow receptors(i.e ., slowly-dissociating) and low-affinity receptorsas fast receptors . Previous studies suggest that high-

1146

Francisco,

affinity receptors mediate NGF responsiveness (Sutteret al ., 1979 ; Bernd and Greene, 1984 ; Green andGreene, 1986 ; Meakin and Shooter, 1991) .gp140"" and p75 NGt'z act as receptors for NGF (Ho-

sang and Shooter, 1985; Green and Greene, 1986 ;Johnson et al ., 1986 ; Radeke et al ., 1987 ; Kaplan etal ., 1991a ; Klein et al ., 1991 ; Meakin et al ., 1992) .p75"° "' binds NGF with low affinity (Johnson et al .,1986 ; Radeke et al ., 1987 ; Mahadeo et al ., 1994) .A role for p75 NGFR in signal transduction has beensuggested (Davies et al ., 1993 ; Barker and Shooter,1994 ; Dobrowsky et al ., 1994 ; Hantzopoulos et al .,1994 ; Verdi et al ., 1994), but this has not been fullyelucidated . One contribution to NGF signaling may bethrough increasing the association rate for NGF bind-ing to gp140 t " (Mahadeo et al ., 1994) . gp140 trk " isthe protein product of the protooncogene trkA (Kaplanet al ., 1991 a ; Klein et al ., 1991) . The tyrosine kinasedomain of gp140WA is activated by NGF binding .Through NGF-mediated tyrosine autophosphorylation(Kaplan et al ., 1991a,b ; Klein et al ., 1991), gp140trk'serves as a substrate for binding of phospholipase C-y 1 (PLC--y l ), SHC and possibly extracellular signal-regulated kinase-I (ErkI ) (Ohmichi et a1 ., 1991 ; Loebet al ., 1992, 1994 ; Stephens et al ., 1994) . Ras activa-tion appears to play an important role in signalingleading to differentiation (Hagag et al ., 1986 ; Szebe-renyi et al ., 1990 ; Thomas et al ., 1992 ; Wood et al .,1992) . High-affinity receptors may consist of hetero-meric complexes of p75"° "' z and gp140"k" (Bothwell,

Received December 23, 1994 ; revised manuscript received March27, 1995 ; accepted April 3, 1995 .

Address correspondence and reprint requests to Dr. W . C . Mobleyat Department of Neurology, M-794, University of California, SanFrancisco, San Francisco, CA 94143-0114, U.S .A .Abbrevialioms used: DME H-21, Dnlhecco's modified Eagle's me-

dium ; EGF, epidermal growth factor ; EGFR, EGF receptor ; Erk,extracellular signal-regulated kinase ; GAM, neutralizing nervegrowth factor antiserum ; NGF, nerve growth factor ; NGS, normalgoat serum ; PAGE, polyacrylamide gel electrophoresis ; PBS, phos-phate-buffered saline ; PLC--y l, phospholipase C- ,y l ; RTA, anti-rattrkA antibody ; SDS, sodium dodecyl sulfate .

1991 ; Hempstead et al ., 1991 ; Mahadeo et al ., 1994),gp 140"" homodimers or oligomers (Klein et al ., 1991 ;Jing et al ., 1992 ; Meakin et al ., 1992), or possiblyboth (Weskamp and Reichardt, 1991) . Whatever theprecise makeup of these receptors, it is clear thatgp140'" activation is key to many aspects of NGF-signal transduction (Loeb and Greene, 1993) .NGF induces both short-term and long-term re-

sponses in PC12 cells . Among the former are eventsintimately associated with signaling as well as induc-tion of immediate early genes (e.g ., cfos, NGFI-A) .Delayed responses include the regulated expression ofgenes whose products are important for neuronal dif-ferentiation (e.g ., tyrosine hydroxylase, tau, synapsin1) (Halegoua et al ., 1991 ; Longo et al ., 1993) . It issomewhat surprising that the genes for p75 NOFR andgp140` are also induced by NGF (Lindsay et al .,1990 ; Miller et al ., 1991 ; Holtzman et al ., 1992b ; Mea-kin, et al ., 1992) . These findings raise the possibilitythat through increased receptor gene expression NGFcould modify its own signaling. Little attention hasfocused on NGF signaling in cells exposed to NGF forlong intervals and on the consequences of increasedreceptor gene expression . We examined trkA mRNA,trkA protein, trkA cross-linking to NGF, and trkA sig-naling in PC 12 cells treated with NGF. Both short- andlong-term changes were detected . Our findings suggestthat NGF-mediated up-regulation of trkA gene expres-sion serves to maintain, and perhaps to enhance, signal-ing in cells chronically exposed to the factor .

EXPERIMENTAL PROCEDURES

Cell culturePC12 cells were maintained in Dulbecco's modified Ea-

gle's medium (DME H-21) supplemented with 10% heat-inactivated horse serum, 5% fetal calf serum, penicillin 100U/ml, and streptomycin 100 gg/ml. PC12 cells not treatedwith NGF are also referred to as "naive" cells. PC12 cellswere "primed" by incubating with mouse NGF [preparedas described (Mobley et al ., 1986)] (50 ng/ml) for at least1 week . During this time they were fed every other day withfresh medium containing NGF. All cells were plated 2 daysin advance of experiments . For binding, cross-linking, andneurite outgrowth experiments, culture plates (Falcon) wereprecoated with rat-tail collagen (Collaborative Biomedical,Bedford, MA, U.S.A .) . Immediately before each experimenton primed cells, the cells were washed three times withNGF-free, fresh tissue culture medium for 2 h at 37°C .

Neurite outgrowthPC 12 eelIs were plated in 12-well dishes at a concentration

of 800 cells per well . Cells were fed with NGF (50 ng/ml)at time 0. Cultures were fixed at 12, 32, 36, and 48 h afterNGF addition by adding 1 ml 2% glutaraldehyde in phos-phate-buffered saline (PBS) . To some wells, a neutralizingNGF antiserum (GAM) (Crutcher et al ., 1993) or controlnormal goat serum (NGS) was added (1 :1,000 final concen-tration) at intervals after NGF addition . Antiserum-con-taining cultures were fixed at 48 h . Vertical strips of eachwell were examined and cells bearing a neurite greater than

NGF REGULATES trkA 114 7

one cell in diameter were scored . Data were expressed asthe percentages of cells bearing neurites .

Immunoprecipitation and western blottingImmunoprecipitation and western blotting wereperformed

as described (Kaplan et al ., 1991b) . PC12 cells were lysedat 4°C in lysis buffer [20 mM Tris-HCI, pH 8.0, 150 mMNaCl, 1 % Nonidet P-40, 10% glycerol, 1 mM sodium ortho-vanadate, and a cocktail of protease inhibitors ( 1 mM phe-nylmethylsulfonyl fluoride, 10 mg/ml benzamidine, 1 mg/mlo-phenanthroline, 0.1 mg/ml each of pepstatin, chymostatin,leupeptin, and aprotinin) (all reagents from Sigma, St . Louis,MO, U.S .A .)] . For Immunoprecipitation with anti-Erkl(Santa Cruz Biotechnology, Santa Cruz, CA, U .S.A .), 0.1 %sodium dodecyl sulfate (SDS) was added to the lysis buffer .Cells were rocked gently in lysis buffer for 20 min. Insolublematerial was removed by centrifugation at 4°C ( 10 min at10,000 g) . Lysates ( 1 ml) were immunoprecipitated whilerocking, overnight at 4°C, with antibodies to trkA (1088; 10pg), PLC-y1 (5 p, g) (Upstate Biotechnology, Lake Placid,NY, U.S .A .), or ErkI (1 Ng) . The anti-trkA antibody 1088was made to a peptide corresponding to the C-terminus (QA-LAQAPPVYLDVLG) of human trkA (Zhou et al ., 1994) .Immunoprecipitates were collected at 4°C by incubating withprotein A-Sepharose beads (Pierce, Rockford, IL, U.S.A .)for 2 h. After washing in lysis buffer and then water, immu-noprecipitates were boiled for 5 min in 50 pl of 2x SDS-polyacrylamide gel electrophoresis (SDS-PAGE) samplebuffer (2% SDS, 10% glycerol, 0.1 M dithiothreitol, 125mM Tris, pH 6.95, 0.1 % bromophenol blue), subjected to7.5% SDS-PAGE, and transferred to nitrocellulose. For ex-amining trkA, blots probed with 1088 or RTA (an antibodymade against extracellular domain of rat trkA, a gift fromD. Clary and L. Reichardt) (Clary et al ., 1994) were in-cubated with 1 pCi 1151-protein A (Amersham, ArlingtonHeights, IL, U.S .A .) . For detecting phosphotyrosine, blotswere incubated with an anti-phosphotyrosine monoclonal an-tibody (4G10; Upstate Biotechnology), then washed andincubated with goat anti-mouse IgG antibodies (BoehringerMannheim, Indianapolis, IN, U .S.A .) . ECL chcmilumines-cence system (Amersham) was used for detection . All blotswere exposed to x-ray film and band intensities were quanti-fied on a densitometer (LKB Ultroscan, Gaithersburg, MD,U.S .A .) . To ensure that we could quantitatively immunopre-cipitate gpl40` and gp110"'"', in preliminary studies weused the same amount of 1088 to immunoprecipitate increas-ing amounts of PC12 cell lysates . The amount of gp110""and gp140' rk" detected increased linearly in proportion to theamount of lysate protein over the range of trkA protein levelsexamined in our experiments .

RNA isolation and northern blot analysisPC12 cells grown to 30% confluence were treated with

mouse NGF (50 ng/ml) at 37 °C for the times indicated.Cells were harvested and total RNA was prepared usingguanidinium isothiocyanate extraction (Holtzman et al .,1992b) . For northern blots, 20 pg of total RNA was electro-phoresed on I % agarose-formaldehyde gels and blotted toGeneScreen (Du Pont, Boston, MA, U.S.A .) as described(Holtzman et al ., 1992b) . Blots were probed with "P-labeledcDNAs (Holtzman et al ., 1992b) for rat trkA (Holtzman etal ., 1992b), rat p75"°"z (gift of E. M. Shooter), or 18SrRNA (Holtzman et al ., 1992b) . After washing at high strin-

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gency, blots were exposed to x-ray films and quantitated asdescribed (Holtzman et al ., 1992a,b) .

NGF binding and internalization studiesStudies were performed to examine NGF binding to sur-

face receptors and to assess NGF internalization . Naive andprimed cells were seeded in six-well collagen-coated platesat a density of 5 X 10 5 cells per well 2 days before assay .Primed cells were washed free of NGF for 2 h at 37'Cimmediately before experiments . Before binding, cells wererinsed two times for a total of 10 min with ice-cold bindingbuffer (PBS containing 1 mg/ml glucose and 1 mg/ml bo-vine serum albumin) and incubated with 2 ml of bindingbuffer containing 100 pM or 2 nM "'I-NGF at 4'C for 2 h .A 2-h incubation was chosen because in preliminary studieswe found that this interval brought binding to steady state .To measure binding to slowly dissociating receptors, cellswere then washed in NGF-free binding buffer at 4'C for 1h . This interval for the wash was shown in preliminary stud-ies to exclude binding to fast-dissociating receptors and tomeasure binding to sites with a very slow rate ofdissociation(mean percent binding of 100 pM 125I-NGF to washed PC 12cells was 47% at 30 min, 46% at 60 min, and 44% at 90min) . For internalization studies, cells bound to NGF atsteady state were incubated at 37'C for various time periodsand chilled to 4°C .

Binding to surface receptors was evaluated using estab-lished methods (Bernd and Greene, 1984) . Cells werewashed three times with 2 ml of ice-cold binding buffer andthen treated for 5 min with 1 ml of ice-cold sodium acetatesolution (0.2 M acetic acid, 0.5 M NaCI) to release thesurface bound ' -5I-NGF (Bernd and Greene, 1984) . Thesolution was then collected . To measure internalized NGF,cells were lysed in 1 ml of 0.2 M NaOH and this solutionwas collected . All samples were assessed for radioactivity ina Beckman y counter 4000 . For all experiments, nonspecificbinding was measured for both the acid releasable and inter-nalized fractions by including 800 nM unlabeled NGF duringbinding . Nonspecific binding was determined to be 18-23%of specific binding for both fractions . All data are reportedas specific binding (i .e ., total minus nonspecific binding) .

NGF cross-linkingPC 12 cells were maintained as indicated . Cells (10'/ 15-

cm dish) were plated 2 days before experiments . Naive cellsand primed cells (washed free of NGF for 2 h at 37 °C withculture medium) were washed with binding buffer at 4'Cfor 10 min and then incubated in binding buffer containingeither 100 pM or 2 nM'25I-NGF, for 2 h at 4°C . To measuredown-regulation of surface gp140" a", cells were warmed to37 °C for l h and then chilled in an ice water bath WC)before cross-linking . To measure binding to slow-dissociat-ing receptors, cells were washed in ligand-free binding bufferat 4'C for I h before cross-linking .To cross-link NGF to gp140"", bis-sulfosuccinimidyl su-

berate (BS') (Pierce) was added to a final concentration of0.8 mM in binding buffer . Samples were incubated at 4'Cfor 30 min, washed, and lysed in lysis buffer, as above . '-s1-

NGF/gp140" complexes were immunoprecipitated with1088 . The immunoprecipitates were boiled in the SDS-PAGE sample buffer for 5 min and subjected to SDS-PAGEusing a gradient-resolving gel (5.0% acrylamide with 0 .1bisacrylamide to 12.0% acrylamide with 0.5% bisacrylam-ide) . Gels were fixed, dried, and exposed to x-ray film or to

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a Phosphorlmager screen . Quantitation was via a MolecularDynamics Phosphorlmager (Molecular Dynamics ; Sun-nyvale, CA, U .S.A .) . In preliminary studies we found thatby using these methods we could measure small amounts ofgpl40'""" (Zhou et al ., 1994) and that the extent of cross-linking to gp140"" increased with increased expression oftrkA .

RESULTS

Induction and maintenance of PC12 cell neuriteoutgrowth required the continuous presence ofNGFNGF activates gp l40" rapidly in PC 12 cells to

induce changes in kinase activities, second messengerproduction, membrane morphology, and immediateearly gene expression (Halegoua et al ., 1991 ; Kaplanet al ., 1991a,b ; Klein et al ., 1991 ; Chao, 1992) . How-ever, many of the NGF effects on neuronal differentia-tion are evident only later . For example, neurite out-growth from PC12 cells is produced after a delay of36-48 h . Late responses to NGF could be induced bysignaling events produced within minutes of initialNGF treatment ; alternatively, they may require pro-longed activation of gp140"' receptors . To examinethese alternatives, PC12 cells were cultured with NGFand neurite outgrowth was assessed at intervals of upto 48 h. To some cultures, a neutralizing GAM wasadded during the course of the experiment to inhibitNGF actions ; an NGS served as control . In the continu-ous presence of NGF, neurites were not detected at 32h but were present at 36 h in 20.7% (n = 3) of cells .About 30% of PC 12 cells had neurites at 48 h . WhenGAM was added at 12 or 32 h, no neurites were de-tected at 48 h . GAM addition at 36 h resulted in retrac-tion of neurites so that by 48 h the level was onlyslightly above that in cultures that did not receive NGF.Even at 40 h, GAM addition reduced neurite outgrowthto 20% by 48 h . These results are evidence that NGFmust be present for >32 h to induce neurites and con-tinuously thereafter to maintain them .

gp140' was tyrosine phosphorylated duringboth brief and prolonged exposure to NGFNGF binding to gp140"' induces receptor dimeriza-

tion and autophosphorylation (Chao, 1992 ; Jing et al .,1992 ; Schlessinger and Ullrich, 1992 ; Loeb andGreene, 1993 ; Stephens et al ., 1994) . A pronouncedincrease in tyrosine-phosphorylated gp140"" is regis-tered within minutes of NGF treatment and persists forat least 6 h (Kaplan et al ., 1991 b) . We asked whethergp140"' tyrosine phosphorylation would also be ap-parent during prolonged NGF treatment . To performthese studies, an antibody (1088) to a peptide corre-sponding to the C-terminus of human TrkA was usedto immunoprecipitate and blot PC12 cell lysates . Pro-teins of 140, 110, and 38 kDa were detected . The pep-tide antigen blocked immunoprecipitation of these pro-teins (Fig . 1) . The 140- and 110-kDa proteins were

FIG . 1 . Analysis of trkA immu-noprecipitates . trkA proteins inPC12 cell lysates were immuno-precipitated using an anti-trkAantibody (1088) in the absence(-) or presence (+) of compet-ing trkA peptide (10 Ng/ml) . Theimmunoprecipitates were ana-lyzed on SIDS-PAGE, transferredto nitrocellulose, probed with1088, and developed using "'I-protein A .

also detected using an antibody to the extracellulardomain of rat trkA (RTA) (Clary et al ., 1994) (datanot shown) . Earlier investigations (Martin-Zanca etal ., 1989 ; Kaplan et al ., 1991a,b ; Klein et al ., 1991 ;Meakin and Shooter, 1991 ; Vetter et al ., 1991 ; Hemp-stead et al ., 1992), indicated that the 140-kDa speciesis the mature form of trkA and serves as the NGFreceptor and that the 110-kDa species is a precursor .Hereafter, they will be referred to as gpl40"a " andgp110"" . The identity of the 38-kDa protein immuno-precipitated with 1088, p38, is uncertain (Fig . 1) .Though detected on blots with 1088, it was not seenby using RTA. One possibility is that it is a truncatedversion of trkA containing all or part of the C-terminalintracellular domain . A protein of identical size wasshown to be reactive with antibodies specific to the C-terminus of trkA and gave rise to a phosphopeptidemap identical to trkA (D . Kaplan, unpublished obser-vations) .To examine tyrosine phosphorylation of gpl40"k",

lysates of untreated and NGF-treated PC 12 cells wereimmunoprecipitated with 1088 and blotted with ananti-phosphotyrosine antibody (4G10) . NGF induceda pronounced increase in phosphorylation at 5 min .Tyrosine phosphorylation of gp140"k" decreased atlater times, but a definite signal was present at 60 min,2 days, and 7 days of treatment (Fig . 2) . Removal ofNGF from the culture medium resulted in rapid loss oftyrosine-phosphorylated gpl40"k" (data not shown) .These data indicate that there is persistence ofgpl40"k" tyrosine phosphorylation in the presence ofNGF. They are evidence that NGF continuously acti-vates gp140` rk" receptors . p38 was also tyrosine phos-phorylated after NGF treatment and the pattern ofphosphorylation after short-term NGF treatment wassimilar to that for gpl40 trk" . It is interesting that after7 days of NGF treatment, the level of tyrosine phos-phorylation on both gp140`1A and p38 was higher thanafter 5 min of treatment .NGF treatment down-regulated surface gp140"k"acutely and decreased trkA protein levels in PC12cellsNGF exposure beyond 5 min led to a clear decrease

in tyrosine-phosphorylated gp140" . Possibilities for

NGF REGULATES trkA 1/49

explaining the decrease in phosphorylation included adecrease in the rate of activation of receptors, an in-crease in the rate of dephosphorylation of activatedreceptors, or both . There are few data for the cellulardisposition of activated gp 140"kA and for the regulationof tyrosine phosphorylation on this receptor . However,in earlier studies it was shown that NGF treatmentinduced down-regulation of gp140"k" at the surface ofPC 12 cells (Hosang and Shooter, 1987) and decreasedthe number of its receptors (Layer and Shooter, 1983) .Ligand-mediated down-regulation of other receptor ty-rosine kinases at the cell surface is linked to degrada-tion of these receptors (Beguinot et al ., 1984 ;Stoscheck and Carpenter, 1984 ; Sorkin and Waters,1993) . Were gp140"' regulated similarly, this couldcontribute to the decreased tyrosine phosphorylationnoted . To address this issue, we attempted first to con-firm that NGF decreased surface gp140"" . For theseexperiments, ' 25I-NGF was bound to its receptors onPC12 cells at 4°C to inhibit membrane traffic . In thecontinued presence of ''S1-NGF, the cells were theneither warmed to 37°C for 60 min or kept at 4°C .125 I-NGF was then cross-linked to gp140 "" at 4°Cby using a membrane-impermeant cross-linking agent.There was a pronounced decrease in cross-linking togp140o-k" under these conditions ; indeed, in warmedcells there was only 16% of the amount detected incells held at 4°C (Fig . 3) . Consistent with earlier find-ings (Hosang and Shooter, 1987), this result indicatedthat NGF down-regulated surface gp140`rk" . The de-crease in cross-linking after warming was comparableto the decrease in tyrosine phosphorylation of gp l40'""during the same interval .

To ask whether NGF treatment would also decreasecellular gpl40 1,k", we measured gpl40"k" at intervalsafter NGF (50 ng/ml) addition . Figure 4A shows thatgp 140 1,kA was unchanged at 5 min, decreased some-what at 30 min, and decreased markedly by 60 min .At 60 min the amount of gp140" k" was 33% of thatpresent before treatment . Thus, NGF treatment resultedin rapid down-regulation of this receptor not only atthe cell surface but also as measured on the whole celllevel . Either decreased synthesis or increased degrada-tion of gp140'rk" could have been responsible for the

FIG . 2. Phosphorylation of gpl40'rkA after NGF treatment . PC12cells were treated with NGF (50 ng/ml) for 0 min, 5 min, 60 min,2 days, and 7 days . Cell lysates were immunoprecipitated using1088, blotted, and then probed with anti-phosphotyrosine anti-body (4G10) . Lanes contain protein from an equal number ofcells .

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FIG . 3 . Cross-linking of ' 25 1-NGF to surface gp140 "A in PC12cells . PC12 cells, naive or NGF-primed, were incubated with 100pM 125 1-NGF at 4°C for 2 h and then warmed to 37°C for 0 or60 min . Cells were then chilled and cross-linking was performedas described in Experimental Procedures . Cells were lysed andlysates were immunoprecipitated with 1088 . The resulting immu-noprecipitates were analyzed by SDS-PAGE . The gel was driedand exposed to x-ray film . After 60 min at 37°C, the amount ofgp140' cross-linked to "'I-NGF dropped to the same level inboth naive and primed cells . There is a band at 200 kDa ; aband of similar molecular mass has been seen in prior studies(Hartman et al ., 1992) . In this band, NGF specifically cross-linked to trkA appears to be present in a higher order complex .Each lane represents protein from an equal number of cells . Theexperiment was done twice with the same results .

decrease in cellular gp140"" . The persistence ofgpl 10""a , a precursor to gp140"", at levels equal tothat in untreated cells for 1 h, suggests that increaseddegradation of gp140" was responsible .

Prolonged NGF exposure increased trkA proteinlevels

After prolonged NGF treatment, we noted an in-crease in the level oftyrosine-phosphorylated gp 140'""(Fig . 2) . To ask whether this could reflect an increasein surface receptors, trkA protein levels were mea-sured . NGF treatment induced a pronounced increasein trkA protein (Fig . 4A and B) . On a per-cell basis,gpl 10t'kA was increased to 3.4-fold (n = 2) the un-treated level at 2 days, and at day 7 it was increasedto 5.1-fold (n = 5) . The increase in gp140"-" was less

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pronounced . At day 2, gp140"" was increased slightlyrelative to the level at 60 min . At day 7, it was in-creased to 3 .0-fold (n = 5) that present in untreatedcells . Total protein in primed cells increased by 28%at 2 days and 60% at 7 days . Therefore, whether ex-pressed on a per cell or per milligram of protein basis,trkA protein was increased in PC12 cells treated withNGF for 7 days . The increase in gp140"' was corre-lated temporally with the increase in tyrosine-phos-phorylated gp140"" but was greater . p38 was alsoincreased by NGF treatment (Fig . 4A) .

Thus, trkA protein levels in PC] 2 cells showed dis-tinct early and late responses to NGF . During the earlyphase, NGF induced a rapid decrease in both surfaceand total cellular gp140"" . During the later phase (2days onward), receptor protein levels increased to ex-ceed pretreatment levels .

NGF increased mRNA for trkA and P75 NGFR

The increase in gpl 10""'' and gp140" after 2 daysof NGF treatment suggested that NGF increased syn-thesis of trkA proteins . In earlier studies, NGF in-creased trkA mRNA in PC12 cells as well as in basalforebrain and caudate-putamen cholinergic neurons(Holtzman et al ., 19926 ; Meakin et al ., 1992) . Toexamine a possible relationship between increasedmRNA and increased trkA protein levels, we investi-gated the time course of NGF induction of trkA inPC12 cells . Total RNA isolated from naive (i .e ., un-treated) PC12 cells and from cells treated with NGF(50 ng/ml) for 4 h, 24 h, 3 days, or 7 days was ana-lyzed by northern blot . trkA mRNA was decreased-40% (n = 4) at 4 h . It was increased to 1 .5-fold (n= 4) the untreated level at 2 days and reached a maxi-mum (3.7-fold, n = 4) at day 7 (Fig . 5A and B),remaining at this level for up to 14 days (data notshown) . NGF is also known to induce p75"c'Fa expres-sion in PC] 2 cells (Miller et al ., 1991) . A differenttime course was seen for NGF effects on p75"°Fe

mRNA . It was increased to 2.0-fold (n = 4) the un-treated level by 8 h (data not shown), 3 .4-fold (n= 4) at 24 h, and remained at about this level at later

FIG . 4 . NGF regulates the levels of gp140',gp110', and p38 . A: trkA proteins were immuno-precipitated with 1088 from lysates of untreatedPC12 cells (0') or cells treated with NGF for 5 min, 30min, 60 min, 2 days, and 7 days . Immunoprecipitateswere analyzed on SDS-PAGE and immunoblottedwith 1088 . An equal number of cells was used foreach lane . B : gpl40'rkA and gp110""A bands werequantitated by densitometry . The level of expressionrelative to untreated cells is plotted (n = 2 for eachpoint) .

FIG. 5 . NGF induces gene expression for trkA and p75"' inPC12 cells . A: PC12 cells were treated with NGF (50 ng/ml) for4 h, 24 h, 72 h, and 168 h . Cells were harvested and total RNAfrom untreated (-) or NGF treated (+) was prepared and ana-lyzed on northern blot; 20 ug was loaded in each lane . Blotswere hybridized with rat trkA cDNA or rat p75"GF" cDNA . The18S rRNA probe was used to control for RNA loading . B: Quanti-tation of NGF effects . The first panel shows the results for PC12cells treated with NGF (50 ng/ml) for 4 h, 1 day, 3 days, 7 days,and 10 days . The second panel shows the effect of NGF removalfrom cells treated for 10 days and then analyzed after 2 or 4days in culture . Total RNA was collected at each time pointand analyzed on northern blot . Data from northern blots werequantitated using densitometry . Each point represents the meanSEM (n = 4) .

tithes (Fig . 5A and B) . We also examined gene expres-sion in cultures containing low levels of serum (2%fetal calf serum and 1 % horse serum) . Under theseconditions, trkA and p75N°`'R mRNA were induced toa greater extent . Maximum induction for trkA mRNAwas 5 .3-fold (n = 3) at day 7 . p75"" mRNA wasincreased after only 4 h, and maximum induction was8 .0-fold (n = 3) at day 7 . For both genes the effectof NGF was transient . After NGF removal, mRNAlevels for both trkA and gp75NGrR fell to control levelswithin 48 h (Fig . 5B) . Corresponding to the increasein mRNA levels, we found by western blot of thesame cultures that 7 days of NGF treatment increasedgp l40"A to 3.0-fold (n = 5) and p75 "GFR to 2 .4-fold(n = 3) that present in untreated cells (level calculated

NGF REGULATES trkA 1151

on a per-cell basis ; data not shown) . These studiesshow that gp110" F' and gp140 1,k1 were increased atthe time that trkA mRNA was increased and that forNGF treatment from day 2 through day 7, increasingmRNA levels were correlated with increasing proteinlevels . They suggest that NGF acted through an in-crease in trkA mRNA to increased trkA protein levels .The same may be true of NGF effects on gene expres-sion for p75 NGPR .

Increased gp140'1" contributed to increased NGFbinding and internalization in primed cellsThe increase in gp l40""' and p75 "` after pro-

longed NGF treatment raised the possibility that NGF-treated cells would have more surface receptors forNGF. This possibility was suggested by earlier studieson PC 12 cells showing that NGF produced an increasein both high-affinity and low-affinity NGF bindingsites with no change in their K� (Bernd and Greene,1984) . We investigated ' 251-NGF binding to naive andprimed (i .e ., NGF treated for 7 days) cells under condi-tions identical to those used previously (Bernd andGreene, 1984), except that all our measurements weremade at 4°C to inhibit membrane traffic and receptorinternalization . Two NGF concentrations were used .At 100 pM NGF, one is below the ED5, for the neuriteoutgrowth response (ED;,, - 400 pM), and at a levelthat is subsaturating for high-affinity receptors (K��, 400 pM) (Bernd and Greene, 1984) . At 2 nM, NGFoccupies most of these receptors and produces maxi-mal neurite outgrowth . We found that '''1-NGF bind-ing to primed cells was increased . Relative to naivecells, on primed cells binding was increased to 1 .8(-- 0.19)-fold (n = 6) at 100 pM and 1 .6 (- 0.09)-fold (n = 3) at 2 nM. High-affinity receptors show aslow rate of dissociation for NGF. At 2 nM, '25I-NGF(2 nM) bound to slowly-dissociating surface receptorswas increased 1 .6 (-±- 0.1)-fold (n = 3) on primedcells ; there was a corresponding increase in fast-disso-ciating binding (i.e ., binding to low-affinity receptors)to 1 .5 ( ± 0.1)-fold on these cells . ''5I-NGF internaliza-tion provides another measure of NGF-receptor func-tion, i .e ., one that reflects the participation of high-affinity receptors (Bernd and Greene, 1984) . After 60-min treatment, '25I-NGF internalization for primedcells was 1 .8 (- 0.1 )-fold (n = 3) that in naive cells .These data for ''5I-NGF binding and internalization areconsistent with earlier observations ( Bernd andGreene, 1984) .To show whether increased trkA receptors contrib-

uted to increased NGF binding, we performed cross-linking studies . Naive and primed cells were incubatedwith '51-NGF at 4°C for 2 h, and then cross-linked atthe same temperature . For primed cells, ' z5I-NGFcross-linked to gpl40"" was increased 44% (n = 2)at 100 pM (Fig . 3) and 100% at 2 nM (n = 2) (Fig .6) . There was a comparable increase in ' 25 I-NGFcross-linking to gp140"-'"' in slowly dissociating recep-

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FIG. 6 . Cross-linking to gp140' in slow-dissociating receptorswas increased in primed cells. ' 251-NGF (2 nM) was bound tonaive and primed PC12 cells for 2 h at 4°C . Cells were eithercross-linked immediateiy to ' 25 1-NGF or washed for 1 h in NGF-free binding buffer at 4'C before cross-linking . Lysates wereimmunoprecipitated with 1088, analyzed by SIDS-PAGE, and ex-posed to x-ray film . Each lane contains protein from an equalnumber of cells . Note the increase in cross-linking of ' 211-NGFto gpl40 'r"A in primed cells, both before and after the wash . Inthis experiment the percentage of NGF cross-linking to gpl40'rkAin slow receptors was 80.6% for naive cells and 90.3% forprimed cells .

tors (Fig . 6) . trkA expression confers on cells the abil-ity to internalize NGF (Jing et al ., 1992 ; Loeb andGreene, 1993) . To ask whether an increase in gp l 40"-kAreceptors could mediate the increase in NGF internal-ization in primed cells, we measured the amount ofthese receptors present at the cell surface before andafter a 1-h incubation at 37'C in the continuous pres-ence of 125I-NGF . After warming, the amount of `l-NGF (100 pM) that could be cross-linked to gp 140i,AAdecreased markedly in both naive and primed cells(Fig . 3) . The net decrease for primed cells was 1 .5-2.0-fold that in naive cells . These studies show thatincreased NGF binding and internalization in primedcells are due, in part, to increased binding to surfacegp 140'rkA .

In NGF-primed cells there was increased tyrosinephosphorylation of gpl40' and PLC-yl but notof Erkl

An increase in tyrosine-phosphorylated gp140"kAafter 7 days of NGF treatment (Fig . 2) suggested thatthere may be increased NGF signaling in these cells .The increase in surface gp140`rAA also suggested thispossibility . To test this, the effect of NGF treatmenton tyrosine phosphorylation ofgp140'rkA was examinedin naive cells and in primed cells washed free of NGFfor 2 h before assay . After treatment for 5, 30, or 120min at 37°C, cell Lysates were immunoprecipitated with1088 and probed with anti-phosphotyrosine antibody(4G10) . As shown in Fig . 7, a relatively high level oftyrosine-phosphorylated gp 140`rkA was present at 5 minin both naive and primed cells . Tyrosine-phosphory-lated gpl40'rkA decreased thereafter in both naive andprimed cells and the decreases followed a similar timecourse . Nevertheless, the level of tyrosine-phosphory-

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FIG . 7 . NGF-mediated tyrosine phosphorylation of trkA was in-creased in primed cells . Naive and primed PC12 cells were un-treated (0) or treated with NGF 50 ng/ml for 5 min, 30 min,or 120 min . Cell lysates were immunoprecipitated with 1088,subjected to SIDS-PAGE, immunoblotted, and the blots probedwith anti-phosphotyrosine antibody (4G10) . Lanes contain pro-tein from an equal number of cells .

lated gp 140"k'' was greater in primed cells at each timepoint tested . As noted above, primed cells contain moreprotein . To normalize for this, the results were ex-pressed per milligram of protein . The ratio of the signalin primed to that in naive cells was 0.9-fold (n = 3)at 5 min, 1 .8-fold (n = 3) at 30 min, and 2.9-fold (n= 3) at 2 h . As before, p38 was phosphorylated inresponse to NGF in both naive and primed cells . Thetemporal pattern was similar to that for gp140'rAA .PLC-yl is a target of NGF-induced tyrosine phos-

phorylation and activation (Kim et al ., 1991 ; Ohmichiet al ., 1991 ; Vetter et al ., 1991 ; Stephens et al ., 1994) .We investigated the level of tyrosine phosphorylationof PLC--y I in naive and primed PC 12 cells . Likegp140"k-A , tyrosine phosphorylation of PLC-TI wasdemonstrable at 5 min and was sustained through 7days of exposure to NGF (Fig . 8, first five lanes) . Inprimed cells washed free of NGF, tyrosine-phosphory-lated PLC-yl decreased to a very low level (Fig . 8,

FIG . 8. NGF-mediated tyrosine phosphorylation of PLC-y1 wasincreased in primed cells . Naive and primed PC12 cells wereuntreated or treated with NGF at 50 ng/ml for 5 min, 30 min, 2h, or 7 days . Cells were lysed and equal amounts of protein wereimmunoprecipitated with anti-PLC-y1 antibody . Immunoprecipi-tated proteins were analyzed on SIDS-PAGE and probed firstwith anti-phosphotyrosine antibody (A) and then with anti-PLC-yi antibody (B) .

FIG . 9. NGF-mediated tyrosine phosphorylation of Erk1 was notincreased in primed cells . Naive and primed PC12 cells wereuntreated or treated with NGF at 50 ng/ml for 5 min, 30 min, 2h, or 7 days . Cells were lysed and equal amounts of protein wereimmunoprecipitated with anti-Erk1 antibody . Immunoprecipi-tated proteins were analyzed on SIDS-PAGE and immunoblottedfirst with anti-phosphotyrosine antibody (A) and then with anti-Erkl antibody (B) .

lane 6) . When primed cells were then reexposed toNGF for 5 min, the level of tyrosine phosphorylationof PLC- ,y I was 1 .7-fold (n = 3) that in naive cells ona per milligram of protein basis (Fig . 8, lane 7) . Anincreased ratio was also present at later time points .The data for tyrosine phosphorylation of gp 140"1a andPLC-,y1 are evidence for increased NGF signalingthrough gpl40"r" in primed cells .

Erkl is a serine-threonine kinase that participatesin NGF signaling pathways leading to differentiation(Loeb et al ., 1992 ; Thomas et al ., 1992 ; Wood et al .,1992) . To ask whether downstream signaling was alsoincreased in primed cells, we examined tyrosine phos-phorylation of Erkl . There was no increase in primedcells (Fig . 9) . However, another immunoprecipitatedprotein that migrated above Erkl did show increasedtyrosine phosphorylation (Fig . 9) . On the basis of ear-lier studies, this protein can be identified as Erk4(Boulton et al ., 1991) .

DISCUSSION

NGF acts through its receptors to evoke the inte-grated responses required for maintenance of neuronalviability and enhanced neuronal differentiation . Defin-ing the biochemical and cellular events that character-ize these responses is an important objective . The dis-covery that trkA is a receptor tyrosine kinase for NGF(Kaplan et al ., 1991a,b ; Klein et al ., 1991 ; Meakinand Shooter, 1991 ; Meakin et al ., 1992) has greatlyfacilitated these studies . Herein we examined acute andprolonged NGF signaling in PC 12 cells . Our data showthat NGF regulated trkA and that its effects were de-pendent on the duration of exposure . Initially there wasinduction of tyrosine phosphorylation on gp140"" .Immediately thereafter there was down-regulation ofsurface and cellular gp140"kA and decreased but persis-tent tyrosine phosphorylation of gpl40"' . Laterevents were characterized by increased receptor geneexpression for both trkA and p75 NGFR

. At 7 days, NGF-

NGF REGULATES trkA 1153

treated cells showed modest increases in binding togp140"'`" and enhanced signaling through this receptor .The data are evidence that NGF regulates trkA at sev-eral levels .NGF had to be present essentially continuously in

the culture medium to induce and maintain neurites onPC12 cells . Our studies are consistent with the needfor continued signaling through surface trkA to inducemorphological differentiation . One manifestation of re-ceptor activation is tyrosine phosphorylation (van derGeer et al ., 1994) . We noted tyrosine phosphorylationof gp140t kA in PC 12 cells within minutes of exposureto NGF. This observation, and the decrease in phos-phorylation over the next hour, agree with earlier find-ings (Kaplan et al ., 1991b ; Qiu and Green, 1991 ;Hempstead et al ., 1992) . It is significant that tyrosine-phosphorylated gp140' rkA was shown to persist, albeitat a relatively low level, in the continued presence ofNGF. These data suggested that NGF in the mediuminduced persistent activation of gp140 nAA . Additionalevidence for continued activation of trkA receptors byNGF was persistent tyrosine phosphorylation of p38,a specific substrate of NGF-receptor activation (Oh-michi et al ., 1992), and of PLC--y I and Erkl . SHC isanother substrate of activated trkA ; others have shownthat SHC phosphorylation persists for at least severalhours in the presence of NGF (Stephens et al ., 1994) .Given recent data for signaling through mutant trkAreceptors (Stephens et al ., 1994), persistent activationof either PLC--y l or SHC may be required to inducePC12 neurites .We noted pronounced and rapid effects of NGF on

the level of surface and cellular trkA . gp140" -" down-regulation by NGF parallels observations for the effectof other polypeptide ligands on their receptor tyrosinekinases (Carpenter, 1987 ; Sorkin and Waters, 1993) .For example, in the case of the epidermal growth factorreceptor (EGFR), EGF addition results in a pro-nounced and rapid decrease in surface receptors andaccelerated receptor degradation (Beguinot et al .,1984 ; Stoscheck and Carpenter, 1984 ; Murthy et al .,1986) . EGFR internalization is via coated pits (Begui-not et al ., 1984 ; Lamaze et al ., 1993) and, thoughcontroversial, receptor tyrosine kinase activity may berequired to achieve highly efficient ligand-mediatedreceptor internalization (Glenney et al ., 1988 ; Chen etal ., 1989 ; Wiley et al ., 1991 ; Lamaze et al ., 1993) .There are few data for the molecular and cellular eventsrequired for internalization and down-regulation ofgp 140'rß .

Delayed responses to NGF include enhanced expres-sion of a number of genes whose products mark differ-entiated neurons (Halegoua et al ., 1991 ; Longo et al .,1993) . The genes for both NGF-receptor proteins ap-pear to be in this class . p75"" mRNA levels wereincreased before trkA mRNA, suggesting differencesin the mechanisms of induced expression . However,three similarities were noted . The first was the need

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for continued NGF treatment to maintain increasedmRNA levels . The second was the corresponding in-crease in the level of the p75 NGFR and trkA proteins .The third was the increased induction of both genesunder low-serum conditions, suggesting the presenceof serum factors that inhibit NGF actions . Thus, as forthe products of other genes that mark differentiatedneurons (Halegoua et al ., 1991), NGF appears to havea significant but not exclusive role in regulating expres-sion of its receptors . Studies on EGF provide prece-dents for our findings (Clark et al ., 1985 ; Kudlow etal ., 1986 ; Bjorge et al ., 1989) . Treatment of humanKB carcinoma cells with EGF increased EGFR mRNAand protein . Nuclear runoff-transcription assaysshowed no increase due to EGF, suggesting the exis-tence of posttranscriptional controls . NGF may in-crease mRNA levels for p75 NGFR through increasedtranscription (Miller et al ., 1991) ; the mechanismleading to increased trkA mRNA has not been estab-lished .NGF treatment for 7 days increased NGF binding to

high- and low-affinity surface receptors and increasedNGF internalization . These findings demonstrated acorrelation between receptor gene expression and re-ceptor binding . Furthermore, we have linked increasedtr" gene expression directly to increased receptorbinding . Relative to the increases in p75 Nc "'' andgp140"-kA , the increase in NGF binding was small .When expressed per cell, a 200% increase in totalgp140'"A'A was associated with smaller increases in NGFbinding to slow-dissociating receptors (60% increase)and cross-linking to gp140" A (100% increase) at thecell surface . These observations suggest that the num-ber of surface gpl40` receptors is not simply a func-tion of the protein level present . Indeed, it is probablethat several processes influence the number of surfaceNGF receptors : synthesis, membrane insertion, inter-nalization, recycling, and degradation . Studies of otherreceptor tyrosine kinases indicate that their ligands canexert control at one or more of these levels (Sorkinand Waters, 1993) . Though further studies are re-quired, our data suggest that NGF may influence thelevel of surface trkA through actions at most of theselevels .

Bernd and Greene (1984) showed an increase inreceptor binding in primed cells . To compare our find-ings with theirs, we used identical conditions for bind-ing . However, we performed all our studies at 4°C, toinhibit internalization of gp140`"kA . In the earlier study,binding was examined at 37°C . Under this condition,surface binding in naive cells plateaued at 5 min andremained at this value through 60 min . At 5 min, therewas little difference between primed and naive cells .Our data can be compared with theirs for binding atthe earliest time points . When we expressed our resultsper milligram of protein, we also saw little change inNGF binding . Conversely, when the binding data areexpressed per cell, in both studies there was a modest

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increase . It is interesting that at 37°C, binding toprimed cells continued to increase and by 60 min athree- to fourfold increase was detected . Therefore, therather large increase in receptors in primed cells wasdue principally to binding events that occurred afterprolonged incubation at 37°C (Bernd and Greene,1984) . Membrane trafficking of gp140`"k", p75 Ni7FR 'or both may explain this finding .We noted enhancement of NGF signaling in primed

cells . Increased signaling was evident in increased ty-rosine phosphorylation of gp140`r1A , PLC--y l, p38, andErk4 . These experiments link increased gp140`"AA toincreased receptor activation . It is interesting that thedifference between primed and naive cells was longlived ; indeed, for gp140"''A , PLC-yl, and Erk4, thedifference was greatest 2 h after NGF addition . HowNGF acted to enhance signaling in primed cells isuncertain . A simple increase in the number of surfacereceptors appears insufficient to explain our findings .Indeed, when binding was adjusted for the proteinlevel, there was little change . Further studies are re-quired to define the link between increased gp140"1Aand increased NGF signaling .Our studies do not allow assessment of the potential

physiological consequences of increased NGF receptorgene expression on NGF signaling . First, PC12 cellsmay not be ideal for addressing such questions . Sec-ond, the most dramatic increases were seen in primedcells washed free of NGF for 2 h . This experimentalparadigm was required to compare binding to signalingdirectly, but it may not reflect a physiological conditionin which cells are continuously exposed to NGF. Oneinterpretation of the data is that increased trkA geneexpression is a response to gp140 1,kA down-regulationthat serves to restore and maintain NGF signaling .Given dramatic down-regulation of gp140 1,kA , and theneed for continued NGF exposure for maintenance ofmature neuronal phenotype, this suggestion is reason-able. Whether increased trkA and p75"°rk expressionwould result in enhanced NGF signaling in vivo isuncertain. It is known that NGF increases trkA and1)75";" expression in basal forebrain cholinergic neu-rons in vivo (Holtzman et al ., 1992b) and that earlyexposure to exogenous NGF enhances the response tolater administration (Johnston et al ., 1987) . The cur-rent study raises the possibility that through regulatingsynthesis, internalization, and degradation of its recep-tors, NGF could produce quantitative as well as quali-tative differences in NGF signaling .

Acknowledgment : We thank Drs. D. Clary and L. Rei-chardt for the gift of rat trkA antibody . This research wassupported by National Institutes of Health grants NS31076,NS24054, and AG 10672 (W.C.M .) . J .Z . was supported bythe Adler Foundation . M.L.G . was supported by a YoungInvestigator Award from the National Alliance for Researchon Schizophrenia and Depression (NARSAD) .

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