Serotonin 5-HT1A Receptor-mediated Erk Activation Requires Calcium/Calmodulin-dependent Receptor Endocytosis*

Gregory J. Della RoccaDagger , Yurii V. Mukhin§, Maria N. Garnovskaya§parallel , Yehia Daaka, Geoffrey J. Clark**, Louis M. Luttrell, Robert J. Lefkowitz, and John R. Raymond§Dagger Dagger

From the Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, the ** NCI, National Institutes of Health, Rockville, Maryland 20857, and the § Department of Medicine (Nephrology), Medical University of South Carolina and the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, South Carolina 29425

    ABSTRACT
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Abstract
Introduction
References

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta -arrestin1 and dynamin attenuated 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.

    INTRODUCTION
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Abstract
Introduction
References

Receptors coupled to heterotrimeric guanine nucleotide-binding (G) proteins, the largest known family of cell surface receptors, mediate cellular responses to many extracellular stimuli, such as neurotransmitters, peptide hormones, odorants, and photons (1). In addition to regulating the generation of soluble second messengers, many G protein-coupled receptors mediate proliferative or differentiative signals in various cultured cell lines and tissues via mitogen-activated protein (MAP)1 kinases (2, 3). Erk1/2 MAP kinases are serine/threonine kinases involved in the activation of nuclear transcription factors. Erk1/2 activity is regulated by threonine/tyrosine phosphorylation, which is controlled by a highly conserved phosphorylation cascade. Phosphorylation of Erk1/2 is catalyzed by the MAP/Erk kinases 1 and 2 (MEK1 and MEK2) that are themselves phosphorylated and activated by MEK kinases such as raf-1 oncogene family proteins. Activation of Raf-1 occurs as a consequence of membrane translocation, which can be mediated by the GTP-bound form of the small G protein, Ras.

Like the epidermal growth factor (EGF) receptor and other receptor tyrosine kinases, many G protein-coupled receptors regulate Ras function via tyrosine phosphorylation. Several Gi-coupled receptors stimulate pertussis toxin-sensitive, Ras-dependent Erk activation through tyrosine phosphorylation of adapter proteins, such as Shc and Gab1, and membrane recruitment of the Ras guanine nucleotide exchange factor, mSos (2, 4). Shc tyrosine phosphorylation and Erk activation by Gi-coupled receptors are sensitive to inhibitors of src family nonreceptor tyrosine kinases (4, 5), suggesting a role for src kinases in mitogenic signaling by G protein-coupled receptors. In some systems, Ca2+/CAM has also been implicated in Gi- and Gq/11-coupled receptor-mediated Erk activation (6-8).

Recent data have also suggested that clathrin-mediated endocytosis plays a crucial, if poorly understood, role in Erk activation via EGF, insulin-like growth factor-1 receptors (9, 10), lysophosphatidic acid (LPA), and beta 2-adrenergic receptors (11, 12). Inhibition of beta 2-adrenergic receptor sequestration with dominant inhibitory mutants of beta -arrestin1 or dynamin impairs Erk activation with no effect on receptor-G protein coupling (12).

5-HT mediates mitogenic effects in many cell types (13, 14). In CHO-K1 cells, the 5-HT1A receptor rapidly activates Erk via a pathway that involves pertussis toxin-sensitive G protein beta gamma subunits, phosphatidylinositol 3'-kinase and src kinase, the Shc and Grb2 adapter proteins, mSos, Ras, and Raf (13, 15). Moreover, 5-HT1A receptors have been colocalized with adenylyl cyclase and G proteins in clathrin-rich brain vesicles (16). In this report, we show that 5-HT1A receptor-mediated Erk activation is sensitive to inhibitors of Ca2+/CAM and clathrin-mediated endocytosis but not to inhibitors of the known CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterases. Rather, the Ca2+/CAM dependence of Erk activation derives from a requirement for CAM in 5-HT1A receptor endocytosis. These data suggest a previously unappreciated role for CAM in the sequestration of G protein-coupled receptors and provide a mechanism for cooperativity between Ca2+/CAM and tyrosine phosphorylation in mitogenic signal transduction.

    EXPERIMENTAL PROCEDURES

Materials-- 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), phorbol 12-myristate 13-acetate (PMA), and LPA were from Sigma. Fluphenazine, W-7, EGF, cyclosporine A, FK506, A23187, and ionomycin were from Calbiochem. Basic fibroblast growth factor (bFGF) was from Upstate Biotechnology (Lake Placid, NY). ML-7, ML-9, HA1077, KN-62, KN-92, vinpocetine, and 8-methoxymethyl isobutylmethylxanthine were from Biomol (Plymouth Meeting, PA).

DNA Constructs-- The cDNAs for FLAG-tagged beta 2-adrenergic receptor and HA-tagged 5-HT1A receptor were cloned in our laboratory (17-19). Constitutively activated mutants V12-Ras and Raf-CAAX were the gifts of C. Der, and DD-MEK was the gift of R. Erikson. Constitutively activated Y530F-Src, in which the regulatory carboxyl-terminal tyrosine residue has been mutated, was prepared as described (20-22). cDNAs encoding the dominant-interfering mutants V53D-beta -arrestin1 and K44A-dynamin were the kind gift of M. Caron. The cDNA encoding the CAM-sequestering protein calspermin was the gift of A. Means. Calspermin cDNA was subcloned into the pcDNA3 vector via a NcoI-XbaI digest of pCaMPL-calsp followed by blunt-end treatment with the Klenow fragment of DNA polymerase I. The insert was placed into EcoRV-digested, calf intestinal alkaline phosphatase-treated pcDNA3. Correct orientation was verified by diagnostic restriction digests.

Cell Culture and Transfection-- HEK-293 cells were maintained in minimum essential medium with Earle's salts (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Inc.) and 50 µg/ml gentamicin (Life Technologies, Inc.) at 37 °C in a humidified 5% CO2 atmosphere. CHO-1A-27 cells (18) were maintained in Ham's F-12 nutrient mixture (Life Technologies, Inc.) supplemented with 10% FBS, 50 µg/ml gentamicin, and 400 µg/ml G418. Transfections were performed on 80-90% confluent monolayers in 100-mm dishes, using calcium phosphate coprecipitation for HEK-293 cells (23) and LipofectAMINE for CHO cells (9 µl/µg of DNA, Life Technologies, Inc.) (15). Empty pcDNA3 vector was added to transfections to keep the total mass of DNA added per dish constant within experiments. Prior to stimulation, cells were incubated overnight in serum-free medium containing 0.1% bovine serum albumin.

Erk1/2 Phosphorylation and Kinase Assay-- Stimulations were carried out at 37 °C in serum-starving medium. Monolayers were then lysed directly using 200 µl/well Laemmli sample buffer. Phosphorylation of Erk1/2 was detected by immunoblotting using rabbit phosphospecific Erk IgG (New England Biolabs) as described previously, except that the bands were visualized with Vistra ECF reagent (Amersham Pharmacia Biotech) (13, 15). Membranes were stripped and reprobed with rabbit anti-Erk2 IgG (Santa Cruz Biotechnology) to quantitate total Erk2.

Alternatively, monolayers were lysed in 500 µl/well RIPA buffer (150 mM NaCl, 50 mM Tris-Cl, pH 8.0, 10 mM EDTA, 1% v/v Nonidet P-40, 0.5% w/v sodium deoxycholate, 1 mM NaF, 1 mM sodium pyrophosphate, 100 µM NaVO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin), and then Erk2 was immunoprecipitated with rabbit anti-Erk2 IgG plus 50 µl of a 50% slurry of protein G plus/protein A-agarose (Calbiochem). Immune complexes were washed twice in ice-cold RIPA buffer and twice in kinase buffer (50 mM HEPES, pH 7.0, 10 mM MgCl2, 1 mM dithiothreitol). Phosphorylation assays were performed as described previously using myelin basic protein as the substrate (6, 13, 15). Both assays of ERK activity yielded virtually identical results.

beta 2-Adrenergic and 5-HT1A Receptor Sequestration-- Sequestration of epitope-tagged beta 2-adrenergic receptor and 5-HT1A receptor was determined as agonist-induced loss of cell surface immunofluorescence using fluorescence-assisted cell sorting (FACS) (17). Cells expressing tagged receptors were exposed to 10 µM isoproterenol (beta 2-adrenergic receptor) or 10 µM 5-HT (5-HT1A receptor) for 30 min at 37 °C before staining of cell surface receptors. Sequestration was defined as the fraction of total cell surface receptors removed from the plasma membrane following agonist treatment.

cAMP Production-- Cells were metabolically labeled with 3 µCi of [3H]adenine/ml for 6 h in medium containing 3% FBS, washed once in phosphate-buffered saline, and incubated in serum-free medium with 1 mM isobutylmethylxanthine for 30 min at 37 °C. Following a 10-min exposure to agonist, reactions were terminated by the addition of 1 ml/well ice-cold stop solution (0.1 mM cAMP, 4 nCi of [14C]cAMP/ml, 2.5% perchloric acid). Cell lysates were neutralized in KOH, and total [3H]cAMP production was assayed by anion exchange chromatography (24, 25).

    RESULTS AND DISCUSSION

Inhibitors of Ca2+/CAM Impair 5-HT1A Receptor-mediated Erk Activation in CHO-1A-27 Cells-- To examine the role of Ca2+/CAM in Gi-coupled receptor-mediated Erk activation, we employed CHO cells stably expressing the 5-HT1A receptor (CHO-1A-27 cells) (18). In this system, we have previously shown that 5-HT stimulation results in rapid, Ras-dependent, activation of Erk2, which is mediated via Gbeta gamma subunits derived from pertussis toxin-sensitive G proteins (13). As shown in Fig. 1A, stimulation of CHO-1A-27 cells with 5-HT resulted in a 7-fold stimulation of Erk2 kinase activity compared with unstimulated cells. Stimulation of endogenous fibroblast growth factor receptors and acute protein kinase C activation with PMA also induced Erk2 activation. Pretreatment with BAPTA, a cell-permeable Ca2+ sequestrant, in Ca2+-free medium inhibited 5-HT1A receptor- and bFGF-stimulated Erk2 activation, with no effect on the protein kinase C-mediated signal.


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Fig. 1.   Effect of Ca2+ sequestration and CAM antagonism on 5-HT1A receptor-mediated Erk activation. A, CHO-1A-27 cells were treated either with vehicle (control) or with BAPTA (25 µM). Cells were then stimulated for 5 min with 5-HT (10 µM), bFGF (10 ng/ml), or PMA (1 µM) prior to determination of Erk activity. Values shown represent means ± S.E. from four separate experiments each performed in duplicate. B, CHO-1A-27 cells were pretreated for 15 min with vehicle (control), W-7 (50 µM), or fluphenazine (Flu, 10 µM). Cells were then stimulated with 5-HT (10 µM), bFGF (10 ng/ml), or PMA (1 µM) for 5 min prior to determination of Erk activity. Values shown represent means ± S.E. from eight separate experiments, each performed in duplicate. C, HEK-293 cells were cotransfected with cDNA encoding HA-tagged 5-HT1A receptor (1 µg/dish) plus either calspermin cDNA (5 µg/dish) or empty vector (control, 5 µg/dish). After 24 h of serum starvation, cells were stimulated with 5-HT (10 µM), EGF (10 ng/ml), or PMA (1 µM) prior to determination of Erk activity. Values shown represent means ± S.E. from two separate experiments, each performed in duplicate. Data for all three panels are expressed as -fold Erk activity in which the values in unstimulated cells dagger were defined as 1.0. NS, not stimulated. dagger , indicates p < 0.05; *, indicates p < 0.01 versus agonist alone (black bars). Reverse Bonferroni correction was used.

To determine whether CAM activity is required for receptor-mediated Erk activation, CHO-1A-27 cells were pretreated with the pharmacologically distinct CAM antagonists, fluphenazine and W-7. As shown in Fig. 1B, both agents impaired 5-HT- and bFGF-stimulated Erk2 activation. To confirm the specificity of the fluphenazine and W-7 effects, the experiments were repeated in HEK-293 cells transiently coexpressing the 5-HT1A receptor with the CAM-binding protein calspermin (26), which has been shown to function as an intracellular CAM sequestrant.2 As shown in Fig. 1C, calspermin expression attenuated Erk1/2 phosphorylation induced by 5-HT1A or endogenous EGF receptors. Neither isoproterenol-stimulated intracellular cAMP production nor 5-HT- and LPA-induced inhibition of forskolin-stimulated cAMP production was affected by W-7 or fluphenazine, indicating that the CAM antagonists had no effect on receptor-G protein coupling (data not shown).

Ca2+/CAM Antagonists Impair 5-HT1A Receptor-mediated Erk Activation at a Point in the Pathway Downstream of Ras Activation-- In many systems, the pathways of Gi-coupled receptor- and receptor tyrosine kinase-mediated Erk activation converge upstream of Ras activation (27). The finding that CAM antagonists affected both receptor tyrosine kinase- and 5-HT1A receptor-mediated Erk activation suggested that Ca2+/CAM was involved in regulating a common component of the pathway. To determine the point in the signaling cascade at which Ca2+/CAM is required, we assayed the effects of fluphenazine and W-7 on Erk activation via constitutively active mutants of Src, Ras, Raf, and MEK. As shown in Fig. 2, the constitutively activated proteins Y530F-Src, V12-Ras, Raf-CAAX, or DD-MEK were sufficient to induce chronic Erk activation in transiently transfected CHO-1A-27 cells. Fluphenazine or W-7 significantly inhibited Y530F-Src- and V12-Ras-induced Erk2 activation, without affecting Raf-CAAX or DD-MEK. Because the Ca2+/CAM inhibitor-induced blockade can be "bypassed" by activated Raf and MEK but not by activated Src and Ras, these data suggest that Ca2+/CAM is required at a point in the signaling cascade at the level of, or downstream of Ras activation, but upstream of Raf and MEK.


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Fig. 2.   Effect of CAM antagonists on stimulation of Erk1/2 phosphorylation in HEK-293 cells by constitutively activated mutants of Src, Ras, Raf, and MEK. HEK-293 cells were transiently transfected with cDNA encoding Y530F-Src (5 µg/dish), V12-Ras (5 µg/dish), Raf-CAAX (5 µg/dish), MEK1-DD (5 µg/dish), or empty vector (5 µg/dish). Erk1 activity in these cells was determined after 24 h of serum starvation. Data are expressed as -fold Erk activity in which the values in unstimulated cells were defined as 1.0. Values shown represent means ± S.E. from five separate experiments, each performed in duplicate. *, indicates p < 0.01 versus agonist alone (black bars). Reverse Bonferroni correction was used.

Clathrin-mediated Endocytosis Is Required for 5-HT1A Receptor-mediated Erk Activation-- A requirement for clathrin-mediated endocytosis has recently been demonstrated for activation of the Erk cascade via several receptors, including the EGF (9) and insulin-like growth factor 1 (10) receptor tyrosine kinases, and the LPA (11) and beta 2-adrenergic (12) G protein-coupled receptors. Specific inhibitors of G protein-coupled receptor endocytosis, including dominant negative mutants of beta -arrestin1 and dynamin, inhibit beta 2-adrenergic receptor-mediated Erk activation. beta 2-Adrenergic receptor stimulation results in the appearance of both receptor and Raf kinase in a clathrin-rich endocytic vesicle compartment (12). These data suggest that an endocytic process may be required to achieve activation of MEK and Erk kinases following the formation of an activated Ras-Raf complex on the plasma membrane.

To determine whether receptor endocytosis was required for 5-HT1A receptor-mediated Erk activation, we examined the effects of dominant negative K44A-dynamin1 or V53D-beta -arrestin1, a beta -arrestin1 mutant that disrupts homologous desensitization and internalization of adrenergic receptors (28), on 5-HT-stimulated Erk1/2 phosphorylation in transiently transfected HEK-293 cells. Fig. 3 shows that both V53D-beta -arrestin1 and K44A-dynamin1 attenuated 5-HT-stimulated Erk1/2 activation by the 5-HT1A receptor. EGF receptor-induced Erk1/2 activation was sensitive to expression of K44A-dynamin1, in accordance with previously published findings (9), but not to expression of V53D-beta -arrestin1.


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Fig. 3.   Effect of endocytosis inhibitors on 5-HT- and EGF-stimulated Erk1/2 activation in HEK-293 cells. Cells were transiently transfected with cDNA encoding the HA-tagged 5-HT1A receptor (1 µg/dish) and cDNA encoding V53D-beta -arrestin1 (5 µg/dish), K44A-dynamin1 (5 µg/dish), or empty vector (control, 5 µg/dish). Serum-starved cells were stimulated with 5-HT (10 µM) or EGF (10 ng/ml) for 5 min before determination of Erk activity. Data are expressed as -fold Erk activity in which the values in unstimulated cells were defined as 1.0. Values shown represent means ± S.E. from three separate experiments each performed in duplicate. NS, not stimulated. dagger , indicates p < 0.05; *, indicates p < 0.01 versus agonist alone (black bars). Reverse Bonferroni correction was used.

Ca2+/CAM Antagonists Impair 5-HT1A Receptor-mediated Erk Activation by Interfering with Receptor Endocytosis-- Ca2+/CAM regulates multiple enzymes, including CAM-dependent protein kinases, myosin light chain kinase, protein phosphatase 2B, and CAM-sensitive phosphodiesterases. In addition, CAM is known to bind to neuronal clathrin light chains (29), to interact with the actin microfilament attachment protein, fodrin, a member of the spectrin family of membrane proteins (30, 31), and to regulate the activity of the G protein receptor kinases GRK5 and -6 (32-34). CAM also plays an as yet undefined role in endocytic events (35-37) and is important for endocytosis in adrenal chromaffin cells (38).

To test whether known enzymatic effectors of CAM participate in 5-HT1A receptor-mediated Erk activation, pharmacologic inhibitors of several CAM effectors were assayed for the ability to antagonize 5-HT-stimulated Erk2 activation in CHO-1A-27 cells. Preincubation for 30 min with the myosin light chain kinase inhibitors ML-7 (20 µM), ML-9 (50 µM), and HA1077 (50 µM); the protein phosphatase PP2B inhibitors cyclosporine A (1 µM) and FK506 (1 µM); the CAM-dependent protein kinase II and IV inhibitors KN-62 (10 µM) and KN-92 (10 µM); and the CAM-sensitive phosphodiesterase type I inhibitors vinpocetine (50 µM) and 8-methoxymethyl isobutylmethylxanthine (20 µM) had no effect upon 5-HT1A receptor-stimulated Erk activation (data not shown).

Because 5-HT1A receptor endocytosis is an obligate step in the Erk activation cascade, we tested the hypothesis that Ca2+/CAM is involved in receptor endocytosis. Agonist-induced sequestration of HA-tagged 5-HT1A and FLAG-tagged beta 2-adrenergic receptors was determined in the presence of fluphenazine, W-7, or hypertonic sucrose. As shown in Fig. 4, both antagonists of Ca2+/CAM impaired the sequestration of 5-HT1A receptors in transiently transfected HEK-293 cells. These results were duplicated for the beta 2-adrenergic receptor (data not shown). The effect of the CAM inhibitors was comparable with that of preincubation in hypertonic medium and expression of K44A-dynamin1 (data not shown), both known inhibitors of clathrin-mediated endocytosis. Coexpression of calspermin with epitope-tagged 5-HT1A receptors also resulted in similar abrogation of agonist-dependent receptor internalization (data not shown).


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Fig. 4.   Effect of CAM antagonists and hypertonic sucrose on agonist-dependent sequestration of the 5-HT1A receptor in HEK-293 cells. Cells were transiently transfected with cDNA encoding the HA-tagged 5-HT1A receptor (1 µg/dish). After 3 h of serum starvation, cells were pretreated with vehicle, hypertonic sucrose (450 mM), fluphenazine (Flu, 10 µM), or W-7 (50 µM) for 15 min. Cells were then stimulated with 5-HT (10 µM) for 35 min prior to determination of HA-5-HT1A receptor cell surface expression by fluorescence-assisted cell sorting. Data are expressed as percent of cell surface receptors expressed in comparison with unstimulated cells. Sequestration was calculated as the percent difference in cell surface fluorescence when compared with unstimulated cells. Values shown represent means ± S.E. from three separate experiments each performed in triplicate. dagger , indicates p < 0.05; *, indicates p < 0.01 versus 5-HT alone (black bar). Reverse Bonferroni correction was used.

Several authors have postulated a role for Ca2+/CAM in the activation of the Erk cascade. Both alpha -adrenergic receptor-mediated Erk activation in HEK-293 cells (6) and angiotensin receptor-mediated Erk activation in vascular smooth muscle cells (8) are sensitive to CAM inhibitors. Simultaneous expression of activated mutants of CaM kinase IV and CaM kinase kinase is sufficient to induce weak activation of Erk in NG-108 cells (39). We find that antagonism of Ca2+/CAM using either pharmacologic inhibitors or the calcium sequestrant protein calspermin disrupts both Erk1/2 activation and agonist-induced endocytosis of the 5-HT1A receptor. The effects of CAM inhibition are not mimicked by pharmacologic inhibition of CAM effectors, including CaM kinase IV. Rather, our data indicate that the effects of CAM antagonists on receptor endocytosis are sufficient to account for the observed inhibition of Erk activation. However, because CAM inhibitors were somewhat more effective in preventing endocytosis than in blocking Erk activation, it is possible that there is an additional endocytosis-independent Erk activation pathway.

The mechanism whereby endocytosis of G protein-coupled receptors contributes to activation of the Erk cascade is unclear. The appearance of both Raf and beta 2-adrenergic receptors in a clathrin-rich light vesicle fraction following agonist exposure suggests that the endocytic process may be required to transduce signals between an activated Ras-Raf complex on the plasma membrane and the MEK and Erk kinases in the cytosol (12). Our finding that CAM antagonists inhibit Erk activation mediated by constitutively active mutants of Src and Ras, but not Raf and MEK, supports such a model by localizing the putative endocytosis-dependent step in the signaling cascade downstream of Ras activation. CAM has been shown to bind to clathrin light (29, 41) and heavy chains (42) and to regulate vesicle recycling by influencing vesicle tubulation (43) and recruitment of clathrin to ligand-receptor complexes (44). Thus, CAM inhibitors could prevent Erk activation by 1) preventing recruitment of receptor-ligand complexes to clathrin, and/or 2) preventing successful activation of Raf by Ras, and/or 3) preventing tubulation and vesicle recycling. Certainly, other mechanisms might also be possible.

The finding that 5-HT1A receptor-mediated activation of the Erk cascade is dependent upon receptor endocytosis indicates a novel dimension in 5-HT receptor signaling. Endocytosis of G protein-coupled receptors has previously been viewed as a consequence of receptor desensitization (40), i.e. uncoupling of receptor from G protein because of G protein-coupled receptor kinase phosphorylation and arrestin binding. Our data suggest that the processes that terminate 5-HT1A receptor coupling to G proteins simultaneously contribute to the transduction of Ras-dependent signals.

    ACKNOWLEDGEMENTS

We thank D. Addison and M. Holben for excellent secretarial assistance. We thank A. R. Means for the gifts of calspermin cDNA and anti-calspermin antibody, and insightful discussion.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants DK52448 (to J. R. R.), HL16037 (to R. J. L.), and DK02352 and DK55524 (to L. M. L.) and by the Medical Research Service of the Department of Veterans Affairs (to J. R. R. and M. N. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by National Institutes of Health Medical Scientist Training Program Grant T32GM-07171.

Recipient of a fellowship from the American Heart Association.

parallel Recipient of a type II merit award from the Department of Veterans Affairs.

Dagger Dagger To whom correspondence should be addressed: 829 Clinical Sciences Bldg., Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Fax: 843-792-8399; E-mail: raymondj{at}musc.edu.

    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; MEK, mitogen and extracellular signal-regulated kinase; EGF, epidermal growth factor; CAM, calmodulin; LPA, lysophosphatidic acid; CHO, Chinese hamster ovary; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PMA, phorbol 12-myristate 13-acetate; bFGF, basic fibroblast growth factor; HA, hemagglutinin; FBS, fetal bovine serum.

2 A. R. Means, personal communication.

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