Rapamycin induces transactivation of the EGFR and increases cell survival.

The mammalian target of rapamycin (mTOR) signaling network regulates cell growth, proliferation and cell survival. Deregulated activation of this pathway is a common event in diverse human diseases such as cancers, cardiac hypertrophy, vascular restenosis and nephrotic hypertrophy. Although mTOR inhibitor, rapamycin, has been widely used to inhibit the aberrant signaling due to mTOR activation that plays a major role in hyperproliferative diseases, in some cases rapamycin does not attenuate the cell proliferation and survival. Thus, we studied the mechanism(s) by which cells may confer resistance to rapamycin. Our data show that in a variety of cell types the mTOR inhibitor rapamycin activates extracellularly regulated kinases (Erk1/2) signaling. Rapamycin-mediated activation of the Erk1/2 signaling requires (a) the epidermal growth factor receptor (EGFR), (b) its tyrosine kinase activity and (c) intact autophosphorylation sites on the receptor. Rapamycin treatment increases tyrosine phosphorylation of EGFR without the addition of growth factor and this transactivation of receptor involves activation of c-Src. We also show that rapamycin treatment triggers activation of cell survival signaling pathway by activating the prosurvival kinases Erk1/2 and p90RSK. These studies provide a novel paradigm by which cells escape the apoptotic actions of rapamycin and its derivatives that inhibit the mTOR pathway.

Transglutaminase-catalyzed transamidation: a novel mechanism for Rac1 activation by 5-hydroxytryptamine2A receptor stimulation.

Transglutaminase (TGase)-induced activation of small G proteins via 5-hydroxytryptamine (HT)(2A) receptor signaling leads to platelet aggregation (Cell 115:851-862, 2003). We hypothesize that stimulation of 5-HT(2A) receptors in neurons activates TGase, resulting in transamidation of serotonin to a small G protein, Rac1, thereby constitutively activating Rac1. Using immunoprecipitation and immunoblotting, we show that, in rat cortical cell line A1A1v, serotonin increases TGase-catalyzed transamidation of Rac1. This transamidation occurs in both undifferentiated and differentiated cells. Treatment with a 5-HT(2A/2C) receptor agonist 2,5-dimethoxy-4-iodoamphetamine, but not the 5-HT(1A) receptor agonist 5-hydroxy-2-dipropylamino tetralin, increases transamidation of Rac1 by TGase. In A1A1v cells, 5-HT(2A) receptors mediate the transamidation reaction because expression of 5-HT(2C) receptors was not detectable and the selective 5-HT(2A) receptor antagonist blocked transamidation. Time course studies demonstrate that transamidation of Rac1 is significantly elevated after 5 and 15 min of serotonin treatment, but returns it to control levels after 30 min. The activity of Rac1 is also transiently increased following serotonin stimulation. Inhibition of TGase by cystamine or small interfering RNA reduces TGase modification of Rac1, and cystamine also prevents Rac1 activation. Serotonin itself is bound to Rac1 by TGase following 5-HT(2A) receptor stimulation as demonstrated by coimmunoprecipitation experiments and a dose-dependent decrease of serotonin-associated Rac1 by cystamine. These data support the hypothesis that Rac1 activity is transiently increased due to TGase-catalyzed transamidation of serotonin to Rac1 via stimulation of 5-HT(2A) receptors. Activation of Rac1 via TGase is a novel effector and second messenger of the 5-HT(2A) receptor-signaling cascade in neurons.

Transgenic avian-derived recombinant human interferon-alpha2b (AVI-005) in healthy subjects: an open-label, single-dose, controlled study.

BACKGROUND/AIMS: This study characterized the safety and pharmacological properties of AVI-005, a novel glycosylated recombinant human interferon-alpha2b produced from the egg whites of chickens transfected with human cDNA. METHODS: 18 healthy volunteers received single subcutaneous rising doses (0.5, 1.66 or 5 million international units, MIU) of AVI-005. A randomized parallel comparator group of 10 subjects received 5 MIU of unglycosylated IFN-alpha2b (Intron A). The pharmacokinetic parameters t1/2, tmax, Cmax, AUC0-24h, Vd, and clearance were compared between AVI-005 and unglycosylated IFN-alpa2b. RESULTS: At equipotent doses, AVI-005 had a larger AUC0-24h than the control interferon. Pharmacodynamic markers ofneopterin and beta2-microglobulin for the two treatments were similar. These markers were increased by AVI-005 in a dose-dependent manner. Pharmacodynamic responses to treatment with AVI-005 were shown by the change in mRNA expression for interferon inducible protein kinase and 2'5'-oligoadenylate synthetase. Adverse events in the two groups were qualitatively and quantitatively similar. CONCLUSION: AVI-005 demonstrates biological activity and pharmaco-kinetic properties in humans that support further development.

Protein associated with Myc (PAM) is a potent inhibitor of adenylyl cyclases.

Using the yeast two-hybrid assay and the second of the two large cytosolic domains of type V adenylyl cyclase (ACV) as bait, we identified a small region (amino acids 1028-1231) in the protein associated with Myc (PAM) as an interaction site for ACV. This small region of PAM as well as purified full-length PAM inhibited the activity of ACV. Additionally, full-length PAM was a very potent inhibitor of ACI and AC activities in S49 cyc(-) cells and HeLa cells with IC(50) values in the pm and low nm range. Moreover, the regulator of chromatin condensation 1-like domain of PAM (amino acids 446-1062) was sufficient and as potent as full-length PAM at inhibiting the activity of ACV. Interestingly, full-length PAM did not inhibit ACII activity that was stimulated by either forskolin of Galpha(s). When endogenous levels of PAM in HeLa cells were decreased using antisense oligodeoxynucleotides, the basal cAMP content was elevated, and the dose-response curve for vasoactive intestinal peptide-elicited cAMP accumulation in HeLa cells was shifted to the left. Therefore, we conclude that PAM is a very potent, novel inhibitor of specific isoforms of AC. Furthermore, the regulator of chromatin condensation 1-like domain of PAM is sufficient to exert the effects of the full-length protein on AC and decreases in endogenous PAM levels in HeLa cells can modulate both basal and agonist stimulated cAMP accumulation.

Molecular biological approaches to unravel adenylyl cyclase signaling and function.

Signal transduction through the cell membrane requires the participation of one or more plasma membrane proteins. For many transmembrane signaling events adenylyl cyclases (ACs) are the final effector enzymes which integrate and interpret divergent signals from different pathways. The enzymatic activity of adenylyl cyclases is stimulated or inhibited in response to the activation of a large number of receptors in virtually all cells of the human body. To date, ten different mammalian isoforms of adenylyl cyclase (AC) have been cloned and characterized. Each isoform has its own distinct tissue distribution and regulatory properties, providing possibilities for different cells to respond diversely to similar stimuli. The product of the enzymatic reaction catalyzed by ACs, cyclic AMP (cAMP) has been shown to play a crucial role for a variety of fundamental physiological cell functions ranging from cell growth and differentiation, to transcriptional regulation and apoptosis. In the past, investigations into the regulatory mechanisms of ACs were limited by difficulties associated with their purification and the availability of the proteins in any significant amount. Moreover, nearly every cell expresses several AC isoforms. Therefore, it was difficult to perform biochemical characterization of the different AC isoforms and nearly impossible to assess the physiological roles of the individual isoforms in intact cells, tissue or organisms. Recently, however, different molecular biological approaches have permitted several breakthroughs in the study of ACs. Recombinant technologies have allowed biochemical analysis of adenylyl cyclases in-vitro and the development of transgenic animals as well as knock-out mice have yielded new insights in the physiological role of some AC isoforms. In this review, we will focus mainly on the most novel approaches and concepts, which have delineated the mechanisms regulating AC and unravelled novel functions for this enzyme.

The C terminus of sprouty is important for modulation of cellular migration and proliferation.

The Drosophila Sprouty (SPRY) protein has been shown to inhibit the actions of epidermal growth factor and fibroblast growth factor. However, the role of mammalian SPRY proteins has not been clearly elucidated. We postulated that human Sprouty2 (hSPRY2) is an inhibitor of cellular migration and proliferation. Indeed, using stably transfected HeLa cells, which expressed hemagglutinin (HA)-tagged hSPRY2 or hSPRY2 tagged at the C terminus with red fluorescent protein, we demonstrated that hSPRY2 inhibits the migration of cells in response to serum, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor. Additionally, hSPRY2 also inhibited the growth of HeLa cells in response to serum. Previously, two C-terminal domains on hSPRY2, which are necessary for its colocalization with microtubules (residues 123-177) or translocation to membrane ruffles (residues 178-194), have been identified (Lim, J., Wong, E. S., Ong, S. H., Yusoff, P., Low, B. C., and Guy, G. R. (2000) J. Biol. Chem. 275, 32837-32845). Therefore, using TAT-tagged hSPRY2 and its mutants, we determined the role of these two C-terminal domains in the inhibition of cell migration and proliferation. Our data show that the deletion of either of these two regions in hSPRY2 abrogates its ability to modulate cell migration in response to different growth factors and proliferation in response to serum. Therefore, we conclude that hSPRY2 inhibits the actions of a number of growth factors, and its C terminus, which is homologous among various SPRY isoforms, is important in mediating its biological activity.

Adenylyl cyclase regulates signal onset via the inhibitory GTP-binding protein, Gi.

Adenylyl cyclase, the enzyme that converts ATP to cAMP, is regulated by its stimulatory and inhibitory GTP-binding proteins, G(s) and G(i), respectively. Recently, we demonstrated that besides catalyzing the synthesis of cAMP, type V adenylyl cyclase (ACV) can act as a GTPase-activating protein for Galpha(s) and also enhance the ability of activated receptors to stimulate GTP-GDP exchange on heterotrimeric G(s) (Scholich, K., Mullenix, J. B., Wittpoth, C., Poppleton, H. M., Pierre, S. C., Lindorfer, M. A., Garrison, J. C., and Patel, T. B. (1999) Science 283, 1328-1331). This latter action of ACV would facilitate the rapid onset of signaling via G(s). Because the C1 region of ACV interacts with the inhibitory GTP-binding protein Galpha(i), we investigated whether the receptor-mediated activation of heterotrimeric G(i) was also regulated by ACV and its subdomains. Our data show that ACV and its C1 domain increased the ability of a muscarinic receptor mimetic peptide (MIII-4) to enhance activation of heterotrimeric G(i) such that the amount of peptide required to stimulate G(i) in steady-state GTPase activity assays was 3-4 orders of magnitude less than without the C1 domain. Additionally, the MIII-4-mediated binding of guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) to G(i) was also markedly increased in the presence of ACV or its C1 domain. In contrast, the C2 domain of ACV was not able to alter either the GTPase activity or the GTPgammaS binding to G(i) in the presence of MIII-4. Furthermore, in adenylyl cyclase assays employing S49 cyc(-) cell membranes, the C1 (but not the C2) domain of ACV enhanced the ability of peptide MIII-4 as well as endogenous somatostatin receptors to activate endogenous G(i) and to inhibit adenylyl cyclase activity. These data demonstrate that adenylyl cyclase and its C1 domain facilitate receptor-mediated activation of G(i).

Cysteine 3 is not the site of in vitro palmitoylation on G(salpha).

Several studies have examined the role of palmitoylation of G protein alpha subunits by nonenzymatic in vitro acylation using palmitoyl-CoA. Here, we investigated nonenzymatic palmitoylation of purified G(salpha) in vitro. GDP-bound G(salpha) was stoichiometrically autoacylated on cysteine residue(s) with micromolar concentrations of palmitoyl-CoA. The acylation led to a complete loss of steady-state GTPase activity and GTPgammaS binding to G(salpha). Mutation of Cys 3 to Ala in G(salpha) did not prevent either palmitoylation or its consequent functional alterations. However, stoichiometric palmitoylation of His(6)-G(salpha) did not alter its GTPase activity or GTPgammaS binding. Isoelectric focusing of tryptic peptides from autoacylated wild type, His(6)-tagged, and C3A mutant of G(salpha) showed that Cys 160 is the site of in vitro palmitoylation. Therefore, we conclude that in vitro palmitoylation of G(salpha) occurs on Cys 160 and this modification decreases the ability of the protein to exchange GTP for GDP; N-terminus elongation of G(salpha) prevents this latter effect without altering palmitoylation.

The juxtamembrane region of the epidermal growth factor receptor is required for phosphorylation of Galpha(s).

We have previously demonstrated that Galpha(s) associates with the juxtamembrane region of the epidermal growth factor (EGF) receptor (EGFR) and that the EGFR can phosphorylate and activate this G protein (H. Poppleton et al., 1996, J. Biol. Chem. 271, 6947-6951; H. Sun et al., 1995, Proc. Natl. Acad. Sci. USA 92, 2229-2233). In this report, we have employed peptides EGFR-13 and EGFR-14 (corresponding to amino acids 645-657 and 679-692 in the EGFR, respectively) which disrupt the association of Galpha(s) with the EGFR to investigate whether or not this region of the EGFR is required for phosphorylation of the G protein. EGFR-13 increased the tyrosine phosphorylation of G(alpha)s by two-fold whereas EGFR-14 decreased the phosphorylation of the G protein. Phosphorylation of EGFR-13 on the threonine residue corresponding to Thr654 of the EGFR obliterated the ability of the peptide to increase Galpha(s) phosphorylation. EGFR-13 and EGFR-14, but not phospho-EGFR-13, competed for the association of the EGFR with Galpha(s). A peptide betaIII-2 corresponding to amino acids Arg259-Lys273 in the beta2-adrenergic receptor which competes for association of Galpha(s) with the EGFR and increases protein tyrosine kinase activity of the EGFR could mimic the effects of EGFR-13. Among the three peptides (EGFR-13, EGFR-14, and betaIII-2) that interfere with association of Galpha(s) to the EGFR, only EGFR-13 and betaIII-2 have been shown to activate the G protein. Polylysine which increases EGFR tyrosine kinase activity but does not interfere with association of Galpha(s) and EGFR also augmented phosphorylation of Galpha(s) by the EGFR. Phosphopeptide mapping demonstrated that EGFR-13 and polylysine increased phosphorylation of Galpha(s) by the EGFR on the same additional sites. Collectively, these data suggest that the interaction of Galpha(s) with residues 645-657 of the EGFR, or a peptide corresponding to this sequence alters the conformation of the G protein and/or the EGFR such that Galpha(s) is readily phosphorylated by the EGFR. The peptide EGFR-14, which does not activate Galpha(s), does not allow for the efficient phosphorylation of the G protein even though it does elevate the intrinsic tyrosine kinase activity of the EGFR. The hyperphosphorylation of Galpha(s) by EGFR is likely to require the contact of the G protein with EGFR-13 region (aa 645-657 in the EGFR) as well as augmentation of EGFR kinase activity.

Regions on adenylyl cyclase that are necessary for inhibition of activity by beta gamma and G(ialpha) subunits of heterotrimeric G proteins.

The two large cytoplasmic domains (C1 and C2) of adenylyl cyclases (AC), when expressed separately and mixed together, reconstitute enzyme activity that can be regulated by various modulators. Therefore, we have used the C1 or its C1a subdomain and C2 regions from type I AC (ACI) and type V AC (ACV) to identify the region on ACI that interacts with beta gamma subunits of heterotrimeric G proteins. In addition, we also used a chimeric C1 domain (VC1aIC1b) in which the C1a region was derived from ACV and the C1b region was from ACI. By mixing the C1 or C1a or VC1aIC1b domains with C2 regions of ACI or ACV, we have shown that the C1a region (amino acids 236-471) of ACI is sufficient to observe beta gamma-mediated inhibition of enzyme activity, which is stimulated by either constitutively active G(salpha) (G(salpha)*) or Ca(2+)/calmodulin (CaM). Although the C1b region and C2 domain of ACI were by themselves not sufficient for inhibition of activity by beta gamma subunits, the presence of both of these regions formed another beta gamma interaction site that was sufficient to observe G(salpha)*- or Ca(2+)/CaM-stimulated activity. Inhibition of AC activity attributable to interaction of beta gamma subunits at either of the two sites was blocked by a peptide (QEHA) that has previously been shown to inhibit the effects of beta gamma on various effectors. Moreover, the C1 region of ACI was sufficient to observe G(ialpha1)-elicited inhibition of Ca(2+)/CaM-stimulated activity. Although the C1a region of ACV was sufficient for inhibition of activity by G(ialpha1), the presence of C1b region from either ACI or ACV increased sensitivity to inhibition by the inhibitory G protein. Thus, the inhibitory influences of G(ialpha1) are mediated on the C1 regions of both ACI and ACV. The effects of beta gamma on ACI can be mediated by interactions with the C1a region and a beta gamma interacting site formed by the C1b and C2 domains of this enzyme.

Transmodulation of epidermal growth factor receptor function by cyclic AMP-dependent protein kinase.

Binding of epidermal growth factor (EGF) to its receptor (EGFR) augments the tyrosine kinase activity of the receptor and autophosphorylation. Exposure of some tissues and cells to EGF also stimulates adenylyl cyclase activity and results in an increase in cyclic AMP (cAMP) levels. Because cAMP activates the cAMP-dependent protein kinase A (PKA), we investigated the effect of PKA on the EGFR. The purified catalytic subunit of PKA (PKAc) stoichiometrically phosphorylated the purified full-length wild type (WT) and kinase negative (K721M) forms of the EGFR. PKAc phosphorylated both WT-EGFR as well as a mutant truncated form of EGFR (Delta1022-1186) exclusively on serine residues. Moreover, PKAc also phosphorylated the cytosolic domain of the EGFR (EGFRKD). Phosphorylation of the purified WT as well as EGFRDelta1022-1186 and EGFRKD was accompanied by decreased autophosphorylation and diminished tyrosine kinase activity. Pretreatment of REF-52 cells with the nonhydrolyzable cAMP analog, 8-(4-chlorophenylthio)-cAMP, decreased EGF-induced tyrosine phosphorylation of cellular proteins as well as activation of the WT-EGFR. Similar effects were also observed in B82L cells transfected to express the Delta1022-1186 form of EGFR. Furthermore, activation of PKAc in intact cells resulted in serine phosphorylation of the EGFR. The decreased phosphorylation of cellular proteins and diminished activation of the EGFR in cells treated with the cAMP analog was not the result of altered binding of EGF to its receptors or changes in receptor internalization. Therefore, we conclude that PKA phosphorylates the EGFR on Ser residues and decreases its tyrosine kinase activity and signal transduction both in vitro and in vivo.

Modulation of the protein tyrosine kinase activity and autophosphorylation of the epidermal growth factor receptor by its juxtamembrane region.

Using peptides epidermal growth factor receptor (EGFR)-13 and EGFR-14, which correspond to residues 645-657 and 679-692, respectively, in the juxtamembrane, cytosolic region of the epidermal growth factor receptor (EGFR) we have investigated the role of specific regions of the receptor in regulating its autophosphorylation and protein tyrosine kinase activity. EGFR-13, but not EGFR-14, increased autophosphorylation (by twofold) of the full-length and two truncated forms (Delta1022-1186 and a constitutively active receptor kinase domain) of the EGFR. EGFR-13 increased the stoichiometry of tyrosine phosphorylation of the full-length receptor from 4.2 to 10.1 mol Pi/mol EGFR and that of EGFRDelta1022-1186 from 1.0 to 2 mol Pi/mol receptor. Increased receptor autophosphorylation in the presence of EGFR-13 cannot solely be attributed to an increase in tyrosine kinase activity because EGFR-14 and polylysine increased tyrosine kinase activity of EGFRDelta1022-1186 and full-length EGFR, respectively, to the same extent as EGFR-13 without any effects on receptor autophosphorylation. Phosphorylation of EGFR-13 (P-EGFR-13) on the threonine residue corresponding to Thr654 in EGFR obliterated the ability of the peptide to increase autophosphorylation and markedly diminished its capacity to increase receptor tyrosine kinase activity. Additionally, EGFR-13, but not EGFR-14 or P-EGFR-13, decreased the migration of the receptor on nondenaturing gels, indicating that EGFR-13 induces some conformational change. Phosphopeptide maps of the EGFR phosphorylated in the presence of EGFR-13 or pp60(c-src) demonstrated that the additional sites phosphorylated in the presence of EGFR-13 were the same as those phosphorylated by pp60(c-src) (i.e., Y803, Y845, Y891, Y920, and Y1101). Thus, we conclude that EGFR-13, but not EGFR-14 or P-EGFR-13, competes to disrupt interactions between amino acids 645-657 and some other region(s) on the EGFR to either alleviate a conformational constraint or alter dimer conformation. This change increases the protein tyrosine kinase activity of the EGFR and provides access to additional tyrosine autophosphorylation sites in the receptor.

Polyamine depletion arrests cell cycle and induces inhibitors p21(Waf1/Cip1), p27(Kip1), and p53 in IEC-6 cells.

The polyamines spermidine and spermine and their precursor putrescine are intimately involved in and are required for cell growth and proliferation. This study examines the mechanism by which polyamines modulate cell growth, cell cycle progression, and signal transduction cascades. IEC-6 cells were grown in the presence or absence of DL-alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, which is the first rate-limiting enzyme for polyamine synthesis. Depletion of polyamines inhibited growth and arrested cells in the G1 phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the level of p53 protein and other cell cycle inhibitors, including p21(Waf1/Cip1) and p27(Kip1). Induction of cell cycle inhibitors and p53 did not induce apoptosis in IEC-6 cells, unlike many other cell lines. Although polyamine depletion decreased the expression of extracellular signal-regulated kinase (ERK)-2 protein, a sustained increase in ERK-2 isoform activity was observed. The ERK-1 protein level did not change, but ERK-1 activity was increased in polyamine-depleted cells. In addition, polyamine depletion induced the stress-activated protein kinase/c-Jun NH2-terminal kinase (JNK) type of mitogen-activated protein kinase (MAPK). Activation of JNK-1 was the earliest event; within 5 h after DFMO treatment, JNK activity was increased by 150%. The above results indicate that polyamine depletion causes cell cycle arrest and upregulates cell cycle inhibitors and suggest that MAPK and JNK may be involved in the regulation of the activity of these molecules.

Interaction of asparagine and EGF in the regulation of ornithine decarboxylase in IEC-6 cells.

Our laboratory has shown that asparagine (ASN) stimulates both ornithine decarboxylase (ODC) activity and gene expression in an intestinal epithelial cell line (IEC-6). The effect of ASN is specific, and other A- and N-system amino acids are almost as effective as ASN when added alone. In the present study, epidermal growth factor (EGF) was unable to increase ODC activity in cells maintained in a salt-glucose solution (Earle's balanced salt solution). However, the addition of ASN (10 mM) in the presence of EGF (30 ng/ml) increased the activity of ODC 0.5- to 4-fold over that stimulated by ASN alone. EGF also showed induction of ODC with glutamine and alpha-aminoisobutyric acid, but ODC induction was maximum with ASN and EGF. Thus the mechanism of the interaction between ASN and EGF is important for understanding the regulation of ODC under physiological conditions. Therefore, we examined the expression of the ODC gene and those for several protooncogenes under the same conditions. Increased expression of the genes for c-Jun and c-Fos but not for ODC occurred with EGF alone. The addition of ASN did not further increase the expression of the protooncogenes, but the combination of EGF and ASN further increased the expression of ODC over that of ASN alone. Western analysis showed no significant difference in the level of ODC protein in Earle's balanced salt solution, ASN, EGF, or EGF plus ASN. Addition of cycloheximide during ASN and ASN plus EGF treatment completely inhibited ODC activity without affecting the level of ODC protein. These results indicated that 1) the increased expression of protooncogenes in response to EGF is independent of increases in ODC activity and 2) potentiation between EGF and ASN on ODC activity may not be due to increased gene transcription but to posttranslational regulation and the requirement of ongoing protein synthesis involving a specific factor dependent on ASN.

Facilitation of signal onset and termination by adenylyl cyclase.

The alpha subunit (Gsalpha) of the stimulatory heterotrimeric guanosine triphosphate binding protein (G protein) Gs activates all isoforms of mammalian adenylyl cyclase. Adenylyl cyclase (Type V) and its subdomains, which interact with Gsalpha, promoted inactivation of the G protein by increasing its guanosine triphosphatase (GTPase) activity. Adenylyl cyclase and its subdomains also augmented the receptor-mediated activation of heterotrimeric Gs and thereby facilitated the rapid onset of signaling. These findings demonstrate that adenylyl cyclase functions as a GTPase activating protein (GAP) for the monomeric Gsalpha and enhances the GTP/GDP exchange factor (GEF) activity of receptors.

Polyamine deficiency alters EGF receptor distribution and signaling effectiveness in IEC-6 cells.

Cell growth and migration are essential processes for the differentiation, maintenance, and repair of the intestinal epithelium. Epidermal growth factor (EGF) is an important factor in the reorganization of the cytoskeleton required for both processes. Because we had previously found significant changes in the cytoskeleton during polyamine deficiency, it was of interest to know whether those changes could prevent EGF from stimulating growth and migration. Polyamine biosynthesis in IEC-6 cells was interrupted by treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase, the primary rate-limiting enzyme of polyamine biosynthesis. DFMO halted cell proliferation and inhibited cell migration, and neither function could be normally stimulated by EGF. Immunocytochemistry of the transferrin receptor (used as a marker for the endocytic pathway) revealed an abnormal distribution of the EGF receptor (EGFR) 10 min after binding EGF. Polyamine deficiency depleted the cells of interior microfilaments, thickened the actin cortex, and prevented the prompt association of EGF-bound EGFR with actin. EGF-stimulated 170-kDa protein tyrosine phosphorylation and the kinase activity of purified membrane EGFR were reduced by 50%. Immunoprecipated EGFR protein concentration, however, was not reduced by polyamine deficiency. All of these changes could be prevented by supplementation with putrescine. Cytoskeletal disruption, reduced EGFR phosphorylation and kinase activity, aberrant intracellular EGFR distribution, and delayed association with actin filaments suggest a partial explanation for the dependence of epithelial cell growth and migration on polyamines.

Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsalpha.

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the alpha subunit of the stimulatory GTP-binding protein of AC (Gsalpha) to stimulate enzyme activity. Concentration-response curves with Gsalpha suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsalpha interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572-F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsalpha-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042-T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsalpha concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042-T1051) in the C2 domain of the enzyme to modulate Gsalpha-elicited stimulation of activity.

Characterization of soluble forms of nonchimeric type V adenylyl cyclases.

Type V adenylyl cyclase (ACV) belongs to the family of Ca2+-inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the alpha subunit of the stimulatory G protein Gs (G(s alpha)) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the alpha subunit of the inhibitory G protein Gi (G(i alpha)) inhibited all forms of the enzyme, indicating that the regions for G(s alpha) and G(i alpha) interaction are preserved in the soluble forms. Ca2+ inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G(s alpha) and forskolin obliterated the Ca2+-mediated inhibition of the full-length and C1-C2 forms of ACV. In 45Ca2+ overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca2+. Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound 45Ca2+. To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G(s alpha) and G(i alpha) is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca2+ and inhibition of enzyme activity.

The juxtamembrane, cytosolic region of the epidermal growth factor receptor is involved in association with alpha-subunit of Gs.

Previously, we have demonstrated that epidermal growth factor (EGF) can stimulate adenylyl cyclase activity via activation of Gs in the heart. Moreover, we have recently shown that Gsalpha is phosphorylated by the EGF receptor protein tyrosine kinase and that the juxtamembrane region of the EGF receptor can stimulate Gs directly. Therefore, employing isolated cardiac membranes, the two-hybrid assay, and in vitro association studies with purified EGF receptor and Gsalpha we have investigated Gsalpha complex formation with the EGF receptor and elucidated the region in the receptor involved in this interaction. In isolated cardiac membranes, immunoprecipitation of EGF receptor was accompanied by co-immunoprecipitation of Gsalpha. In the yeast two-hybrid assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met644-Trp707) associated with Gsalpha. However, interactions of these regions of the EGF receptor with constitutively active Gsalpha were diminished in the two-hybrid assay. Employing purified proteins, our studies demonstrate that the EGF receptor, directly and stoichiometrically, associates with Gsalpha (1 mol of Gsalpha/mol of EGF receptor). This association was not altered in the presence or absence of ATP and therefore, was independent of tyrosine phosphorylation of either of the proteins. Peptides corresponding to the juxtamembrane region of the receptor decreased association of the EGF receptor with Gsalpha. However, neither the C-terminally truncated EGF receptor (Delta1022-1186) nor a peptide corresponding to residues 985-996 of the receptor altered association with Gsalpha, thus indicating the selectivity of the G protein interaction with the juxtamembrane region. Interestingly, peptides corresponding to N and C termini of Gsalpha did not alter the association of Gsalpha with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active Gsalpha poorly associated with the EGF receptor, in vitro experiments with purified proteins also demonstrated that activation of Gsalpha by guanosine 5'-3-O-(thio)triphosphate decreased the association of G protein with the EGF receptor. Thus we conclude that the juxtamembrane region of the EGF receptor, directly and stoichiometrically, associates with Gsalpha and that upon activation of Gsalpha this association is decreased.

Water transport in collecting ducts of Japanese quail.

Previously, we reported that the countercurrent urine concentration mechanism in birds appears to operate by recycling of a single solute (NaCl), in which the thick ascending limb of looped nephrons provides an energy source. To determine the importance of the medullary collecting duct (MCD) in the countercurrent multiplier system, we examined in isolated and perfused MCDs from Japanese quail, Coturnix coturnix, the osmotic and/or diffusional water permeability and whether arginine vasotocin (AVT) regulates water permeability. We noted that dark cells that possess electron-dense cytoplasm and numerous mitochondria and light mucus-secreting cells exist in the cortical collecting duct (CD), whereas only mucus-secreting cells are present in the MCDs. The volume flux (Jv) in the MCDs from intact birds and that from the water-deprived birds were nearly zero; after exposure to a hyperosmotic bath and AVT (2 x 10(-5) M), the Jv was significantly higher in water-deprived birds. The diffusional water permeability (Pdw) was moderately high in MCDs bathed in an isosmotic bath in which the Pdw was increased slightly by AVT (10(-5) M, bath) and more markedly (10(-5) M) by forskolin (Fsk), whereas 1,9-dideoxy Fsk (an inactive analogue) showed no effect. Furthermore, the basal adenosine 3',5'-cyclic monophosphate (cAMP) levels were higher in the medulla than in the cortex and were stimulated only slightly by AVT (10(-5) M) and markedly by Fsk (10(-4) M) in both the cortex and medulla. These results in the C. coturnix CD indicate the following. 1) Two types of cells are present; whereas dark cells resemble mammalian intercalated cells morphologically, it is not certain whether mucus-secreting cells are equivalent to principal cells. 2) AVT increases Pdw via a cAMP mechanism; the relatively high basal Pdw and minor effect of AVT on Jv and Pdw suggest, however, that diffusional water movement across the MCD may occur without significant direct control by AVT.