Activation In Vitro of Somatostatin Receptor Subtypes 2, 3, or 4 Stimulates Protein Tyrosine Phosphatase Activity in Membranes from Transfected Ras-Transformed NIH 3T3 Cells: Coexpression with Catalytically Inactive SHP-2 Blocks Responsiveness

Dean B. Reardon, Paul Dent, Steven L. Wood, Ting Kong and Thomas W. Sturgill

Howard Hughes Medical Institute and the Center for Cell Signaling Departments of Internal Medicine and Pharmacology University of Virginia Charlottesville, Virginia 22908


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Somatostatin receptors (sstr) subtypes 1–5 were transiently expressed in NIH 3T3 cells stably transformed with Ha-RasG12V to assess the ability of each receptor to stimulate protein tyrosine phosphatase (PTPase) activity in vitro. Treatment of membranes from sstr2-, sstr3-, or sstr4-expressing cells with somatostatin-14 plus guanyl-5'-yl imidodiphosphate (GMPPNP) increased PTPase activity, and this stimulation was pertussis toxin-sensitive. Somatostatin alone, GMPPNP alone, or somatostatin plus GDP were ineffective under these conditions. sstr1 and sstr5 failed to increase PTPase activity although both receptors were expressed, as assessed by appearance of high-affinity binding sites for [125I-Tyr11]somatostatin-14. Somatostatin plus GMPPNP stimulated PTPase activity in vitro when sstr2 was coexpressed with wild type PTP1B or a Cys to Ser (C/S), catalytically inactive PTP1B or with wild type SH2-domain containing PTPase SHP-2. However, coexpression with catalytically inactive C/S SHP-2 abrogated this response. Thus, three of the five cloned sstr’s can couple to activate PTPase in this cellular background. Abrogation of the response by C/S SHP-2 strongly suggests, but does not prove, a role for SHP-2 in the mechanism.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Somatostatin is a cyclic neuroendocrine peptide that mediates diverse physiological actions in the central nervous system and in peripheral tissues. Originally discovered as GH release inhibitory factor and isolated from the hypothalamus, somatostatin is now understood to function not only as an endocrine factor or neurotransmitter, but also as a paracrine or autocrine factor (1, 2). The diversity of somatostatin’s actions arise in part from cell type-specific expression of five distinct somatostatin receptors designated sstr1-5, all structurally typical of G protein-coupled receptors, and each capable of coupling to multiple effectors by specific activation of subsets of pertussis toxin-sensitive G proteins (3, 4).

Somatostatin and selected analogs have antiproliferative effects in vivo and in vitro that, if better understood, could foster development of new cancer therapies (5). Addition of somatostatin inhibits proliferation of several cell lines in culture (6). Removal of somatostatin from cultures of mixed splenic lymphocytes by expression of an antisense oligonucleotide, or from cultures of NIH 3T3 cells expressing sstr2 by addition of antisomatostatin antibody, enhances proliferation (1, 2). These observations and others indicate a direct effect of somatostatin to inhibit cellular proliferation.

Activation of a membrane-associated protein tyrosine phosphatase (PTPase) has been studied as one possible mechanism for the growth inhibition. Schally and co-workers (7) discovered that somatostatin stimulated a PTPase in plasma membranes from MIA PaCa-2 cells (7). The stimulated PTPase dephosphorylated either an endogenous substrate, epidermal growth factor receptors 32P-labeled on tyrosine by autophosphorylation, or an exogenous substrate, [32P]pTyr-histone. Stork and co-workers (8) implicated G proteins in the activation mechanism by demonstrating that PTPase activation by either guanyl-5'-yl imidodiphosphate (GMPPNP) or somatostatin was blocked by pertussis toxin.

More recently, we demonstrated that GMPPNP stimulated a PTPase in membranes from NIH 3T3 cells transformed with oncogenic Ha-Ras (9). PTPase activation appeared to be mediated by G{alpha}i/o-subunits for two reasons. G{alpha}i/o, but not Gß{gamma}, subunits copurified with a stimulated PTPase from GMPPNP-treated membranes after solubilization and Superose 6 chromatography. Activated G{alpha}i/o-subunits, but not Gß{gamma}-subunits, reconstituted PTPase activity upon addition to fractions from control membranes. To test whether somatostatin receptors could couple to these G proteins to stimulate PTPase activity, we transiently expressed human somatostatin receptor 3 (sstr3) in these cells (10). Somatostatin plus GMPPNP activated a PTPase in membrane preparations from these transfected cells, indicating that sstr3 is capable of stimulating this effector in this background.

The number and identity of PTPases regulated, directly or indirectly, by G proteins is unknown, but recent reports suggest that the Src homology 2 (SH2) domain-containing PTPase SHP-1 (11) may be modulated by somatostatin (2, 12, 13). PTPase activity and a protein band immunoreactive to an anti-SHP-1 antibody were circumstantially present in partially purified preparations of somatostatin receptors from rat acinar membranes (12); this anti-SHP-1 antibody would likely not distinguish SHP-1 from SHP-2. Stable expression of sstr2 in NIH 3T3 cells increases specific cellular content of SHP-1 mRNA and protein (2). SHP-1 activity was also increased 3- to 4-fold in subconfluent sstr2-expressing cells growing in complete medium, assayed by specific immune complex assay (2). More recently, somatostatin analog SMS 201–995 (Octreotide) was shown to cause translocation of SHP-1 to the particulate fraction (100,000 Xg) and reciprocal depletion from the supernatant fraction prepared from MCF-7 breast carcinoma cells (13). Octreotide also increased PTPase activity in the particulate fraction, and the majority was recovered with anti-SHP-1 antibody (13). These effects of Octreotide were maximal at 4 h and were specific for SHP-1 and not SHP-2 (13).

Here, we extend our investigations by testing the ability of each of the five cloned somatostatin receptors to promote PTPase activity in membranes from Ras-transformed NIH 3T3 cells. In addition, we tested whether coexpression of a dominant-negative mutant of SHP-2 could interfere with PTPase activation in vitro. Our findings raise some interesting mechanistic possibilities.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Transient Expression of sstr2A, sstr3, and sstr4 Confers PTPase Activation by Somatostatin and GMPPNP
Somatostatin receptors types 1–5 were tested for qualitative ability to stimulate PTPase activity in vitro. The rationale used deserves emphasis. The different receptors were transiently expressed in NIH 3T3 cells stably transformed with Ha-RasG12V. Somatostatin receptors are not detectably expressed in this cellular background without transient or stable expression of sstr1-5 (2, 10). Membranes were isolated by flotation upon a 39% (wt/vol) sucrose cushion and washed by dilution and pelleting. Portions of the membranes were then treated with combinations of guanine nucleotides and somatostatin, pelleted, and assayed for PTPase activity using [[32P]pTyr]RCM-lysozyme (9) as substrate. It was practical to study no more than two receptors at a time. Thus, determination of whether sstr1, sstr2A, sstr4, or sstr5 were capable of stimulating PTPase activity was always made alongside sstr3 as a positive control.

We previously chose sstr3 for study (10) because its third intracellular loop has a small insertion that makes it distinct from the other cloned somatostatin receptors (3). The third intracellular loop of G protein-coupled receptors is often important in specific binding and activation of G proteins (14). Activation of sstr3 stimulated PTPase activity in vitro (Fig. 1AGo), consistent with our previous report (10). Treatment of membranes with somatostatin or somatostatin plus GDP did not significantly alter PTPase activity in comparison to GDP. GMPPNP alone usually caused no significant increase in PTPase activity under these conditions. However, combined treatment with somatostatin plus GMPPNP reproducibly increased PTPase activity 1.5- to 1.8-fold in comparison to GDP. No stimulation occurred using membranes from cells transfected with a control plasmid. Although several factors (transfection efficiencies < 100%; a background of nonstimulated PTPases; nonoptimal activation conditions) likely contribute to make the fold stimulation small, the effect was sufficiently large to allow qualitative determination of the capacity of sstr1-5 to stimulate PTPase activity when studied in multiple experiments.



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Figure 1. Activation of Somatostatin Receptor Subtypes 2, 3, and 4 Stimulates Membrane-Associated PTPase Activity in Vitro

Panel A, Membranes were isolated from Ras-transformed NIH 3T3 cells expressing specific sstr subtypes, and assayed (2 µg total protein) for PTPase activity after treatment (see Materials and Methods) for 10 min at 37 C with GDP, GDP plus somatostatin, GMPPNP, GMPPNP plus somatostatin, or with somatostatin alone (final concentrations: guanine nucleotides, 0.1 mM; somatostatin-14, 1 µM). Ordinate: PTPase activity, expressed as percent release of total 32P in the assay. Data are the averages of the mean of triplicate determinations of n = 3 experiments. Bars, SD. SS14, Somatosta tin-14. Panel B, Binding of [125I-Tyr11]-somatostatin-14 to membranes (200 µg total protein) in the presence (0.1 µM) or absence of unlabeled somatostatin-14 (see Materials and Methods). Typical data from one experiment are shown.

 
Transient expression of sstr2A or sstr4 as well as sstr3 promoted stimulation of PTPase by somatostatin plus GMPPNP in vitro (Fig. 1AGo). Activation of PTPase by somatostatin plus GMPPNP correlated with induction of high-affinity binding sites for [125I-Tyr11]somatostatin-14 (Fig. 1BGo). No detectable stimulation by somatostatin plus GMPPNP occurred when cells were transfected with a control plasmid (data not shown and Ref. 10). PTPase activation by somatostatin was blocked by prior overnight treatment of the transfected cells with pertussis toxin (data not shown), indicating dependence upon Gi/o, in agreement with the study of Pan et al. (8).

Transient expression of sstr1 or sstr5 did not promote in vitro PTPase activation by somatostatin in multiple experiments in which expression of sstr3 was studied in parallel as a positive control (Fig. 2Go). This inability of sstr1 and sstr5 to support PTPase activation was not merely due to failure of receptor expression because membranes from sstr1- and sstr5-expressing cells contained high-affinity binding sites for the radioligand (Fig. 2Go).



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Figure 2. Somatostatin Receptor Subtypes 1 and 5 Do Not Couple to PTPase Activation in Vitro in Membranes Isolated from Receptor-Transfected Ras-Transformed Cells

Panel A, PTPase activity of membranes treated in vitro as in Fig. 1AGo. Data are from n = 3 experiments. Panel B, Binding of [125I-Tyr11]-somatostatin-14. Data are from a typical experiment.

 
Coexpression of Catalytically Inactive SHP-2 Abrogates Stimulation of PTPase Activity by Somatostatin
One motivation for study of SH2 domain- containing PTPases has been mentioned: the suggestions in the somatostatin literature that SHP-1 is involved (2, 12, 13). A second motivation was provided by reports that document an inhibition of insulin-stimulated mitogen-activated protein kinase by expression of catalytically inactive SHP-2 (15, 16) and an enhancement of epidermal growth factor-stimulated mitogen-activated protein kinase by expression of wild type (wt) SHP-1 (17). Genetic evidence from Drosophila also indicates that the SH2 domain containing phosphatase corkscrew (CSW) is required for signaling by Ras and Raf (18).

pCMV constructs for expression of active and inactive SHP-2 (15), identical except for absence (wt SHP-2) or presence (C/S SHP-2) of an inactivating Cys to Ser mutation, were obtained from J. Pessin (University of Iowa, Iowa City, IA) (15). As controls, we used pCMV constructs for expression of active (wt) and inactive (C/S) PTP1B (15). The total amount of pCMV DNA transfected per dish from any source was held constant in all coexpression experiments to be described.

We studied effects of phosphatase coexpression on sstr2 because the earlier reports suggesting a link between SH2 domain-containing phosphatases used sstr2 (2, 12, 16). Somatostatin stimulated PTPase activity in membranes prepared from cells transfected with pCMV-sstr2A (Fig. 3Go). Somatostatin also stimulated PTPase activity in membranes cotransfected with pCMV constructs for PTP1B or C/S PTP1B, although the magnitude of effect was reduced to a similar extent in each case (Fig. 3Go). This reduction may be due to untoward competition for transcription or translation factors in the instance where two proteins are being expressed. Cotransfection with a pCMV construct for wt SHP-2 also reduced the magnitude of PTPase stimulation, but did not abrogate the somatostatin response (Fig. 4Go).



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Figure 3. Somatostatin Receptor 2 Activates PTPase in Vitro when Coexpressed with Either Wild Type PTP1B or Catalytically Inactive C/S PTP1B

Membranes (2 µg) were assayed for PTPase activity after treatment with guanine nucleotides and/or somatostatin-14 as in Fig. 1AGo. Ras-transformed NIH 3T3 cells were cotransfected with expession construct for sstr2A (5 µg) and 10 µg of either control pCMV6b vector, pCMV-wt PTP1B, or pCMV-C/S PTP1B. n = 3.

 


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Figure 4. Coexpression of Catalytically Inactive SHP-2 with sstr2A Blocks in Vitro Activation of Membrane-Associated PTPase

Panel A, Ras-transformed NIH 3T3 cells were cotransfected with expession construct for sstr2A (5 µg) and 10 µg of either control pCMV6b vector, wt SHP-2, or C/S SHP-2. Membranes (2 µg) were assayed for PTPase activity after treatment with guanine nucleotides and/or somatostatin-14 as in Fig. 1AGo. n = 3. Panel B, An example of the raw data, as counts per min released, from one experiment. Average of triplicate PTPase assays.

 
In contrast, no stimulation of PTPase activity by somatostatin was observed in membranes prepared from cells cotransfected with the pCMV plasmid for expression of C/S SHP-2 (Fig. 4Go). This was a consistent finding. It was observed in multiple experiments using different cells and with different preparations of plasmid DNA. The wt and C/S constructs are identical except for the point mutation, and thus both proteins should be transcribed and translated with equal efficiency. Equal quantities of either DNA construct, prepared by twice banding in cesium chloride, were used. Care was also taken in plating and transfecting. Recovery of membrane protein was equivalent. Nevertheless, we compared expression of wt and C/S SHP-2 proteins by Western blotting, using myc epitopes placed at the NH2-termini (15). Both proteins were expressed as expected (Fig. 5Go). The variability in expression observed was random and was not skewed to favor C/S SHP-2.



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Figure 5. C/S SHP-2 Is Not Preferentially Expressed in Comparison to SHP-2

Ras-transformed NIH 3T3 cells were transfected with 10 µg of either wt SHP-2 or C/S SHP-2 expression, and whole-cell lysates were analyzed in duplicate lanes for expression by Western blotting (see Materials and Methods). Results from three separate experiments are shown.

 
Experiments were also performed to ensure that coexpression with C/S SHP-2 did not prevent expression of sstr2. Duplicate plates were transfected for binding assays with iodinated somatostatin-14. Specific binding was detected when sstr2 plasmid was transfected with or without wt SHP-2 or C/S SHP-2 plasmids, as expected. No specific binding was detected with control vector. Expressed as percent specific binding of total counts per min in the assay (200 µg total protein), the values for sstr2 were 1 ± 0.5 (range) for sstr2, 1 ± 0.4 for sstr2 plus wt SHP-2, and 1.6 ± 0.4 for sstr2 plus C/S SHP-2. The difference in specific binding was higher for coexpression with C/S SHP-2 in comparison to coexpression with wt SHP-2 in one set and was reversed in the other. Thus, the loss of PTPase response to somatostatin by coexpression with the C/S mutant was not due to loss of receptor expression.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of sstr2A, sstr3, or sstr4 (but not sstr1 or sstr5) conferred capacity for stimulation of PTPase activity by somatostatin in vitro in isolated membranes. The cellular background of expression of G proteins or PTPases in v-Ras-transformed NIH 3T3 fibroblasts could affect this profile, and thus the coupling could be different in other cell types. This caveat given, these data help to clarify some ambiguities in the literature.

Buscail et al. (16) tested sstr1-5 for ability to stimulate PTPase activity in whole cell homogenates from Chinese hamster ovary cells (CHO), engineered to specifically express a given receptor, and treated in situ with the somatostatin analog RC-160 for 15 min (16). The substrate used was tyrosine-phosphorylated poly(Glu, Tyr). The clearest results were obtained with CHO-sstr2 cells wherein RC-160 stimulated PTPase activity 110% above control. RC-160 stimulated PTPase activity from CHO-sstr1 cells very modestly (30% at 1 µM) but had no effect on PTPase activity in cells expressing sstr3, sstr4, or sstr5 (16). These results confirmed a previous study of sstr1 and sstr2 (17) from the same authors, using similar assays, wherein RC-160 or somatostatin stimulated PTPase activity 2-fold in COS-7 cells transiently expressing sstr2, and only modestly stimulated (1.3-fold) PTPase activity in COS-7 cells expressing sstr1.

Using different methodologies, Florio et al. (18) studied PTPase regulation in CHO-K1 cells stably expressing sstr1 or sstr2. Crude membranes were prepared by differential centrifugation from cells treated in situ with somatostatin for 20–120 min, suspended in a HEPES buffer containing 1% NP-40, and assayed for PTPase activity with a radiolabeled c-src peptide substrate. Somatostatin did not affect PTPase activity in CHO-K1-sstr2 cells and stimulated PTPase activity 1- to 6-fold in CHO-K1-sstr1 cells after 2 h.

Our study and those just discussed can not be directly compared because of experimental differences. However, our results confirm an ability of sstr2A to regulate PTPase activity, as reported by Susini and colleagues (16, 17). In addition, we show that sstr3 and sstr4 can couple to PTPase activity. The negative data obtained for sst3 and sstr4 in the Buscail study (16) may be due to the cellular background or a lower sensitivity for detecting activated PTPase. We observed no stimulation of PTPase activity by somatostatin with sstr1, and Buscail et al. (16, 17) observed only very small increases for sstr1 in their studies. Thus, our data and those of Buscail et al. (16, 17) may be substantially in agreement with respect to sstr1. Our data and Buscail’s data (16, 17) conflict strongly with that of Florio et al. (18) for sstr2. Both Florio et al. (18) and Buscail et al. (16) studied PTPase activation by sstr2 in CHO cell lines. One possibility is that different phosphatase phenomena were being studied; we and Buscail et al. (16, 17) used principally 10- to 15-min stimulations with somatostatin whereas Florio et al. (18) used principally 2-h stimulations.

The laboratories of Susini and Srikant have proposed that the SH2-domain containing PTPase SHP-1, and not SHP-2, is regulated by somatostatin, albeit by different mechanisms. Susini and co-workers have shown that sstr2 expression can up-regulate SHP-1 transcription in NIH 3T3 cells (2) and have furthermore hypothesized that SHP-1 binds to activated somatostatin receptors and is activated in consequence (12). More recently, Srikant and colleagues (13) demonstrated that somatostatin induced translocation of SHP-1 from the cytosolic to the membrane fraction in breast cancer cells and proposed that translocation per se accounts for an observed increase in SHP-1 activity in the particulate fraction.

SHP-1 and SHP-2 are closely related nontransmembrane PTPases of 65,000 and 68,000 Mr (19, 20). The principal features of each are two nonidentical SH2 domains at the NH2 terminus and a catalytic domain located at the COOH terminus. Corresponding SH2 domains and the catalytic domains of SHP-1 and SHP-2 are 50–70% similar by alignment (20), but are sufficiently different that their functions could be distinct (20). Strong evidence indicates that SHP-1 and SHP-2 are restrained by intramolecular interactions of their SH2 domains with the COOH-terminal portion of their catalytic domains (21) and, furthermore, that both can be activated in vitro by liganding of their SH2 domains (19, 20, 21). However, the molecular details of their regulation are still poorly understood, both as to the molecular segments involved and as to the individual roles of the SH2 domains.

We used coexpression of catalytically inactive SHP-2 with sstr2 to test whether an SH2 domain-containing phosphatase was involved in our system. We chose to study SHP-2 because expression of SHP-1 is principally confined to hematopoietic cells (19). C/S SHP-2 is catalytically inactive but otherwise capable of binding phosphotyrosine via its SH2-domains and capable of binding substrates. If an SH2 domain-containing phosphatase were involved, we reasoned that C/S SHP-2 would be expressed in greater amounts than endogenous SHP-2 and thus might compete for any pool of SHP-2 in our membrane preparations. There it would behave as a "dominant-negative," blocking PTPase stimulation.

The result that C/S SHP-2 blocks PTPase activation was surprising to us. SHP-2 is a non-transmembrane phosphatase, generally accepted to be cytosolic and to be recruited to membrane receptors or docking proteins such as insulin receptor substrate 1 by phosphotyrosine:SH2-domain interactions that activate its enzymatic activity (19). The PTPase that is activated by somatostatin and guanine nucleotides in our membrane preparations is either membrane-associated and copurifying, or present as a contaminant from the cytosolic fraction. Translocation is not involved. Suzuki et al. (22) performed a careful study of the cellular localization of SHP-2 in rat brain, utilizing both immunohistochemistry and cellular disruption and fractionation. SHP-2 was mainly observed in the synaptic plasma membrane fraction and could be immunoprecipitated in association with a 100-kDa tyrosine-phosphorylated protein. SHP-2 can be detected by Western blotting in our partially purified membranes (our unpublished data), but its manner of association and significance are unknown. The strong evidence supporting one modality of regulation of SHP-2, by liganding of its SH-2 domains, presents a bias against existence of another, undiscovered mechanism involving G proteins, but such could exist. As yet, we have not observed activation in vitro of purified, bacterially expressed glutathione-S-transferase-SHP-2 fusion protein by purified, activated brain G{alpha}i/o-subunits. Other component(s) may be required. Alternatively, C/S SHP-2 may indirectly affect protein-protein interactions in situ that negate in vitro activation of another PTPase. Thus, although our results simply taken are consistent with identification of SHP-2 as a somatostatin-regulated phosphatase, more work will be required to test this hypothesis.

Regulation of SHP-2 by Gi/o by a conventional SH2 mechanism is also possible because Gß{gamma} oligomers stimulate the activity in situ of a tyrosine kinase (23), possibly c-Src (24), that phosphorylates Shc on tyrosine residues. Moreover, SHP-2 can be found in association with c-Src (25). However, the conditions we used herein for PTPase activation, omitting ATP and including millimolar concentrations of GMPPNP, would be expected to preclude phosphorylation reactions. Thus, phosphorylation is not likely to be involved in the in vitro activation of PTPase activity by somatostatin in the experimental system we describe.

A 115-kDa tyrosine phosphorylated protein has been strongly implicated in SHP-2 function. An unidentified protein recognized by anti-pTyr antibodies can be precipitated from insulin-treated cells in association with SHP-2 (26). Daughter of Sevenless, DOS, the likely homolog of this protein in Drosophila, has been identified as a pleckstrin-homology domain containing protein of unknown function, possibly an adapter or docking function (27, 28). pTyr-DOS binds CSW by SH2 domain interactions and serves as substrate for CSW in vitro (27). By epistasis arguments, DOS functions in parallel with CSW and downstream of the tyrosine kinase Sevenless and upstream of, or in parallel with, Ras in the pathway to specify photoreceptor development (27, 28). Interestingly, a portion of DOS is plasma membrane-associated, and this fraction is increased by Sevenless signaling (28).

In summary, evidence from several laboratories demonstrates that somatostatin activates a PTPase(s) that we currently view as unidentified. SH2-domain containing phosphatases are candidates (2, 12, 13), supported here by the demonstration that expression of C/S SHP-2 blocks PTPase activation. Regulation of PTPase activity by somatostatin may occur rapidly, by G protein-dependent pathways (Refs. 2, 8, 10 , and data herein) or by long-term alteration of transcription and expression (2). Somatostatin receptor subtypes 2, 3, and 4 can function to activate PTPase activity in vitro in membranes from v-Ras-transformed NIH 3T3 cells. Elucidation of the PTPases and mechanisms involved is critically important to full understanding of G protein-mediated signaling.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Somatostatin-14 and guanine nucleotides were purchased from Sigma-Aldrich (St. Louis, MO) and Boehringer-Mannheim (Mannheim, Germany), respectively. NIH 3T3 cells transformed with Ha-Ras (G12V) were obtained from L. Feig (Tufts University, Boston, MA). [125I-Tyr11]-somatostatin-14 was an Amersham (Arlington Heights, IL) product. Expression constructs for human sstr1, murine sstr2A, human sstr3, human sstr4, and human sstr5 along with parental control vectors pCMV6b and pCMV6c were provided by G. I. Bell (Howard Hughes Medical Institute, University of Chicago, Chicago, IL). pCMV constructs (15) for expressing PTP1B wt, PTP1B C/S, and myc epitope-tagged wild type and C/S SHP-2 were kindly provided by J. Pessin (University of Iowa, Iowa City, IA).

Cell Culture and Transfection
NIH 3T3 cells stably transformed with Ha-RasG12V were routinely grown (37 C; 10% CO2 atmosphere) in DMEM containing 10% (vol/vol) FCS and used throughout. Cells (at ~60% confluency in 100-mm dishes) were transfected using CaPO4 precipitation [15–20 µg expression construct(s), 0.125 M CaCl2, 25 mM BES [N,N-bis(2-hydroxyethyl)-2-aminoethane-sulfonic acid (Sigma-Aldrich))], 140 mM NaCl, 0.75 mM Na2HPO4, pH 6.95] and diluted 1:10 dropwise onto cells in dishes containing fresh media. Cells were incubated overnight at 37 C in 5% CO2, washed extensively in PBS to remove precipitated DNA, and allowed to recover 24–48 h in complete media at 37 C in 10% CO2.

Preparation of Membranes from Transfected Cells
Transfected cells were subjected to serum starvation for 2 h before membrane isolation. Cells were washed in PBS, then suspended in 5 ml of buffer A [25 mM HEPES, pH 7.6 at 4 C, 10 mM EDTA, 10 mM EGTA, 5 mM benzamidine, 0.1% (vol/vol) 2-mercaptoethanol, 1 mM fresh phenylmethylsulfonyl fluoride, 40 µg/ml each of Na-p-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, leupeptin, pepstatin A, aprotinin, and 1 mg/ml soybean trypsin inhibitor] containing 8.6% (wt/vol) sucrose. Suspended cells were subjected to 30 passes (4 C) in a motorized Teflon-glass homogenizer. The homogenate was layered onto buffer A containing 39% (wt/vol) sucrose, and centrifuged (30 min, 4 C) at 30,000 x g in an SW41 rotor (Beckman, Palo Alto, CA). Membranes collected at the interface were washed by dilution to 25 ml with 25 mM Tris, pH 7.9 (4 C), and centrifugation (30 min, 4 C) at 40,000 x g in a 50.2Ti rotor. Membranes were suspended to ~1 mg/ml total protein in buffer B [buffer A containing 8.6% (wt/vol) sucrose and the above indicated protease inhibitors], and used immediately.

Stimulation of Membranes with Guanine Nucleotides and Somatostatin
Membranes were treated with GDP to produce a basal state by incubation with 1 mM GDP at 22 C and for a further 20 min after adjustment of Mg to 5 mM in excess of total chelator concentrations. Membranes were recovered for use by centrifugation at 100,000 x g in an Airfuge (Beckman); after traces of the supernatant were carefully removed, membranes were resuspended to 1 mg/ml protein in buffer B, adjusted to 5 mM total Mg+2 in excess of chelators. Portions (20 µl) were placed on the walls of tubes containing appropriate amounts of concentrated stocks of guanine nucleotides and/or somatatostatin. Reactions (37 C) were initiated by tapping the tubes to mix membranes with stimulators. After 10 min, portions (2 µl) were removed and assayed in triplicate for PTPase activity.

PTPase Assay
PTPase activity was assayed with [[32P]pTyr]RCM-lysozyme as substrate as described (10). Data were expressed as percentage release of total 32P in the assay.

Ligand Binding and Western Blotting
The binding assay for iodinated somatostatin was modified from the work of Rens-Domiano et al. (29). Cells were homogenized in [50 mM Tris, pH 7.4 at 4 C, 1 mM EGTA, 5 mM MgCl2, 10 µg/ml leupeptin and pepstatin A, 0.5 µg/ml aprotinin, 200 µg/ml bacitracin]. The particulate fraction was isolated by centrifugation (45,000 x g, 15 min, 4 C). Crude membranes were resuspended in homogenization buffer. Membrane protein (200 µg) was incubated (30 min, 25 C) with [125I-Tyr11]-somatostatin-14 (50,000 cpm) with or without nonlabeled competing somatostatin-14 (100 nM). Bound radioligand was determined after filtration and washing as described (13).

For Western analysis, SHP-2 expression constructs were used to transfect cells as detailed above. Whole-cell lysate (100 µg) dissolved in SDS sample buffer was electrophoresed (10% gel) and transferred to nitrocellulose (0.45 µm). The myc-epitope-tagged SHP-2 protein was detected by enhanced chemiluminescence (Amersham) using anti-myc (1:2,000, Santa Cruz Biochemical, Santa Cruz, CA) as 1 C antibody and horseradish peroxidase-linked anti-mouse (1:5000, Bio-Rad) as 2 C antibody.

Note Added in Proof.

GnRH stimulates PTPase activity upon addition, with GTP-{gamma}-S, to plasma membranes from ovarian carcinomas (30).


    ACKNOWLEDGMENTS
 
We appreciate the generous help of J. Pessin and G. Bell. We thank Kirsten Wentz and Jackie Endriss for many plasmid purifications, Konrad Zeller for technical assistance, and Corky Harrison for manuscript preparation.


    FOOTNOTES
 
Address requests for reprints to: Thomas W. Sturgill, M.D., Ph.D., Howard Hughes Medical Institute, Room 7021, Multistory Building, University of Virginia Health Sciences Center, Box 577, Charlottesville, Virginia 22908.

This work was supported by the Howard Hughes Medical Institute and NIH Grant DK-41077 (to T.W.S.).

Received for publication October 14, 1996. Revision received March 19, 1997. Accepted for publication April 17, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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