CCK(B)/gastrin receptor mediates synergistic stimulation of DNA synthesis and cyclin D1, D3, and E expression in Swiss 3T3 cells.

In order to develop a model system for identifying signaling pathways and cell cycle events involved in gastrin-mediated mitogenesis, we have used high efficiency retroviral-mediated transfection of cholecystokinin (CCK)(B)/gastrin receptor into Swiss 3T3 cells. The retrovirally-transfected CCK(B)/gastrin receptor binds 125I-CCK-8 with high affinity (Kd = 1.1 nM) and is functionally coupled to intracellular signaling pathways including rapid and transient increase in Ca2+ fluxes, protein kinase C-dependent protein kinase D activation, and MEK-dependent ERK1/2 activation. In the presence of insulin, CCK-8 or gastrin induced a 66.5 +/- 8.8-fold (mean +/- SEM, n = 24 in eight independent experiments) increase in cellular DNA synthesis, reaching a level similar to that achieved by stimulation with a saturating concentration of fresh serum, and much greater than the response to each agonist added alone. CCK-8 also induced a striking increase in the expression of cyclins D1, D3, and E and hyperphosphorylation of Rb acting synergistically with insulin. Similar effects were observed when CCK(B)/gastrin receptor was activated in the presence of EGF or bombesin. Our results demonstrate that activation of CCK(B)/gastrin receptor retrovirally-transfected into Swiss 3T3 induces a potent synergistic effect on DNA synthesis, accumulation of cyclins D1, D3, and E and hyperphosphorylation of Rb in combination with insulin, EGF, or bombesin. Thus, the CCK(B)/gastrin receptor transfected into Swiss 3T3 cells provides a novel model system to elucidate mitogenic signal transduction pathways and cell cycle events activated via this receptor.     

CCK-4-Induced Calcium Mobilization in T Cells Is Enhanced in Panic Disorder

Abstract: We investigated the effects of brain cholecystokinin (CCK) receptors on the intracellular calcium concentration and protein kinase C in human T cells. CCK-4 produced a transient increase in calcium in the absence of extracellular calcium. CCK-B agonists stimulated calcium mobilization in a dose-dependent manner in T cells. CCK-B antagonists suppressed CCK-4-induced calcium mobilization more potently than CCK-A antagonist. The recovery of desensitization of the CCK-4-induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A. The responsiveness to CCK-4 was also reduced by phorbol 12,13-dibutyrate (PDBu), and this effect of PDBu was abolished completely by preincubation with staurosporine. CCK-4-induced calcium mobilization was too small to attribute the desensitization to the protein kinase C transduction pathway. T cells from patients with untreated panic disorder exhibited significantly higher cholecystokinin-4-induced calcium mobilization than those from healthy controls or patients with treated panic disorder. These results suggest that cholecystokinin-B receptor function in T cells of patients with panic disorder is enhanced. Cholecystokinin-4-induced calcium mobilization in T cells may be state dependent and useful as a biological marker of panic disorder.     

Basic Amino Acids at the C-Terminus of the Third Intracellular Loop Are Required for the Activation of Phospholipase C by Cholecystokinin-B Receptors

Abstract: In common with other G_q protein-coupled receptors, the third intracellular loop of the cholecystokinin-B (CCK-B) receptor contains three basic amino acids (K333/K334/R335) at the C-terminal segment. To determine the importance of these conserved basic residues in G_q-protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS-7 cells and recording Ca²⁺-activated Cl^? currents from Xenopus oocytes. Site-directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK-B receptors were expressed in COS-7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK-8) failed to induce inositol phosphate formation in COS-7 cells and evoke Ca²⁺-activated Cl^? currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single-point mutations resulted in a significant reduction in CCK-8-induced inositol phosphate formation and CCK-8-activated Ca²⁺-dependent Cl^? currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK-8-simulated inositol phosphate formation and the amplitude of CCK-8-evoked Ca²⁺-dependent Cl^? currents. Radioligand-binding studies showed that the above-mentioned mutations did not affect the affinity for CCK-8 and receptor expression level in COS-7 cells. These findings suggest that basic amino acids at the C-terminus of the third cytoplasmic loop are required for the signal transduction by CCK-B receptors.     

Characterization of the novel CCK analogs JMV-180, JMV-320, and JMV-332 in H345 cells

The interaction of the novel CCK analogs JMV-180, JMV-320, and JMV-332 with CCK-B/gastrin receptors on small cell lung cancer (SCLC) cells was investigated. JMV-180, JMV-320, and JMV-332 potently inhibited specific binding of ¹²⁵I-CCK-8 to CCK-B/gastrin receptors expressed on the SCLC cell line NCI-H345 (H345) with IC₅₀ values of 4.9, 1.8, and 7.0 nM, respectively. JMV-320 and JMV-332 stimulated intracellular calcium ([Ca²⁺]_i) release in a dose-dependent manner in cells preloaded with indo-1. JMV-180 did not stimulate [Ca²⁺]_i but inhibited the [Ca²⁺]_i release elicited by 10 nM CCK-8 in a dose-dependent manner. These data indicate that JMV-320 and JMV-332 function as CCK-B/gastrin receptor agonists while JMV-180 functions as a CCK-B/gastrin receptor antagonist in H345 cells.     

EGF receptor function is required in late G(1) for cell cycle progression induced by bombesin and bradykinin

We examined therole of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinaseactivation in G protein-coupled receptor (GPCR) agonist-inducedmitogenesis in Swiss 3T3 and Rat-1 cells. Addition of EGFR tyrosinekinase inhibitors (e.g., tyrphostin AG-1478) abrogated bombesin-inducedextracellular signal-regulated kinase (ERK) activation in Rat-1 cellsbut not in Swiss 3T3 cells, indicating the importance of cell contextin determining the role of EGFR in ERK activation. In strikingcontrast, treatment with tyrphostin AG-1478 markedly (~70%)inhibited DNA synthesis induced by bombesin in both Swiss 3T3 and Rat-1cells. Similar inhibition of bombesin-induced DNA synthesis in Swiss3T3 cells was obtained using four structurally different inhibitors ofEGFR tyrosine kinase. Furthermore, kinetic analysis indicates that EGFRfunction is necessary for bombesin-induced mitogenesis in mid-lateG₁ in both Swiss 3T3 and Rat-1 cells. Our results indicatethat EGFR kinase activity is necessary in mid-late G₁ forpromoting the accumulation of cyclins D1 and E and implicate EGFRfunction in the coupling of GPCR signaling to the activation of thecell cycle.

     

Leu13-psi(CH2NH)Leu14]bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells

The pseudopeptide [Leu13-psi(CH2NH)Leu14]bombesin blocks bombesin-stimulated mitogenesis in Swiss 3T3 cells in a competitive and reversible manner, but not that of other mitogens. It inhibits the mobilization of intracellular Ca2+ and activation of protein kinase C by bombesin-like peptides. It acts at receptor level, as shown by inhibition of [125I]GRP binding and reduction in cross-linking of the Mr 75-85,000 receptor-associated protein. Thus [Leu13-psi(CH2NH)Leu14]bombesin is a specific bombesin receptor antagonist in Swiss 3T3 cells which blocks long-term growth promoting effects of bombesin-like peptides.     

Stable expression of high affinity NK1 (substance P) and NK2 (neurokinin A) receptors but low affinity NK3 (neurokinin B) receptors in transfected CHO cells.

Stable CHO cell clones which selectively express all three rat tachykinin receptors were established by transfection. The binding of radiolabled substance P and neurokinin A (substance K) to CHO clones expressing the NK1 and NK2 receptors, respectively, were saturatable and of high affinity (Kd = 0.17 nM (NK1); 3.4 nM (NK2)). Scatchard analysis of the binding data indicated for both receptors binding to a single population of binding sites, and competition binding studies showed that the binding specificities of the receptors corresponded to those of classical NK1 and NK2 receptors. In contrast, the binding of eledoisin to the NK3 receptor expressed in the transfected CHO cells was of low affinity (IC50 = 240 nM) compared to the high affinity of the receptor found when it was transiently expressed in COS-7 cells (IC50 = 8 nM). However, in both cases the receptor exhibited the specificity of a classical NK3 receptor. The established cell clones may provide an important tool for further analysis of the molecular mechanisms involved in binding, activation, and coupling of receptors for tachykinin peptides.     

Unsulfated CCK-8 not blocked by proglumide or CR 1409 in the mouse abdominal irritant-induced stretching assay: possible central site of action

Previous studies have shown that unsulfated cholecystokinin octapeptide (CCK-8-U) shares with the sulfated octapeptide (CCK-8-S) and the carboxyl-terminal tetrapeptide (CCK-4) the ability to block abdominal irritant-induced stretching when administered intracerebroventricularly. The effects of CCK-8-U, however, are not naloxone-reversible and do not appear upon systemic administration. To assess the hypothesis that the antistretching effects of CCK-8-U are mediated by central-type (CCK-B), rather than peripheral-type (CCK-A) receptors, the present experiments examined the reversal of these effects by CR 1409, a CCK receptor antagonist with in vitro selectivity for CCK-A receptors, and by proglumide. Both antagonists, when administered ICV, blocked the antistretching effects of CCK-8-S and CCK-4 (ICV), but not those of CCK-8-U. CR 1409 was approximately 40 times more potent against CCK-8-S by the ICV route than subcutaneously, indicating a likely action on CCK-A receptors in the brain. The effects of morphine, bombesin and neurotensin (ICV) were not blocked by CR 1409, indicating specificity for CCK receptor-mediated effects. The antistretching effects of CCK-8-U do not appear to be mediated by CCK-A receptors, and the possibility of a CCK-B receptor site of action must be considered.     

Alpha beta T cell receptor and CD3 transduce different signals in immature T cells. Implications for selection and tolerance

Ligand binding to alpha beta TCR has different consequences in thymocytes at different developmental stages, causing alternatively positive selection, clonal deletion, or activation. These various functional consequences may be due to changes in the signaling properties of the receptor complex during development. In this report we show that alpha beta TCR engagement on immature thymocytes has different effects on intracellular free calcium concentrations than alpha beta TCR engagement on mature T cells. In contrast, CD3 engagement on immature thymocytes and mature T cells has the same effect on intracellular free calcium, suggesting that altered signal transduction in immature thymocytes may be due to inefficient alpha beta TCR-CD3 coupling. These studies also suggest that in certain T cell populations, activation events resulting from ligation of CD3 may not accurately reflect the activation events resulting from ligation of the physiologic receptor, alpha beta TCR.     

Tachykinin neurokinin 3 receptor signaling in cholecystokinin-elicited release of oxytocin and vasopressin

Neurokinin 3 receptor (NK3R) signaling has an integral role in the stimulated oxytocin (OT) and vasopressin (VP) release in response to hyperosmolarity and hypotension. Peripheral injections of cholecystokinin (CCK) receptor agonists for the CCK-A (sulfated CCK-8) and CCK-B (nonsulfated CCK-8) receptors elicit an OT release in rat. It is unknown whether NK3R contributes to this endocrine response. Freely behaving male rats were administered an intraventricular pretreatment of 250 or 500 pmol of SB-222200, a specific NK3R antagonist, or 0.15 M NaCl before an intraperitoneal or intravenous injection of CCK-8 (nonsulfated or sulfated) or 0.15 M NaCl. Blood samples were taken before intraventricular treatment and 15 min after intraperitoneal or intravenous injection, and plasma samples were assayed for OT and VP concentration. Intraperitoneal injection of both nonsulfated and sulfated CCK-8 significantly increased plasma OT levels and had no effect on plasma VP levels. Intravenous injection of sulfated CCK-8 stimulated an increase in plasma OT levels and did not alter plasma VP levels. However, intravenous injection of nonsulfated CCK-8 stimulated a significant increase in plasma levels of both OT and VP. No other studies have demonstrated CCK-8-stimulated release of VP in rat. NK3R antagonist did not alter baseline levels of either hormone. However, pretreatment of NK3R antagonist significantly blocked the CCK-stimulated release of OT in all CCK treatment groups and blocked VP release in response to intravenous injection of nonsulfated CCK-8. Therefore, central NK3R signaling is required for OT and VP release in response to CCK administration.     

Vasoactive intestinal contractor, a novel peptide, shares a common receptor with endothelin-1 and stimulates Ca2+ mobilization and DNA synthesis in Swiss 3T3 cells

Vasoactive intestinal contractor peptide (VIC), a novel member of the endothelin family, stimulated a rapid increase in the intracellular Ca2+ concentration in fura-2-loaded Swiss 3T3 cells. Sequential addition of VIC and endothelin-1 (ET1) demonstrated the induction of both homologous and heterologous desensitization. VIC was as potent as ET1 in displacing the binding of 125I-ET1 and in stimulating mitogenesis in Swiss 3T3 cells. These findings suggest that VIC and ET1 share a common receptor in Swiss 3T3 cells.     

Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells

We recently reported that store-operated Ca²⁺ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca²⁺ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca²⁺ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca²⁺ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca²⁺ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskeleton; calcium entry     

Gangliosides Inhibit Platelet-Derived Growth Factor-Stimulated Receptor Dimerization in Human Glioma U-1242MG and Swiss 3T3 Cells

Abstract: We previously showed that gangliosides inhibit DNA synthesis in Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) in a dose-responsive manner. This correlated with the inhibitory effects of several gangliosides (except GM3) on tyrosine phosphorylation of the PDGF receptor (PDGFR). [³⁵S]Methionine-labeled Swiss 3T3 cells were incubated either with or without gangliosides and stimulated with PDGF, and proteins were cross-linked with bis(sulfosuccinimidyl) suberate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that two protein bands (170 and 350 kDa) were specifically immunoprecipitated with an anti-PDGFR antibody. Using both Swiss 3T3 and human glioma U-1242MG cells, western blots with anti-PDGFR and anti-phosphotyrosine antibodies confirmed that these bands were the PDGFR monomer and dimer, respectively, and that phosphotyrosine was present in these bands only after cells were stimulated with PDGF. Of the gangliosides tested, GM1, GM2, GD1a, GD1b, GD3, and GT1b, but not GM3, inhibited the formation of the 350-kDa band. These results demonstrate that all gangliosides tested, except GM3, probably inhibit PDGF-mediated growth by preventing dimerization of PDGFR monomers. Loss of more complex gangliosides in human gliomas would permit unregulated activation of the PDGFR, contributing to uncontrolled growth stimulation. We propose that ganglioside inhibition of receptor dimerization is a novel mechanism for regulating and coordinating several trophic factor-mediated cell functions.     

Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for the G protein-coupled receptor GPR4

Sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC) are bioactive lipid molecules involved in numerous biological processes. We have recently identified ovarian cancer G protein-coupled receptor 1 (OGR1) as a specific and high affinity receptor for SPC, and G2A as a receptor with high affinity for LPC, but low affinity for SPC. Among G protein-coupled receptors, GPR4 shares highest sequence homology with OGR1 (51%). In this work, we have identified GPR4 as not only another high affinity receptor for SPC, but also a receptor for LPC, albeit of lower affinity. Both SPC and LPC induce increases in intracellular calcium concentration in GPR4-, but not vector-transfected MCF10A cells. These effects are insensitive to treatment with BN52021, WEB-2170, and WEB-2086 (specific platelet activating factor (PAF) receptor antagonists), suggesting that they are not mediated through an endogenous PAF receptor. SPC and LPC bind to GPR4 in GPR4-transfected CHO cells with K(d)/SPC = 36 nm, and K(d)/LPC = 159 nm, respectively. Competitive binding is elicited only by SPC and LPC. Both SPC and LPC activate GPR4-dependent activation of serum response element reporter and receptor internalization. Swiss 3T3 cells expressing GPR4 respond to both SPC and LPC, but not sphingosine 1-phosphate (S1P), PAF, psychosine (Psy), glucosyl-beta1'1-sphingosine (Glu-Sph), galactosyl-beta1'1-ceramide (Gal-Cer), or lactosyl-beta1'1-ceramide (Lac-Cer) to activate extracellular signal-regulated kinase mitogen-activated protein kinase in a concentration- and time-dependent manner. SPC and LPC stimulate DNA synthesis in GPR4-expressing Swiss 3T3 cells. Both extracellular signal-regulated kinase activation and DNA synthesis stimulated by SPC and LPC are pertussis toxin-sensitive, suggesting the involvement of a G(i)-heterotrimeric G protein. In addition, GPR4 expression confers chemotactic responses to both SPC and LPC in Swiss 3T3 cells. Taken together, our data indicate that GPR4 is a receptor with high affinity to SPC and low affinity to LPC, and that multiple cellular functions can be transduced via this receptor.     

Heparan sulfates from Swiss mouse 3T3 and SV3T3 cells: O-sulfate difference

A difference in the extent of sulfation between the heparan sulfate isolated from Swiss 3T3 mouse cells and that from Swiss 3T3 cells transformed by the DNA virus SV40 has been reported previously. This variance is manifested by different chromatographic and electrophoretic properties. Heparan sulfates from the two cell types were treated with nitrous acid under conditions that gave selective deaminative cleavage of glucosaminyl residues with sulfated amino groups in order to define the nature of the difference in sulfation further. The O-sulfate containing fragments from the heparan sulfates were compared by gel filtration and ion-exchange chromatography. The results showed that the 3T3 heparan sulfate contains 8% more O-sulfate than does the SV3T3 heparan sulfate. Analysis of uronic acids revealed that both types of heparan sulfates contain 45% L-iduronic acid and 55% D-glucuronic acid. These and other observations indicate that the primary difference in sulfation between the 3T3 and SV3T3 heparan sulfates lies in the extent of O-sulfation.     

Growth factor-mediated K+ channel activity associated with human myeloblastic ML-1 cell proliferation

ML-1 cell proliferation is dependent on the presence of serumgrowth factors. Removing serum from the culture medium results ingrowth arrest and promotes differentiation. In this study, we foundthat a 4-aminopyridine-sensitiveK⁺ channel was highly expressed inproliferating ML-1 cells and significantly diminished inG₁-arrested ML-1 cells induced by serum deprivation but was restored within 30 min in these cells withaddition of 10% fetal bovine serum (FBS) or 5 ng/ml epidermal growthfactor (EGF). Intracellular adenosine 3',5'-cyclicmonophosphate (cAMP) levels, but not guanosine 3',5'-cyclicmonophosphate, were significantly increased in serum-deprived cellsstimulated by FBS or EGF, and the effects of FBS and EGF on the channelactivation were mimicked by exogenous cAMP. In inside-out patches,K⁺ channel activity wassignificantly increased by the cAMP-dependent protein kinase catalyticsubunit, whereas the effect of EGF on K⁺ channel activation was blockedby Rp-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphothioate. Together, our resultsdemonstrate that serum growth factors stimulateK⁺ channel activity inproliferation of ML-1 cells through protein kinase-inducedphosphorylation and suggest an important molecular mechanism for serumgrowth factor-stimulated mitogenesis in ML-1 cells.

     

Augmentation of lymphocyte responses by monoclonal antibodies to the gangliosides GD3 and GD2: the role of protein kinase C, cyclic nucleotides, and intracellular calcium

Previous studies have shown that MAb's against the gangliosides GD3 and GD2 may augment T cell responses to a variety of stimuli. We present evidence that antiganglioside MAb's, like PHA, increase intracellular cGMP and protein kinase C yet have no effect on intracellular Ca2+. Stimulation of T cells with MAb's to GD3 was associated with increased cGMP levels, particularly in the CD8+ T cell subset which showed the highest degree of potentiation by the MAb's. Augmentation of T cell responses by the MAb's to GD3 and GD2 was also mimicked by activation of PKC with phorbol esters but both agents together produced marked synergistic effects on cell division, suggesting they had different but complementary modes of action. Furthermore, use of neomycin to inhibit PKC activation only partially reversed the augmentation of proliferative responses by the antiganglioside MAb's. It did however inhibit the MAb-induced increase in IL2 production and IL2 receptor (Tac) expression. These studies suggest therefore that the potentiation of IL2 production by the MAb's against GD2 and GD3 was due to enhanced activation of PKC whereas their augmentation of proliferative responses appeared to be due to effects on late events in T cell activation and was associated with both increased cGMP levels and activation of PKC.     

HER3 intracellular domains play a crucial role in HER3/HER2 dimerization and activation of downstream signaling pathways

Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3’s function as an allosteric activator and its role in downstream signaling.