The angiotensin II AT2 receptor is an AT1 receptor antagonist

The vasopressor angiotensin II activates AT(1) and AT(2) receptors. Most of the known in vivo effects of angiotensin II are mediated by AT(1) receptors while the biological functions of AT(2) receptors are less clear. We report here that the AT(2) receptor binds directly to the AT(1) receptor and thereby antagonizes the function of the AT(1) receptor. The AT(1)-specific antagonism of the AT(2) receptor was independent of AT(2) receptor activation and signaling, and it was effective on different cells and on human myometrial biopsies with AT(1)/AT(2) receptor expression. Thus, the AT(2) receptor is the first identified example of a G-protein-coupled receptor which acts as a receptor-specific antagonist.     

Phosphorylation of tyrosine 319 of the angiotensin II type 1 receptor mediates angiotensin II-induced trans-activation of the epidermal growth factor receptor

Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR.     

Identification of a second egfr gene in Xiphophorus uncovers an expansion of the epidermal growth factor receptor family in fish

The epidermal growth factor receptor (EGFR) gives name to a family of receptors formed by four members in mammals (EGFR, ErbB2, ErbB3, and ErbB4). Members of this family can be activated to become potent oncogenes, and many human and animal tumors express qualitatively or quantitatively altered receptors from this group. We have isolated and characterized a second egfr gene in the melanoma model fish Xiphophorus. Both Xiphophorus egfra and egfrb duplicates are co-orthologs of the mammalian egfr gene. Database analysis showed that not only egfr but also erbB3 and erbB4 are present as duplicates in some fish species. They originated from ancient duplication events that might be consistent with the hypothesis of a fish-specific genome duplication. In Xiphophorus, the egfrb gene underwent a second duplication that generated the melanoma-inducing oncogene Xmrk. The study and comparison of some of the functional characteristics of both Xiphophorus EGF receptors, including expression profile, ligand-binding abilities, and intracellular signal transduction revealed that Xiphophorus Egfra not only shares common features with Egfrb and the human EGFR but also shows significant differences in its functional characteristics. The mechanism of maintenance of these duplicates remains to be clarified.     

Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells

The epidermal growth factor receptor (EGFR) is a cyto-skeleton-binding protein. Although purified EGFR can interact with acting in vitro and normally at least 10% of EGFR exist in the insoluble cytoskeleton fraction of A431 cells, interaction of cytosolic EGFR with actin can only be visualized by fluorescence resonance energy transfer when epidermal growth factor presents in the cell medium. Results indicate that the correct orientation between EGFR and actin is important in the signal transduction process.     

Delineating the functional map of the interaction between nimotuzumab and the epidermal growth factor receptor

Molecular details of epidermal growth factor receptor (EGFR) targeting by nimotuzumab, a therapeutic anti-cancer antibody, have been largely unknown. The current study delineated a functional map of their interface, based on phage display and extensive mutagenesis of both the target antigen and the Fv antibody fragment. Five residues in EGFR domain III (R353, S356, F357, T358, and H359T) and the third hypervariable region of nimotuzumab heavy chain were shown to be major functional contributors to the interaction. Fine specificity differences between nimotuzumab and other anti-EGFR antibodies were revealed. Mapping information guided the generation of a plausible in silico binding model. Knowledge about the epitope/paratope interface opens new avenues for the study of tumor sensitivity/resistance to nimotuzumab and for further engineering of its binding site. The developed mapping platform, also validated with the well-known cetuximab epitope, allows a comprehensive exploration of antigenic regions and could be expanded to map other anti-EGFR antibodies.     

An electrostatic engine model for autoinhibition and activation of the epidermal growth factor receptor (EGFR/ErbB) family

We propose a new mechanism to explain autoinhibition of the epidermal growth factor receptor (EGFR/ErbB) family of receptor tyrosine kinases based on a structural model that postulates both their juxtamembrane and protein tyrosine kinase domains bind electrostatically to acidic lipids in the plasma membrane, restricting access of the kinase domain to substrate tyrosines. Ligand-induced dimerization promotes partial trans autophosphorylation of ErbB1, leading to a rapid rise in intracellular [Ca(2+)] that can activate calmodulin. We postulate the Ca(2+)/calmodulin complex binds rapidly to residues 645--660 of the juxtamembrane domain, reversing its net charge from +8 to -8 and repelling it from the negatively charged inner leaflet of the membrane. The repulsion has two consequences: it releases electrostatically sequestered phosphatidylinositol 4,5-bisphosphate (PIP(2)), and it disengages the kinase domain from the membrane, allowing it to become fully active and phosphorylate an adjacent ErbB molecule or other substrate. We tested various aspects of the model by measuring ErbB juxtamembrane peptide binding to phospholipid vesicles using both a centrifugation assay and fluorescence correlation spectroscopy; analyzing the kinetics of interactions between ErbB peptides, membranes, and Ca(2+)/calmodulin using fluorescence stop flow; assessing ErbB1 activation in Cos1 cells; measuring fluorescence resonance energy transfer between ErbB peptides and PIP(2); and making theoretical electrostatic calculations on atomic models of membranes and ErbB juxtamembrane and kinase domains.     

Modeling the epidermal growth factor -- epidermal growth factor receptor l2 domain interaction: implications for the ligand binding process

Signaling from the epidermal growth factor (EGF) receptor is triggered by the binding of ligands such as EGF or transforming growth factor alpha (TGF-alpha) and subsequent receptor dimerization. An understanding of these processes has been hindered by the lack of structural information about the ligand-bound, dimerized EGF receptor. Using an NMR-derived structure of EGF and a homology model of the major ligand binding domain of the EGF receptor and experimental data, we modeled the binding of EGF to this EGF receptor fragment. In this low resolution model of the complex, EGF sits across the second face of the EGF receptor L2 domain and EGF residues 10-16, 36-37, 40-47 bind to this face. The structural model is largely consistent with previously published NMR data describing the residues of TGF-alpha which interact strongly with the EGF receptor. Other EGF residues implicated in receptor binding are accounted by our proposal that the ligand binding is a two-step process with the EGF binding to at least one other site of the receptor. This three-dimensional model is expected to be useful in the design of ligand-based antagonists of the receptor.     

Heparin-binding epidermal growth factor and its receptor ErbB4 mediate implantation of the human blastocyst

The mechanisms that mediate implantation of the human embryo remain poorly understood and represent a fundamental problem in reproductive biology. Candidate molecules that mediate and facilitate implantation have been identified in animal studies, and include heparin binding epidermal growth factor. Here we demonstrate a potential function for the transmembrane form of heparin-binding epidermal growth factor in mediating blastocyst attachment to the endometrium, in two different novel in vitro models for human implantation. Furthermore, we demonstrate specific localisation of the heparin-binding epidermal growth factor receptor ErbB4, on the surface of the trophectoderm in peri-implantation human blastocysts. Our data lead the way for further dissection of the molecular mechanisms of implantation of the human embryo, and have implications for infertility, in vitro fertilization and contraception.     

Growth stimulation of non-small cell lung cancer cell lines by antibody against epidermal growth factor receptor promoting formation of ErbB2/ErbB3 heterodimers

Antibodies are the most rapidly expanding class of human therapeutics, including their use in cancer therapy. Monoclonal antibodies (mAb) against epidermal growth factor (EGF) receptor (EGFR) generated for cancer therapy block the binding of ligand to various EGFR-expressing human cancer cell lines and abolish ligand-dependent cell proliferation. In this study, we show that our mAb against EGFRs, designated as B4G7, exhibited a growth-stimulatory effect on various human cancer cell lines including PC-14, a non-small cell lung cancer cell line; although EGF exerted no growth-stimulatory activity toward these cell lines. Tyrosine phosphorylation of EGFRs occurred after treatment of PC-14 cells with B4G7 mAb, and it was completely inhibited by AG1478, a specific inhibitor of EGFR tyrosine kinase. However, this inhibitor did not affect the B4G7-stimulated cell growth, indicating that the growth stimulation by B4G7 mAb seems to be independent of the activation of EGFR tyrosine kinase. Immunoprecipitation with anti-ErbB3 antibody revealed that B4G7, but not EGF, stimulated heterodimerization between ErbB2 and ErbB3. ErbB3 was tyrosine phosphorylated in the presence of B4G7 but not in the presence of EGF. Further, the phosphorylation and B4G7-induced increase in cell growth were inhibited by AG825, a specific inhibitor of ErbB2. These results show that the ErbB2/ErbB3 dimer functions to promote cell growth in B4G7-treated cells. Changes in receptor-receptor interactions between ErbB family members after inhibition of one of its members are of potential importance in optimizing current EGFR family-directed therapies for cancer.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Role of the third intracellular loop of the angiotensin II receptor subtype AT2 in ligand-receptor interaction

Angiotensin II (Ang II) receptor subtypes AT1 and AT2 share 34% overall homology, but the least homology is in their third intracellular loop (3rd ICL). In an attempt to elucidate the role of the 3rd ICL in determining the similarities and differences in the functions of the AT1 and the AT2 receptors, we generated a chimeric receptor in which the 3rd ICL of the AT2 receptor was replaced with that of the AT1 receptor. Ligand-binding properties and signaling properties of this receptor were assayed by expressing this receptor in Xenopus oocytes. Ligand-binding studies using [125I-Sar1-Ile8] Ang II, a peptidic ligand that binds both the AT1 and the AT2 receptor subtypes, and 125I-CGP42112A, a peptidic ligand that is specific for the AT2 receptor, showed that the chimeric receptor has lost affinity to both ligands. However, IP3 levels of the oocytes expressing the chimeric receptor were comparable to the IP3 levels of the oocytes expressing the AT1 receptor, suggesting that the chimeric receptors could couple to phospholipase C pathway in response to Ang II. We have shown previously that the nature of the amino acid present in the position 215 located in the fifth transmembrane domain (TMD) of the AT2 receptor plays an important role in determining its affinity to different ligands. Our results from the ligand-binding studies of the chimeric receptor further support the idea that the structural organization of the region spanning the 5th TMD and the 3rd ICL of the AT2 receptor has an important role in determining the ligand-binding properties of this receptor.     

Molecular cloning, characterization, and distribution of the gerbil angiotensin II AT2 receptor

We isolated a cDNA clone encoding the gerbil AT2 receptor (gAT2) gene from a gerbil adrenal gland cDNA library. The full-length cDNA contains a 1,089-bp open reading frame encoding 363 amino acid residues with 90.9, 96.1, and 95.6% identity with the human (hAT2), rat (rAT2), and mouse AT2 (mAT2) receptors, respectively. There are at least seven nonconserved amino acids in the NH2-terminal domain and in positions Val196, Val217, and Met293, important for angiotensin (ANG) II but not for CGP-42112 binding. Displacement studies in adrenal sections revealed that affinity of the gAT2 receptor was 10-20 times lower for ANG II, ANG III, and PD-123319 than was affinity of the rAT2 receptor. The affinity of each receptor remained the same for CGP-42112. When transfected into COS-7 cells, the gAT2 receptor shows affinity for ANG II that is three times lower than that shown by the hAT2 receptor, whereas affinities for ANG III and the AT2 receptor ligands CGP-42112 and PD-123319 were similar. Autoradiography in sections of the gerbil head showed higher binding in muscles, retina, skin, and molars at embryonic day 19 than at 1 wk of age. In situ hybridization and emulsion autoradiography revealed that at embryonic day 19 the gAT2 receptor mRNA was highly localized to the base of the dental papilla of maxillary and mandibular molars. Our results suggest selective growth-related functions in late gestation and early postnatal periods for the gAT2 receptor and provide an essential basis for future mutagenesis studies to further define structural requirements for agonist binding.     

Mechanical stretch stimulates protein kinase B/Akt phosphorylation in epidermal cells via angiotensin II type 1 receptor and epidermal growth factor receptor

Mechanical stress is known to modulate fundamental events such as cell life and death. Mechanical stretch in particular has been identified as a positive regulator of proliferation in skin keratinocytes and other cell systems. In the present study it was investigated whether antiapoptotic signaling is also stimulated by mechanical stretch. It was demonstrated that mechanical stretch rapidly induced the phosphorylation of the proto-oncogene protein kinase B (PKB)/Akt at both phosphorylation sites (serine 473/threonine 308) in different epithelial cells (HaCaT, A-431, and human embryonic kidney-293). Blocking of phosphoinositide 3-OH kinase by selective inhibitors (LY-294002 and wortmannin) abrogated the stretch-induced PKB/Akt phosphorylation. Furthermore mechanical stretch stimulated phosphorylation of epidermal growth factor receptor (EGFR) and the formation of EGFR membrane clusters. Functional blocking of EGFR phosphorylation by either selective inhibitors (AG1478 and PD168393) or dominant-negative expression suppressed stretch-induced PKB/Akt phosphorylation. Finally, the angiotensin II type 1 receptor (AT1-R) was shown to induce positive transactivation of EGFR in response to cell stretch. These findings define a novel signaling pathway of mechanical stretch, namely the activation of PKB/Akt by transactivation of EGFR via angiotensin II type 1 receptor. Evidence is provided that stretch-induced activation of PKB/Akt protects cells against induced apoptosis.     

Cross-talk between the receptor tyrosine kinases Ron and epidermal growth factor receptor

Heterogeneous receptor-receptor interactions may play a role in intracellular signaling. Accordingly, the interaction of two dissimilar tyrosine kinase receptors, Ron and epidermal growth factor receptor (EGFR) was investigated. The functional interaction of Ron and EGFR in cell scatter and oncogenic transformation was investigated in vivo. Transfection of a dominant negative form of EGFR into human embryonic kidney cells stably expressing Ron (293-Ron) dramatically reduced the scatter response induced by the Ron ligand hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL). The scatter response of the 293-Ron cells was also attenuated by treatment of the cells with the specific EGFR inhibitor AG 1478. Co-transfection of Ron and dominant-negative EGFR, or co-transfection of EGFR and a dominant-negative form of Ron reduced focus formation in NIH/3T3 cells. Western analysis of NIH/3T3 cells overexpressing murine Ron and expressing endogenous levels of EGFR was used to demonstrate that Ron and EGFR co-immunoprecipitate. Stimulation of the cells in vitro with the Ron ligand HGFL or with the EGFR ligand epidermal growth factor (EGF) appeared to induce phosphorylation of both receptors. Co-immunoprecipitation and phosphorylation of phosphatidyl inositol 3-kinase (PI3-K) was also observed. This novel finding of a functional and biochemical interaction between Ron and EGFR suggests that heterologous tyrosine kinase receptor interactions may play a role in cellular processes such as scatter and transformation.     

Differential and synergistic effects of epidermal growth factor receptor antibodies on unliganded ErbB dimers and oligomers

Antibodies directed against the epidermal growth factor receptor (EGFR) offer a potentially powerful therapeutic approach against cancers driven by the EGFR pathway. EGFR antibodies are believed to halt cell surface activation by blocking ligand-induced receptor tyrosine kinase activation, i.e., ligand binding, a change in conformation, or the monomer-dimer transition. In this work, we demonstrate that wild-type EGFR and the truncated de2-7-EGFR (tumor-associated mutant) formed unliganded homo-oligomers and examined the effects of two clinically relevant antibodies on the conformation and quaternary state of these ligand-free EGFR oligomers on the surface of cells. The EGFR antibodies were mAb528, a ligand-blocking antibody that binds domain III, and mAb806, a conformationally sensitive antibody that binds near the dimer interface in domain II. We used a model cellular system, BaF/3 cells, with GFP-tagged receptors in the absence of interference from secreted ligands or other erbB receptor members. Different antibody-mediated effects (conformational transition, receptor cross-linking, or receptor dissociation) were distinguished by combining two complementary biophysical techniques: image correlation spectroscopy (submicrometer scale clustering) and homo-Forster resonance energy transfer (association and/or conformation on a 1-10 nm scale). mAb528 cross-linked EGFR into an inactive EGFR dimer of dimers but had no effect when added to de2-7-EGFR oligomers. mAb806 had a minor effect on EGFR dimers as expected from its poor binding to a conformationally shielded epitope on wtEGFR but bound de2-7-EGFR oligomers, causing a conformational change in the intracellular C-terminal GFP-tagged tail. The combination of the two antibodies had synergistic effects, increasing the level of cross-linking of de2-7-EGFR, but did not lead to enhanced cross-linking of EGFR. The results reveal new modes of receptor-antibody interactions for EGFR and de2-7-EGFR.