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.

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.

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.

Transcriptional activation of the minimal human Proalpha1(I) collagen promoter: obligatory requirement for Sp1.

A construct containing human Proalpha1(I) collagen gene promoter/enhancer-driven chloramphenicol acetyltransferase (CAT), pCOL-KT, failed to be expressed significantly in Sp1-deficient Schneider Drosophila line 2 (SL2) cells. However, CAT expression was induced 200-fold in SL2 cells co-transfected with pCOL-KT and pPACSp1, an Sp1-expression vector driven by the Drosophila actin 5C promoter. Elimination of the four potential Sp1-binding sites from pCOL-KT (pCOL-KTDeltaI), by removal of the first intron, did not abrogate Sp1-mediated induction of CAT. Even more significantly, a minimal Proalpha1(I) collagen promoter (-100 to +117 bp), containing a TATA box (-28 to -25 bp) and one putative Sp1-binding site (-87 to -82 bp), elicited strong Sp1-induced transactivation. Furthermore, mutation of the Sp1 motif in the minimal Proalpha1(I) collagen promoter-CAT construct abolished Sp1-induced expression of the reporter gene. Purified Sp1 protein bound specifically to DNA fragments of the Proalpha1(I) minimal promoter encompassing the putative Sp1-binding site; Sp1 binding could be competed out by a double-stranded oligonucleotide containing the wild-type Sp1 sequence, while an oligonucleotide containing a mutated Sp1 site failed to compete. Based on these results, we postulate that Sp1 plays an obligatory role in the transcriptional activation of the human Proalpha1(I) collagen gene. Additionally, we propose that a bona fide Sp1 motif, located most proximal to the TATA box, is necessary and sufficient for Sp1-mediated activation of the minimal Proalpha1(I) collagen promoter.

Interleukin 1 alpha and an interleukin 1 beta fragment are somnogenic.

The somnogenic activity of interleukin 1 beta (IL-1 beta) has previously been established. Interleukin 1 alpha (IL-1 alpha) is a distinct gene product that possesses similar biological activities. We report here that IL-1 alpha, like IL-1 beta, has the capacity in rabbits to enhance non-rapid-eye-movement sleep, electroencephalographic slow-wave (0.5-3.5 Hz) voltages, and body temperatures and to inhibit rapid-eye-movement sleep. After IL-1 alpha, sleep remained episodic, and at the doses used no abnormal behavior was observed. Several synthetic IL-1 alpha and IL-1 beta peptides were also tested in vivo for somnogenic and pyrogenic activity and in vitro for their ability to stimulate prostaglandin E2 (PGE2) production by fibroblasts and proliferation of T-cells. Only IL-1 beta-(208-240) enhanced non-rapid-eye-movement sleep and body temperature, although both IL-1 beta-(208-240) and IL-1 alpha-(223-250) stimulated PGE2 production; both of these peptides failed to stimulate T-cell production. In contrast, four other IL-1 peptides were nonpyrogenic and somnogenically inactive yet stimulated T-cell proliferation. We conclude that the components of IL-1 required for sleep and temperature activities are different from those required for T-cell proliferation.

Stimulation of glycosaminoglycan synthesis in cultured human dermal fibroblasts by interleukin 1. Induction of hyaluronic acid synthesis by natural and recombinant interleukin 1s and synthetic interleukin 1 beta peptide 163-171.

Hyaluronic acid (HA) is believed to play a critical role in wound healing and in morphogenesis. Factors controlling the production of HA by fibroblasts in normal and pathological states are not completely understood. In this report we have observed that natural human interleukin (IL-1)1 beta and human recombinant (hrIL)-1 alpha and beta are potent stimulators of HA production by fibroblasts in vitro. Hyaluronic acid is the major species of glycosaminoglycan (GAG) stimulated by IL-1 in fibroblasts. PGE2 does not appear to be involved directly in this IL-1 effect on fibroblasts, but stimulation of HA production by IL-1 is dependent on protein synthesis. The synthetic human IL-1 beta peptide 163-171 (Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys), which has been previously shown to stimulate thymocyte proliferation but not fibroblast PGE2 production, is also able to stimulate fibroblast HA production. The synthesis and secretion of IL-1 by mononuclear phagocytes at sites of inflammation and immune reactions in vivo could potentially serve as a signal for fibroblasts to synthesize HA, which in turn could serve to facilitate and modulate reparative and immune processes by virtue of its ability to alter cell-cell, cell matrix, and cell-membrane receptor interactions.