CCK2 (CCK(B)/gastrin) receptor mediates rapid protein kinase D (PKD) activation through a protein kinase C-dependent pathway

Addition of gastrin or cholecystokinin octapeptide (CCK-8) to cultures of Rat-1 cells stably transfected with the CCK2 (CCK(B)/gastrin) receptor induced protein kinase D (PKD) activation that was detectable within 1 min and reached a maximum ( approximately 10-fold) after 2.5 min of hormonal stimulation. Half-maximal PKD activation for both CCK-8 and gastrin was achieved at 10 nM. Treatment with various concentrations of the selective PKC inhibitors Ro 31-8220 or GF-I potently blocked PKD activation induced by subsequent addition of CCK-8 in a concentration-dependent fashion. Our results indicate that PKC-dependent PKD activation is a novel early event in the action of gastrin and CCK-8 via CCK2 receptors.     

Protein kinase D potentiates DNA synthesis induced by Gq-coupled receptors by increasing the duration of ERK signaling in swiss 3T3 cells

Protein kinase D (PKD) potentiates cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the mechanism(s) involved has not been elucidated. Here, we examined whether PKD overexpression in Swiss 3T3 cells regulates the activation/inactivation kinetics of the extracellular-regulated protein kinase (ERK) in response to the mitogenic GPCR agonists bombesin and vasopressin. Addition of bombesin or vasopressin to Swiss 3T3 cells overexpressing PKD induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of either control Swiss 3T3 cells or Swiss 3T3 cells expressing a kinase-inactive PKD mutant. In contrast, the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not increased. Furthermore, bombesin or vasopressin promoted a striking increase in phosphorylation (at Ser-374) and accumulation of c-Fos (the c-fos proto-oncogene product) in Swiss 3T3 cells overexpressing wild-type (but not kinase-inactive) PKD. Inhibition of the sustained phase of ERK/RSK activation abrogated the increase in c-Fos accumulation and DNA synthesis induced by bombesin or vasopressin in PKD-overexpressing cells. Our results demonstrate that PKD selectively potentiates mitogenesis induced by bombesin or vasopressin in Swiss 3T3 cells by increasing the duration of MEK/ERK/RSK signaling.     

Epidermal growth factor and bombesin differ strikingly in the induction of early responses in Swiss 3T3 cells

Swiss 3T3 cells express receptors for both the polypeptide epidermal growth factor (EGF) and the tetradecapeptide bombesin and respond mitogenically to these substances. These cells thus provide a system to analyze potential signal transduction pathways involved in mitogenic stimulation. Here we have determined and compared the early ionic responses elicited by EGF and bombesin and their relation to diacylglycerol (DG) and inositolphosphate (InsPn) production. Whereas EGF fails to cause any significant change in intracellular Ca2+, bombesin effectively induces prompt and transient Ca2+ mobilization from intracellular stores. Further support of the idea that these receptors utilize distinct signalling pathways comes from the measurements of cytoplasmic pH (pHi). As in most target cells, EGF induces a delayed (1 min) but sustained intracellular alkalinization that reaches a new steady state after approximately 10 min. Bombesin, in contrast, elicits a biphasic response; within seconds, a rapid but transient rise in pHi is observed, followed by a further slower sustained alkalinization. Inhibition of the Na+/H+ exchanger prevents both EGF as well as bombesin-induced alkalinization. However, under these conditions, bombesin evokes a rapid and sustained acidification related to the Ca2+ response. Apparently, bombesin initiates a Ca2(+)-dependent acidifying process immediately after binding of the hormone to its receptor. Furthermore, we could demonstrate that the bombesin-induced alkalinization depends on protein kinase C activation whereas the EGF response does not. Determination of the total DG and InsPn accumulation revealed that EGF is ineffective in stimulating phospholipase C-mediated production of these second messengers. In contrast, bombesin causes a rapid DG and InsPn production coinciding with the Ca2+ response and the first phase of the rise in pHi followed by a slower DG accumulation coinciding with the second alkalinization phase. Our results show that in Swiss 3T3 cells the bombesin receptor activates the hydrolysis of inositol lipids as a mechanism of signal transduction, which consequently causes changes in Ca2+i and pHi. Clearly, the EGF receptor utilizes different pathways to evoke mitogenesis and stimulates Na+/H+ exchange independently of DG production and protein kinase C activation.     

Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.     

Distinguishing bombesin receptor subtypes using the oocyte assay.

Physiological responses to mammalian bombesin-like peptides were studied in Xenopus oocytes injected with mRNA isolated from Swiss 3T3 cells and rat esophagus in order to identify and characterize bombesin receptor subtypes. Both groups respond similarly to either gastrin releasing peptide or neuromedin B, but only the response to neuromedin B in oocytes expressing the esophagus mRNA is not blocked by a specific gastrin releasing peptide receptor antagonist, des-Met-[D-Phe6]Bn(6-13) ethyl ester. Complete desensitization of gastrin releasing peptide-evoked responses in oocytes expressing esophagus mRNA does not abolish neuromedin B-evoked responses. A single application of neuromedin B abolishes responses to subsequently applied gastrin releasing peptide in oocytes expressing esophagus, but not Swiss 3T3, mRNA. RNA blot hybridization studies using a Swiss 3T3 gastrin releasing peptide receptor cDNA probe show no detectable hybridization in esophagus mRNA samples. These data suggest that a gastrin releasing peptide receptor is expressed in the esophagus and that it is distinct from that expressed in Swiss 3T3 cells and may represent a third subtype of mammalian bombesin receptor.     

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.

     

CCK-B receptors produce similar signals but have opposite growth effects in CHO and Swiss 3T3 cells

Rat cholecystokinin-B (CCK-B) receptors were transfected intoChinese hamster ovary (CHO)-K1 (CHO-CCK-B) and Swiss 3T3 (Swiss 3T3-CCK-B) cells, and the effects of receptor activation on cell proliferation and intracellular signaling were investigated. CCK octapeptide (CCK-8) treatment had no effect on cell growth in quiescentCHO-CCK-B cells but inhibited DNA synthesis, proliferation, and colonyformation when the cells were grown in fetal bovine serum (FBS). Incontrast, CCK-8 stimulated DNA synthesis in quiescent Swiss 3T3-CCK-Bcells and had no effect when the cells were grown in FBS. Thesedifferences in growth responses were not due to differences in thelevel of receptor expression, as similar numbers of receptors werepresent in both cell types. To determine whether the different growtheffects were due to differences in receptor coupling to common secondmessenger pathways, we investigated the effects of CCK-8 on severalknown intracellular signals. In both cell types, CCK-8 stimulatedincreases in intracellular Ca²⁺concentration and polyphosphoinositide hydrolysis with similar potencies and efficacies. CCK-8 also stimulated arachidonate release from both cell types, although the potency was higher in the CHO cells.Adenosine 3',5'-cyclic monophosphate generation wasobserved at high agonist concentrations in both cell types and was much greater in cells with higher receptor density. In summary, receptor activation had opposite effects on growth parameters in CHO and Swiss3T3 cells, but only minor differences were observed in the characteristics of CCK-B receptor coupling to specific second messengers in the two cell types. Thus cellular context is a principal determinant of the biological effects of CCK-B receptor activation, anddifferences in biological responses may occur independently of majordifferences in receptor coupling.

     

Protein kinase D2 potentiates MEK/ERK/RSK signaling, c-Fos accumulation and DNA synthesis induced by bombesin in Swiss 3T3 cells

Protein kinase D (PKD) plays an important role in mediating cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the function of other isoforms of the PKD family has been much less explored. Here, we examined whether PKD2 overexpression in Swiss 3T3 cells facilitates DNA synthesis and the activation of the extracellular regulated protein kinase (ERK) pathway in response to the mitogenic GPCR agonist bombesin. We show that PKD2 overexpression markedly potentiated the ability of this agonist to induce DNA synthesis. Addition of bombesin to Swiss 3T3 cells overexpressing PKD2 also induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of control cells. In contrast, neither DNA synthesis nor the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD2-independent pathways, was increased. Furthermore, bombesin promoted a striking accumulation of c-Fos protein in cells overexpressing PKD2. Our study demonstrates that PKD2, like PKD, facilitates mitogenesis and supports the hypothesis that an increase in the duration of the ERK signaling leading to accumulation of immediate gene products is one of the mechanisms by which isoforms of the PKD family enhance re-initiation of DNA synthesis by Gq-coupled receptor activation.     

Bombesin induction of c-fos and c-myc proto-oncogenes in Swiss 3T3 cells: significance for the mitogenic response

Bombesin is a potent mitogen for Swiss 3T3 cells and acts synergistically with insulin and other growth factors. We show here that addition of bombesin to quiescent Swiss 3T3 cells causes a striking increase in the levels of c-fos and c-myc mRNAs. Enhanced expression of c-fos (122 +/- 14-fold) occurred within minutes of peptide addition followed by increased expression of c-myc (82 +/- 16-fold). The concentrations of peptide required for half-maximal increase in the levels of c-fos and c-myc mRNAs were 1.0 and 0.9 nM, respectively. The peptide [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P which inhibits the binding of bombesin to its receptor and bombesin-stimulated DNA synthesis in Swiss 3T3 cells blocked the increase in c-fos and c-myc mRNA levels promoted by bombesin. Down-regulation of protein kinase C by long-term exposure to phorbol esters prevented c-fos and c-myc induction by bombesin. This and other results indicate that the induction of these proto-oncogenes by bombesin could be mediated by the coordinated effects of protein kinase C activation and Ca2+ mobilization. The marked synergistic effect between bombesin and insulin was used to assess whether the increase in the induction of c-fos and c-myc is an obligatory event in cell activation. In the presence of insulin, bombesin stimulated DNA synthesis at subnanomolar concentrations but had only a small effect on c-fos and c-myc mRNA levels. This apparent dissociation of mitogenesis from proto-oncogene induction was even more dramatic in 3T3 cells with down-regulated protein kinase C. In these cells bombesin stimulated DNA synthesis in the presence of insulin but failed to enhance c-fos and c-myc mRNA levels at comparable concentrations. Thus, the induction of c-fos and c-myc may be a necessary step in the mitogenic response initiated by ligands that act through activation of protein kinase C but the expression of these proto-oncogenes may not be an obligatory event in the stimulation of mitogenesis in 3T3 cells by mitogens that utilise other signalling pathways.     

Insulin reduces the requirement for EGFR transactivation in bombesin-induced DNA synthesis

The binding of bombesin to its cognate G-protein coupled receptor stimulates quiescent Swiss 3T3 cells to re-initiate DNA synthesis and cell division. Addition of a non-mitogenic concentration of insulin dramatically potentiates bombesin-induced cell proliferation. We examined whether bombesin-induced EGFR transactivation mediates synergistic cell proliferation induced by bombesin and insulin. Treatment with selective EGFR tyrosine kinase inhibitors blocked EGFR transactivation, DNA synthesis, the transition of cells from quiescence into the cell cycle, and the expression of cyclins D1 and E induced by bombesin alone. In contrast, the inhibitors prevented cell cycle progression to a much lesser degree in cells stimulated with the combination of bombesin and insulin. Our results indicate that EGFR transactivation does not mediate synergistic cell proliferation induced by bombesin and insulin, and imply that insulin compensates for the requirement for EGFR transactivation in bombesin-induced DNA synthesis.     

Importance of sulfation of gastrin or cholecystokinin (CCK) on affinity for gastrin and CCK receptors

We investigated the importance of sulfation of gastrin or cholecystokinin (CCK) on influencing their affinity for gastrin or CCK receptors by comparing the abilities of sulfated gastrin-17 (gastrin-17-II), desulfated gastrin-17 (gastrin-17-I), CCK-8 and desulfated CCK-8 [des(SO3)CCK-8] to interact with CCK or gastrin receptors on guinea pig pancreatic acini. For inhibiting binding of 125I-gastrin to gastrin receptors, gastrin-17-II (Kd 0.08 nM) greater than CCK-8 (Kd 0.4 nM) greater than gastrin-17-I (Kd 1.5 nM) greater than des(SO3)CCK-8 (Kd 28 nM). For inhibiting binding of 125I-Bolton Hunter-labeled CCK-8 to CCK receptors the relative potencies were: CCK-8 much greater than des(SO3)CCK-8 = gastrin-17-II greater than gastrin-17-I. Each peptide interacted with both high and low affinity CCK binding sites. The relative abilities of each peptide to interact with high affinity CCK receptors showed a close correlation with their abilities to cause half-maximal stimulation of enzyme secretion. These results demonstrate that, in contrast to older studies, sulfation of both CCK and gastrin increase their affinities for both gastrin and CCK receptors. Moreover, the gastrin receptor is relatively insensitive to the position of the sulfate moiety, whereas the CCK receptor is extremely sensitive to both the presence and exact position of the sulfate moiety.     

Reduction of epidermal growth factor receptor affinity by heterologous ligands: evidence for a mechanism involving the breakdown of phosphoinositides and the activation of protein kinase C

The tetradecapeptide bombesin converts epidermal growth factor (EGF) receptors on Swiss 3T3 cells from a high affinity state (KD = 9.8 X 10(-11)M) to a lower affinity state (KD = 1.8 X 10(-9)M). This conversion occurs when the cells are incubated with bombesin at 37 degrees C but not when incubated at 4 degrees C. Previously, a number of other (chemically unrelated) cell growth-promoting peptides and polypeptides have been shown to induce a similar indirect, temperature-dependent reduction of EGF receptor affinity. We have now demonstrated that hormones and growth factors which cross-regulate EGF receptor affinity in Swiss 3T3 cells have a common ability to stimulate the breakdown of phosphoinositides in these cells. We propose that the reduction of EGF receptor affinity is a consequence of the activation of protein kinase C by the diacylglycerol generated by this breakdown. In support of this proposal we have found that exogenously added diacylglycerol reduces the affinity of the Swiss 3T3 cell EGF receptor.     

The effects of antagonists of receptors for gastrin, cholecystokinin and bombesin on growth of gastroduodenal mucosa and pancreas.

The effects of gastrin, cholecystokinin (CCK) and bombesin on the DNA synthesis, as a biochemical indicator of trophic action in the gastroduodenal mucosa and the pancreas have been examined in rats fasted for 48 h and in rats refed for 16 h with or without administration of specific receptor antagonists for bombesin, gastrin and CCK. Bombesin and gastrin administered three times daily for 48 h in fasted rats significantly increased the rate of DNA synthesis as measured by the incorporation of [3H] thymidine into DNA in each tissue tested. CCK significantly increased DNA synthesis in the duodenal mucosa and pancreatic tissue, but not in the gastric mucosa. The stimulation of DNA synthesis induced by bombesin in the gastroduodenal mucosa and pancreas was abolished by bombesin/GRP receptor antagonist, RC-3095. RC-3095 did not affect DNA synthesis stimulated by gastrin and CCK in these tissues. L-365,260, a receptor antagonist for gastrin suppressed the DNA synthesis induced by gastrin but not by CCK or bombesin in the gastrointestinal mucosa and pancreas. L-364,718 a specific antagonist for CCK receptors was effective only against CCK stimulated duodenal mucosa and pancreatic growth. Refeeding of 48 h fasting rats strongly enhanced the DNA synthesis in all tissues tested, and this effect was significantly reduced in the gastroduodenal mucosa by blocking only gastrin receptors (with L-365,260) and that in the duodenal mucosa and the pancreas by antagonizing of CCK receptors (with L-364,718). Antagonism of bombesin receptors (with RC-3095) did not significantly affect the stimulation of DNA synthesis induced by refeeding in all tissues tested. This study indicates that the stimulation of DNA synthesis can be achieved by exogenous gastrin, CCK and bombesin acting through separate receptor but that only gastrin and CCK play the major role in the postprandial stimulation of the growth of gastroduodenal mucosa and pancreatic tissue.     

Signaling pathways mediating gastrin's growth-promoting effects.

In addition to its fundamental role in stimulating gastric acid secretion, the peptide hormone gastrin induces growth-promoting effects on diversity of target cells. Various mechanisms, including endocrine, paracrine, and autocrine, have been proposed for gastrin's growth-promoting actions. The mitogenic effects of gastrin are mediated by specific cell surface receptors activated after gastrin binding. The functionally defined receptors for gastrin include cholecystokinin A (CCKA) receptor, which is discriminating for sulfated CCK8; cholecystokinin B (CCKB)/gastrin receptor, which binds gastrin17 sulfated, and nonsulfated CCK8 with nearly equal affinities; cholecystokinin C (CCKC), which is a low-affinity gastrin binding protein; and novel, high-affinity receptors selective for amidated gastrin, processing intermediates of gastrin, or both. The signaling pathways mediating gastrin's stimulation of the CCKB/gastrin receptor have been progressively outlined, and the pathways mediating other receptors have been slowly emerging. Engagement of the gastrin receptor initiates various biochemical and molecular events, including recruitment and activation of tyrosine kinases, activation of the phospholipase C signaling pathway leading to phosphoinositide breakdown, intracellular calcium mobilization and protein kinase C stimulation, activation of the mitogen-activated protein kinase pathway, and induction of early response genes. Current emphasis is on understanding the functional significance of processing intermediate forms of gastrin, and the receptor subtypes and pathways that promote the trophic/mitogenic effects of the different molecular forms of gastrin.     

Early events elicited by bombesin and structurally related peptides in quiescent Swiss 3T3 cells. I. Activation of protein kinase C and inhibition of epidermal growth factor binding

Addition of bombesin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an Mr 80,000 cellular protein (designated 80k). The effect was both concentration and time dependent; enhancement in 80k phosphorylation could be detected as early as 10 s after the addition of peptide. Recently, a rapid increase in the phosphorylation of an 80k cellular protein after treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact fibroblasts (Rozengurt, E., A. Rodriguez-Pena, and K. A. Smith, 1983, Proc. Natl. Acad. Sci. USA., 80:7244-7248; Rozengurt, E., A. Rodriguez-Pena, M. Coombs, and J. Sinnett-Smith, 1984, Proc. Natl. Acad. Sci. USA., 81:5748-5752). The 80k phosphoproteins generated in response to bombesin and to phorbol 12,13-dibutyrate were identical as judged by one- and two-dimensional PAGE and by peptide mapping after partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with phorbol 12,13-dibutyrate, which leads to the disappearance of protein kinase C activity, blocked the ability of bombesin to stimulate 80k. Bombesin also caused a rapid (1 min) inhibition of 125I-labeled epidermal growth factor (125I-EGF) binding to Swiss 3T3 cells. The inhibition was both concentration and temperature dependent and resulted from a marked decrease in the affinity of the EGF receptor for its ligand. Peptides structurally related to bombesin, including gastrin-releasing peptide, also stimulated 80k phosphorylation and inhibited 125I-EGF binding; both effects were selectively blocked by a novel bombesin antagonist. These results strongly suggest that these responses are mediated by specific high-affinity receptors that recognize the peptides of the bombesin family in Swiss 3T3 cells. While an increase in cytosolic Ca2+ concentration does not mediate the bombesin inhibition of 125I-EGF binding, the activation of protein kinase C in intact Swiss 3T3 cells by peptides of the bombesin family may lead to rapid inhibition of the binding of 125I-EGF to its cellular receptor.     

Effect of cholecystokinin receptor antagonists, MK-329 and L-365,260, on cholecystokinin-induced acid secretion and histidine decarboxylase activity in the rat

To elucidate the regulatory mechanism of acid secretion by cholecystokinin (CCK) in vivo, we compared the effects of CCK and gastrin on acid secretion and histidine decarboxylase (HDC) activity. We also examined the effects of MK-329, a specific antagonist for pancreatic-type CCK receptor, and L-365,260, a specific antagonist for gastrin-type CCK receptor, on the action of CCK. Graded doses of CCK or gastrin were intravenously infused into conscious rats with gastric fistula. Gastrin-17 I infusion up to 10 nmol/kg/h resulted in dose-related increases in acid secretion. CCK-8 infusion also caused an increase in acid secretion. However, it reached a peak with 0.3 nmol/kg/h CCK-8 and attenuated with higher concentrations of CCK-8. This attenuating effect of a higher dose of CCK was reversed by MK-329, but not by L-365,260. Both CCK and gastrin were potent in increasing fundic HDC activity, and the effect of CCK on HDC activity was significantly inhibited by L-365,260, but not by MK-329. Taken together, the present study suggests that CCK and gastrin stimulate histamine formation via a gastrin-type CCK receptor, and the attenuating action of CCK with higher concentrations on acid secretion in vivo is mediated by a pancreatic-type CCK receptor.     

Cholecystokinin receptor mediated hydrolysis of inositol phospholipids in guinea pig gastric glands

CCK-octapeptide (CCK-8) (EC50 = 0.5 nM), in the presence of Li+, increased 3H-inositol phosphate (IP) accumulation in guinea pig gastric glands prelabeled with 3H-inositol. CCK-8 desulfate, human gastrin I and pentagastrin were much less potent than CCK-8. Antagonists of CCK receptors such as proglumide, dibutyryl-c-GMP and CBZ-Tyr (SO3H)-Met-Gly-Trp-Met-AspNH2 shifted the CCK dose response curve to the right. However, histamine (H1 and H2), cholinergic, substance P and alpha- and beta-adrenergic receptor antagonists had no effect on 3H-IP accumulation induced by CCK. The results suggest that CCK receptor activation in gastric glands leads to an enhanced breakdown of inositol phospholipids which may relate to calcium mobilization and pepsinogen secretion.     

Insulin induces epidermal growth factor (EGF) receptor clustering and potentiates EGF-stimulated DNA synthesis in swiss 3T3 cells: a mechanism for costimulation in mitogenic synergy

In many cellular systems, activation with more than one ligand can produce a cellular response that is greater than the sum of the individual responses to the ligands. This synergy is sometimes referred to as coactivation. In Swiss 3T3 fibroblasts, activation of the epidermal growth factor (EGF) receptor produces a weak induction of DNA synthesis. Insulin has no stimulatory effect on this response. However, in combination, EGF and insulin synergize to cause a large induction of S phase. The underlying cellular biochemistry of this effect has been examined. The data indicate that phospholipase C activation is a major component of agonist-induced DNA synthesis. In contrast, activation of p70 S6 kinase by single agonists was inversely related to their ability to stimulate DNA synthesis. Therefore, it was examined whether stimulation of Swiss 3T3 cells with insulin causes changes in the subcellular distribution of EGF receptors and phospholipase Cgamma1 that could potentially explain the observed synergy or costimulation. It was found that insulin effectively induced the accumulation of EGF receptors on the actin arc of cells without activation of the EGF receptor. In contrast, EGF, when added for several hours, did not cause accumulation of the EGF receptor at this site. However, both EGF and insulin stimulated the accumulation of phospholipase Cgamma1 at the actin arc, which was coincident with the EGF receptor in the case of insulin- stimulated cells. Therefore, it is suggested that the insulin-induced coclustering of the EGF receptor with phospholipase Cgamma1 at the actin arc may allow for greater efficiency of signal transduction, resulting in the synergy observed for these two hormones in stimulation of DNA synthesis.