Activation of Trk Neurotrophin Receptor Signaling by Pituitary Adenylate Cyclase-activating Polypeptides*

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that acts through G protein-coupled receptors, exerts neuroprotective effects upon many neuronal populations. However, the intracellular signaling mechanisms that account for PACAP’s trophic effects are not well characterized. Here we have tested the possibility that PACAP uses neurotrophin signaling pathways. We have found that PACAP treatment resulted in an increase in TrkA tyrosine kinase activity in PC12 cells and TrkB activity in hippocampal neurons. The activation of TrkA receptors by PACAP required at least 1 h of treatment and did not involve binding to nerve growth factor. Moreover, PACAP induced an increase in activated Akt through a Trk-dependent mechanism that resulted in increased cell survival after trophic factor withdrawal. The increases in Trk and Akt were blocked by K252a, an inhibitor of Trk receptor activity. In addition, transactivation of TrkA receptors by PACAP could be inhibited with PP1, an inhibitor of Src family kinases or BAPTA/AM, (1,2-bis(2-aminophe-noxy)ethane- N ,

phic factor families. Each represents small families of proteins that are essential for the development of the vertebrate nervous system. The effects of these factors on cell survival, differentiation, and cell death events depend upon binding to transmembrane receptors and stimulation of downstream signaling cascades characterized by increased protein tyrosine phosphorylation. Neurotrophins utilize Trk receptor tyrosine kinases, and glial-derived neurotrophic factor family members signal through the c-Ret receptor tyrosine kinase, whereas CNTF utilizes JAK tyrosine kinase activity linked to the CNTF receptor complex (1).
It has become increasingly apparent that many other secreted proteins are also capable of providing neuroprotective or neurotrophic actions. The pituitary adenylate cyclase-activating polypeptide (PACAP) belongs to a family of peptides, including secretin, glucagon, and vasoactive intestinal peptide (VIP) (2,3). PACAP exists in two active forms of 38 (PACAP38) and 27 amino acids (PACAP27). Although PACAP was originally isolated from the hypothalamus, it is distributed throughout the central nervous system, including the hippocampus, olfactory bulb, and cerebral cortex (4 -9).
The biological effects of PACAP are mediated by seven-transmembrane-spanning receptors. Two G protein-coupled receptors exist for PACAP. The type 1 PACAP receptor, PAC1, displays high specificity for PACAP (10), whereas type II PACAP receptors, VPAC1 and VPAC2, recognize VIP as well as PACAP with equal affinities (11,12). The PAC1 receptor is expressed in many areas of the developing and adult nervous system (7,(13)(14)(15)(16). Prominent expression of PAC1 has been observed in the cerebral cortex, cerebellum, hippocampus, and basal forebrain.
PACAP also acts as a mediator in injury responses in the nervous system. Both PACAP and the PAC1 receptor are induced after cortical lesion in the hippocampal subregions (9) and following axotomy of motor neurons in facial nerve (28). Brain damage caused by ischemic injury can be reversed following intravenous administration of PACAP (29,30). In addition, neuronal cell death induced by excess glutamate (24) or 6-hydroxydopamine treatment of dopaminergic neurons (22) could be reversed with low concentrations of PACAP. These observations indicate that PACAP may be neuroprotective for neurons following axotomy, injury, or cytotoxic insults.
The ability of PACAP to provide a neurotrophic effect upon populations of neurons that are dependent upon neurotrophins such as NGF and BDNF suggests that PACAP may also use similar signal transduction mechanisms. Indeed, PACAP increases the survival of sensory and sympathetic neurons to the same extent as NGF (26,27) and rescues NGF-dependent cholinergic neurons in vivo after fimbria-fornix lesion (23). Here we have explored the possibility that PACAP induces neurotrophin signaling pathways that lead to trophic effects. Surprisingly, we find that the Trk tyrosine kinase receptor is activated as a result of PACAP treatment. The activation occurred in the absence of neurotrophin binding and was observed in PC12 cells, as well as primary cultures of hippocampal neurons. The activation of Trk receptors by PACAP provides a mechanism to account for the neuroprotective effects by this neuropeptide.

EXPERIMENTAL PROCEDURES
Materials-PACAP38, PACAP27, and VIP were purchased from Peninsula Laboratories (San Carlos, CA), PP1 was from Alexis Biochemicals (San Diego, CA), and K252a, A23187, MK-801, and BAPTA/AM were obtained from Calbiochem. NGF was obtained from Harlan Bioproducts (Indianapolis, IN) and BDNF from PeproTech (Rocky Hill, NJ). All other compounds were from Sigma Chemical Co. Anti-pan-Trk rabbit antiserum raised against the C-terminal region of the Trk receptor was from Barbara Hempstead (#45, Weill Medical College, New York, NY) and from Santa Cruz Biotechnology (B-3). Anti-NGF antibody was obtained from Chemicon. Anti-phosphotyrosine and anti-Akt antibodies were from Santa Cruz Biotechnology. Anti-Shc antibodies were from Upstate Biotechnology and Transduction Laboratories. Anti-phospho-Akt, anti-MAPK, anti-phospho-MAPK, and anti-phosphoTrk (674/675) antibodies were from New England BioLabs.
Immunoprecipitation and Immunoblotting-PC12 (615) cells (31), were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 5% horse serum, supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM glutamine plus 200 g/ml G418. Cells were placed in serum-free media overnight before experiments. Cell lysates from PC12 (615) cells or hippocampal cells were incubated in lysis buffer (1% Nonidet P-40) for 4 h to overnight at 4°C with anti-pan-Trk polyclonal antibody followed by incubation with protein A-Sepharose beads. Equivalent amounts of protein were analyzed for each condition. The beads were washed four times with lysis buffer, and the immune complexes were boiled in SDS-sample buffer and loaded on SDS-PAGE gels for immunoblot analysis. The immunoreactive protein bands were detected by enhanced chemiluminescence (Amersham Biosciences, Inc.).
In Vitro Kinase Assay-Following the indicated treatments, PC12 (615) cells were washed once with ice-cold PBS and lysed in a buffer containing 1% Nonidet P-40, 10 mM Tris, pH 8.0, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 20 mM ␤-glycerophosphate, 10 mM NaF, 1 mM sodium orthovanadate, 2 g/ml aprotinin, 1 g/ml leupeptin, and 25 g/ml phenylmethylsulfonyl fluoride. Clarified lysates were immunoprecipitated with anti-pan-Trk antibodies for 2 h and then incubated with protein G-agarose for 1 h. The beads were washed twice with lysis buffer, once with a high salt buffer (10 mM Tris, pH 7.5, plus 750 mM NaCl), and twice with wash buffer (10 mM Tris, pH 7.5, plus 10 mM MnCl 2 ). Immunoprecipitates were then incubated with kinase assay solution containing 10 mM Tris, pH 7.5, 10 mM MnCl 2 , 5 M ATP, 10 Ci of [␥-32 P]ATP, and 1 g of GST-TrkA(JM) protein for 20 min at room temperature. Production of recombinant GST-TrkA(JM), a fusion protein containing 75 amino acids of the rat TrkA juxtamembrane region (aa 448 -552), has been previously described (32). Kinase reactions were terminated by adding Laemmli buffer followed by boiling. Proteins were resolved by SDS-PAGE (10%) and then transferred onto polyvinylidene difluoride (Millipore). Phosphorylation of GST-TrkA(JM) and immunoprecipitated TrkA was analyzed by PhosphorImager analysis, and the amount of TrkA protein in the same sample was visualized by immunoblotting with anti-panTrk antibodies. Both the extent of phosphorylation and the amount of Trk protein were quanti-fied by densitometric analysis with ImageQuaNT. Intensity of the bands was quantified relative to the untreated samples.

125
I-NGF Binding Analysis-For equilibrium binding studies, 125 I-NGF was prepared as described previously (33). PC12 cells stably overexpressing TrkA (2 ϫ 10 5 cells) and 3T3 cells expressing TrkA (2 ϫ 10 5 cells) were incubated with 125 I-NGF in the absence and presence of PACAP38 (1 M) for 30 min at 25°C. The cells were then washed twice with PBS, and 125 I-NGF was stripped with an acid solution (0.2 M acetic acid, 0.5 M NaCl). Nonspecific binding was assessed by adding unlabeled NGF at a final concentration of 1000 ng/ml and represented Ͻ20% of total binding. Specific binding was defined as total binding minus nonspecific binding. All conditions were carried out in triplicate, and the S.E. was calculated.
Hippocampal Cell Cultures-Dissociated primary cultures of hippocampal neurons from embryonic day 18 (E18) rats were prepared from timed-pregnant Sprague-Dawley rats as described previously (34). Fetuses were removed under sterile conditions and kept in PBS on ice for microscopic dissection of the hippocampus. The meninges were removed, and the tissue was placed in Neurobasal media (Invitrogen). The tissue was briefly minced with fine forceps and then triturated with a fire-polished Pasteur pipette. Cells were counted and plated on culture wells coated with 0.01 mg/ml poly-D-lysine overnight in Neurobasal media containing B27 supplement and L-glutamine (0.5 mM). Experiments were conducted 7-10 days after plating. Prior to treatment, cells were placed in serum-free media for 5 h. MK-801 (1 mM) was added to the serum-free media to decrease the contribution of N-methyl-D-aspartate-mediated cell death.
Cell Death Assay-Hippocampal neurons were maintained in Neurobasal media containing B27 supplement and 0.5 mM L-glutamine for 10 days. B27 was then removed, and PACAP38 (10 -100 nM) or BDNF (100 ng/ml) or IGF-1 (100 ng/ml) was added to the media. MK-801 (1 M) was added to all conditions to decrease the contribution of Nmethyl-D-aspartate-mediated cell death. After 48 h, cell death was assessed by measuring lactate dehydrogenase (LDH) released from injured cells into the media by using the Cytox 96 cytotoxicity assay kit (Promega). LDH values were normalized by subtracting the LDH released by cells maintained in BDNF (100 ng/ml) and scaled to complete killing (ϭ100%) reference induced with treatment with 30 M A23187 for 24 h, a condition that resulted in cell death of all cells (35).

RESULTS
PACAP is produced in two forms, a predominant 38-amino acid (PACAP38) and a 27-amino acid species (PACAP27). These two forms of PACAP are equally effective in elevating cAMP levels in PC12 cells (36). These effects are mediated by PAC1 receptors expressed on PC12 cells. To test whether PACAP exerts any downstream signaling effects through tyrosine phosphorylation, we evaluated the activity of TrkA receptors. After treatment with PACAP, TrkA receptors were immunoprecipitated from PC12 cell lysates and then probed with an anti-phosphotyrosine antibody. Activated TrkA receptors of 110 and 140 kDa were observed with both PACAP38 and PACAP27 treatment of PC12 cells (Fig. 1).
Both PACAP forms increased tyrosine-phosphorylated TrkA receptors at similar nanomolar concentrations, in contrast with treatment with VIP, which required much higher (micromolar) concentrations. VIP typically displays 1000-fold lower affinity for PAC1 receptors (37). In addition, VPAC1 and VPAC2 receptors typically bind both PACAP and VIP with similar affinities (38,39). The differential dose response by PACAP and VIP in PC12 cells is consistent with the action of PAC1 receptors in mediating the effects of PACAP upon TrkA receptor activity.
A time course of PACAP action indicated that the increase in TrkA activation was slow and required at least 90 min (Fig. 2), which is considerably delayed compared with NGF treatment. The activation of TrkA receptors could be inhibited by 100 nM K252a, a well-established inhibitor of Trk tyrosine kinases. This concentration of K252a blocks NGF activation of TrkA receptors and the subsequent biological effects of neurotrophins, without affecting other receptor tyrosine kinases, such as the EGF and fibroblast growth factor (40). Furthermore, PACAP did not stimulate tyrosine phosphorylation of EGF receptors in these cells at either short (10 min) or longer time points (2-3 h), nor did it transactivate c-Ret receptors in Neuro2a-20 cells (data not shown).
To confirm that tyrosine phosphorylation of Trk receptors reflects stimulation of Trk tyrosine kinase activity, an in vitro kinase assay with immunopurified TrkA from PC12 cells was performed. PACAP was able to stimulate TrkA kinase activity by 2-fold as measured by receptor autophosphorylation (Fig.  3A). A similar increase was observed in the phosphorylation of a GST fusion protein, GST-TrkA(JM), containing the juxtamembrane region of the TrkA receptor. This substrate contains the NPQY sequence, which becomes phosphorylated upon receptor activation and serves as the docking site for the Shc adaptor protein. In addition, PACAP was shown specifically to lead to phosphorylation of the activation loop tyrosine residues of the TrkA kinase domain as determined with phospho-Trk (674/675) site-specific antibody (Fig. 3B). These results suggest that PACAP increases Trk tyrosine kinase activity, rather than simply increasing tyrosine phosphorylation of the receptor.
The effects of PACAP might be explained by a direct interaction of PACAP with the Trk receptors. However, binding experiments indicate that PACAP does not compete with NGF for binding to the TrkA receptor. No displacement of 125 I-NGF binding was observed when an excess of PACAP38 was added to PC12 cells overexpressing TrkA (Table I). Because PC12 cells express the p75 neurotrophin receptor, which also binds NGF, similar experiments were conducted in 3T3 cells expressing only TrkA receptors. Excess concentrations of PACAP38 did not displace 125 I-NGF binding to 3T3 cells expressing only TrkA (Table I).
It is also possible that PACAP treatment leads to the production of NGF by PC12 cells that could act in an autocrine fashion to stimulate TrkA receptors. This possibility was discounted by a lack of effect of anti-NGF antibody (1 g/ml) on PACAP38's activation of TrkA (Fig. 4). On the other hand, the same concentration of anti-NGF antibody was effective in blocking the effects of NGF added exogenously to PC12 cells. Taken together with the dose responses with PACAP seen above, these results are consistent with the involvement of PAC1 receptors in mediating the increase in Trk receptor activity.
Downstream Signaling-An immediate downstream consequence of Trk receptor activation is phosphorylation of the adaptor protein, Shc. PACAP treatment of PC12 cells leads to tyrosine phosphorylation of Shc in a time-dependent manner that resembles the delayed time course needed for Trk receptor activation (Fig. 5A). This event was slower than the response seen with a 10-min treatment with NGF.
After activation of PACAP, type I receptors gives rise to rapid increases in MAPK activity in PC12 cells (41) and in cerebellar granular cells (42). Consistent with these previous studies, PACAP38 activated MAPK in PC12 cells within 10 min, as assayed with a phospho-MAPK antibody (Fig. 5B). However, PD98059, an established MEK inhibitor, did not affect Trk receptor activation (data not shown). After 10 min of treatment, MAPK activity remained sustained for several hours. The prolonged activation of MAPK activity by PACAP is reminiscent of the effects of NGF upon Ras and MAPK activities (43,44). The persistent activation of MAPK also corresponds to the time course of Trk autophosphorylation seen above with PACAP. Interestingly, in contrast to Trk activity, MAPK activity was only partially inhibited by K252a. Thus, activation of MAPKs can be achieved either through PAC1 receptor signaling or through Trk receptor activation.
Another pathway influenced by receptor tyrosine kinases is phosphatidylinositol 3-kinase/Akt. The activity of Akt is widely accepted as a signaling mechanism for neuronal cell survival (45). In PC12 cells, PACAP38 was able to activate Akt as detected by a phospho-specific antibody. The time course of Akt activation was very similar to TrkA autophosphorylation induced by PACAP in that phosphorylated Akt was observed well after an hour of treatment. In contrast, NGF gave a robust increase of activated Akt after 10 min, which was not observed with 10 min of PACAP38 treatment. The increase in Akt activity was eliminated by pretreatment with 100 nM K252a (Fig.  5B). These results indicate that PACAP's activation of Akt by PACAP is Trk-dependent. This response has not been previ-ously associated with PACAP action and provides an explanation for the neuroprotective effects of PACAP.
Mechanism of Transactivation-The mechanisms by which G protein-coupled receptors are linked to the activation of receptor tyrosine kinases are incompletely understood. Previously, we found that adenosine binding to the A 2A adenosine receptor in PC12 cells also gave rise to TrkA activation (46). One activity that may mediate G protein-coupled activation of mitogenic receptor tyrosine kinases are members of the Src family kinases (47,48). To investigate whether an Src family member is involved in the activation of Trk receptors by PACAP, the inhibitor PP1 was used (49). Treatment of PC12 cells with 1 M PP1 resulted in a marked decrease in the level of tyrosine-phosphorylated TrkA elicited by PACAP (Fig. 6). Increasing concentrations of PP1 produced a progressively stronger inhibition of PACAP, without affecting NGF-induced TrkA phosphorylation. These results suggest that the regulation of TrkA activity by PACAP may be mediated by a Src family member.
An early neuronal response attributable to PACAP action is a change in cytosolic-free calcium concentration. Using fura-2 as a calcium-sensitive fluorescent dye, primary cultures of hippocampal neurons responded with increased mobilization of intracellular calcium with doses of PACAP38 in the nanomolar range (50). To assess whether a calcium-dependent step is required for transactivation of Trk receptors by PACAP signaling, PC12 cells were treated with BAPTA/AM, an intracellular calcium chelator. Treatment of PC12 cells with 20 M BAPTA/AM led to a complete inhibition of transactivation of Trk receptors by PACAP38 (Fig. 7). In contrast, an extracellular calcium chelator, EGTA, as well as a general chelator, EDTA, had no effect upon TrkA tyrosine kinase activity. All three chelators did not affect NGF phosphorylation of TrkA receptors (data not shown). These results suggest that transactivation of Trk receptors requires an intracellular calciumdependent step, in addition to intracellular tyrosine phosphorylation events.
Trophic Effects of PACAP Signaling-The distinctive increases of Trk receptor kinase and Akt activities in PC12 cells suggest that neurotrophic effects of PACAP may be explained by transactivation events. To determine if this mechanism occurs in primary neurons, we established hippocampal neuronal cultures from E18 rat embryos, a well-defined neuronal cell model. Hippocampal neurons express the TrkB receptor and respond to BDNF, its principal ligand. These neurons also express PAC1, VPAC1, and VPAC2 receptors (9, 50) and not TrkA receptors. Treatment with 10 nM PACAP38 produced an   increase in phosphorylated TrkB receptors in hippocampal neurons (Fig. 8). This response was similar to the activation of TrkB by BDNF administration, except for the longer time course by PACAP. The increase in phosphorylated TrkB receptors was also effectively blocked with 100 nM K252a. A similar concentration of VIP, however, did not activate TrkB receptors, indicating the specificity of PACAP38 effects were through the PAC1 receptor. Therefore, TrkB, as well as TrkA receptors, can be activated by signaling through PAC1 receptors. These results extend the generality of PACAP effects to primary neurons from the central nervous system (CNS).
Previous studies indicated that PACAP could prevent ischemic-induced apoptosis of hippocampal neurons (29,51). To test whether increases in Trk receptor activity are responsible for the trophic abilities of PACAP, we assessed the ability of PACAP to maintain survival of hippocampal neurons grown in the absence of BDNF (Fig. 9). Hippocampal neuron survival was promoted by either BDNF or other growth factors, such as insulin-like growth factor-1 (IGF-1). Withdrawal of BDNF or IGF-1 from hippocampal neurons produced rapid cell death. However, treatment with PACAP effectively rescued over 60% of the cells (Fig. 9). Protection from cell death of these neurons by PACAP was dose-dependent and occurred in a range used in other primary culture experiments (19, 22-24, 26, 27, 42, 52). The administration of PACAP at nanomolar concentration was able to reverse cell death in hippocampal neurons initiated by withdrawal of trophic support by BDNF.
The action of PACAP required TrkB receptor activity, because K252a (100 nM) eliminated the positive effects of PACAP under the same conditions that blocked the activation of TrkB receptors (Fig. 8). Likewise, a similar dose of K252a reversed the survival effects of BDNF but not of IGF-1 in this deprivation assay (Fig. 9), indicating specificity of this inhibitor for the TrkB receptor tyrosine kinase. The ability of PACAP to induce FIG. 5. Effect of PACAP on Shc phosphorylation, and MAPK and Akt activation. A, PC12 cells (615) were treated with PACAP38 (10 nM) for various times or NGF (100 ng/ml) for 10 min. Lysates were prepared and immunoprecipitated with anti-Shc rabbit antiserum (Upstate Biotechnology). Immunocomplexes were analyzed by immunoblotting with anti-phosphotyrosine antibody (pY99). Immunoprecipitation of Shc was then confirmed by immunoblotting of the immunocomplex with Shc antiserum (Transduction Laboratories). B, PC12 cells (615) were treated with PACAP38 (10 nM) for various times in the presence or absence of K252a (100 nM). As a positive control, NGF (5 ng/ml) was added to cells for 10 min. The cells were subsequently harvested in lysis buffer and then subjected to immunoblotting with either anti-phospho-MAPK (pMAPK) or anti-phospho-Akt (pAkt). The blot was reprobed with anti-MAPK to verify equal protein loading (middle blot). TrkB receptor activity concomitantly with this survival response suggests that previous studies describing PACAP's neurotrophic effects in hippocampal cultures, as well animal injury models involving hippocampal neurons, may have resulted from the activation of Trk neurotrophin receptors. These results therefore provide a signaling mechanism for how PACAP may lead to neuroprotection through downstream engagement of the Trk neurotrophin receptor system. DISCUSSION The mechanism by which PACAP promotes survival of neuronal cells has not been well understood. Here we report a signaling mechanism to account for the ability of PACAP to exert neuroprotective effects in neuronal cells. Through crosstalk with Trk receptor tyrosine kinases, PACAP is capable of activating the phosphatidylinositol 3-kinase/Akt cascade, resulting in a survival response in hippocampal cells. The activation of Akt by PACAP has not been previously observed. This response is similar to the effect of NGF and BDNF upon their Trk receptors, except that PACAP's action on tyrosine phosphorylation is more delayed. Interestingly, the time course of Akt activation is quite similar to the pattern of Trk autophosphorylation induced by PACAP. It should be noted that the dose and time course of PACAP are also consistent with many studies in which PACAP has been shown to prevent naturally occurring neuronal cell death. This raises a question concerning the extent of cross-talk between PACAP and neurotrophin receptors and their physiological consequences.
Neurotrophin receptors and PACAP receptors have considerable overlap in their CNS and PNS distribution. In the CNS, PACAP exerts neuroprotection in many neuronal populations found in cerebral cortex, hippocampus, and cerebellum (17,19,20,22,24,29). The majority of these neuronal populations also express either TrkB or TrkC receptors (53). In the basal forebrain, PACAP enhances the survival of cholinergic neurons that co-express TrkA and PAC1 receptors (23). In the PNS, PACAP responsiveness occurs in neurotrophin-dependent sensory populations (27), which are predominantly Trk receptorpositive. It is well established that many sensory neurons lose their dependence for different neurotrophins after development. The survival of adult neurons may rely upon other factors with trophic properties, such as PACAP. Furthermore, targeted mutations of neurotrophin factors in mice do not produce losses in neuronal populations in the CNS (54), suggesting that other factors may substitute for neurotrophins during brain and spinal cord development.
A number of examples of transactivation of mitogenic growth factor receptors in response to G protein-coupled receptor signaling have now been reported. In each case, increased tyrosine phosphorylation and dimerization of receptor tyrosine kinases occurs, followed by association of receptors with tyrosine-phosphorylated adaptor proteins and Ras-dependent activation of MAPKs (47,55). Hence, MAPKs may be activated by a direct pathway through G protein receptor signaling and a transactivation pathway involving receptor tyrosine kinases (56). The mechanisms that direct and regulate these activation events are not well understood.
In the present study, we have demonstrated that PACAP isoforms induce tyrosine phosphorylation of the NGF TrkA and the BDNF TrkB receptors in a dose-dependent manner. In particular, PACAP leads to the stimulation of Trk tyrosine kinase activity (Fig. 3). A distinctive feature of this transactivation event between PACAP's G protein-coupled receptor and the Trk receptor tyrosine kinases is the relative slow time course of activation. In contrast to the activation of EGF receptors by angiotensin, bradykinin, or isoproterenol, which occurs rapidly (5-15 min), increased Trk receptor activity by PACAP required a longer time course of more than 60 min. This was also observed in studies with adenosine, which transactivated Trk receptors through a G protein-coupled receptor mechanism (46). It is formally possible that G protein-coupled receptorstimulated tyrosine phosphorylation of Trk receptors involves autocrine production of neurotrophins, similar to what has been reported with EGF (57). However, this possibility has been discounted by the lack of effect of anti-NGF antibodies upon PACAP-dependent Trk activation in PC12 cells (Fig. 4). Therefore, in these cell systems, PACAP-induced transactivation of Trk receptors does not result via an autocrine/paracrine mechanism involving the release of NGF or BDNF proteins. In addition, it is interesting that the initial phosphorylation of Trk receptors appears to be the 110-kDa intermediate species followed by the phosphorylation of the mature 140-kDa species (Figs. 1 and 2). This suggests that PACAP signaling may initially target Trk receptors in an intracellular compartment.
Through G protein-coupled receptor signaling, PACAP can activate adenylate cyclase and stimulate cAMP formation, phospholipase C, and MAPK activities (13,35,42). We have shown that the PACAP-induced phosphorylation of the Trk receptor was inhibited by an intracellular calcium chelator, BAPTA-AM, but was not affected by an extracellular calcium chelator, EGTA. Elevation of intracellular calcium levels can stimulate a number of signaling events, including the MAPK pathway. This result raises the possibility that calcium levels may play a critical role in the transactivation of Trk receptors, as has been shown for the EGF receptor (58). In this regard, the activation of Trk receptor activity by PACAP was found to be independent of either protein kinase C activity or protein kinase A phosphorylation by the use of pharmacological inhibitors (data not shown). In addition to the dependence upon calcium mobilization, the transactivation of Trk receptors appears to involve Src tyrosine kinases. Src family nonreceptor tyrosine kinases are involved in many MAPK activation events mediated by G protein-coupled receptors (47,55). Src family members may participate in regulating the catalytic activity of Trk receptors or internalization and trafficking of Trk receptors. Growing evidence indicates that multisubunit complexes exist between G protein-coupled receptors and receptor tyrosine kinases (59). Such complexes may specify local and long range signaling in axons and cell bodies of neurons during retrograde transport of Trk receptors.
We have demonstrated a novel signaling pathway between PACAP's G protein-coupled receptor, PAC1, and the Trk neu- FIG. 9. Trophic effects of PACAP38 in hippocampal neurons deprived of BDNF. Primary cultures of E18 hippocampal neurons were prepared as described under "Experimental Procedures." Upon B27 withdrawal, BDNF (50 ng/ml), IGF-1 (100 ng/ml), or various concentrations of PACAP38 were added in the absence or presence of K252a (100 nM). LDH levels were quantitated, and percent cell death was calculated as described under "Experimental Procedures." All bars depict mean Ϯ S.E. from four independent experiments. rotrophin receptors that provides a mechanism for neuroprotection by PACAP. This cross-talk with Trk receptor tyrosine kinases may also explain other functions attributed to PACAP. For example, mutations in the Drosophila amnesiac gene, a PACAP homolog (60,61), and in the PAC1 receptor gene in mice (62) both lead to deficits in associative learning. These phenotypes are similar in nature to hippocampal learning defects found in mice with a defective TrkB receptor gene (63). It has not escaped our attention that PAC1 receptor-mediated signaling involves activation of TrkB receptors in the hippocampus. Thus, transactivation of Trk receptor tyrosine kinases by G protein-coupled receptors may therefore be a general mechanism to propagate signals for cell survival as well as synaptic plasticity.