N-terminal Immunoglobulin-like Domain Research Articles

Abstract 5199: Preclinical activity of the FGFR2-targeted thorium-227 conjugate in preclinical models of colorectal, gastric and triple-negative breast cancer

Abstract FGFR2 is a transmembrane tyrosine kinase receptor, consisting of three extracellular N-terminal immunoglobulin-like domains which are involved in ligand-binding as well as in receptor dimerization. Ligand-independent activation of FGFR2 signaling either via genomic amplification, gene fusion events, mRNA overexpression, or mutations has been observed e.g. in gastric cancer, colorectal cancer (CRC), and triple-negative breast cancer (TNBC). As such, FGFR2 has been described to be involved in cancer progression, promotion of oncogenesis, neoangiogenesis, as well as resistance to targeted therapies. Overexpression of FGFR2 and relatively low levels of cell surface expression of FGFR2 in normal human tissues renders FGFR2 an attractive candidate to explore targeted alpha therapy (TAT). We describe the generation of a high energy, alpha-particle emitting FGFR2 targeted thorium-227 conjugate (FGFR2-TTC). The FGFR2-TTC consists of a fully human FGFR2 binding IgG1 antibody (BAY 1179470) cross-reactive with mouse FGFR2, covalently linked via an amide bond to a chelator moiety (3,2 HOPO), enabling radiolabeling with the alpha particle emitting thorium-227 (227Th). In vitro cytotoxicity experiments with FGFR2-TTC demonstrated potency in the sub-picomolar range compared to a non-targeting control-TTC and a correlation between decrease in cell viability and increasing number of anti-FGFR2 antibodies bound per cell (ABC counts) in a panel of FGFR2-positive cancer cell lines. Upon treatment of cells with FGFR2-TTC, the DNA damage response marker protein γH2AX was up-regulated indicating that the mode-of-action involves induction of DNA double strand breaks. Furthermore, induction of the immunogenic cell death marker calreticulin was observed Biodistribution studies of the FGFR2-TTC in mouse models, evaluated by whole body autoradiography and acquisition of gamma-spectra specific for thorium-227, demonstrated specific accumulation of thorium-227 in FGFR2-positive tumors and very limited signal in murine organs and tissues. FGFR2-TTC exhibited in vivo tumor growth inhibition after a single dose in mouse xenograft models of CRC (NCI-H716) and gastric cancer (SNU-16). In addition, FGFR2-TTC showed anti-tumor activity in the aggressive murine syngeneic orthotopic 4T1 TNBC model. In summary, FGFR2-TTC has been established as a promising targeted alpha therapy (TAT) for efficacious and selective delivery of alpha emitter-based radiotherapy in several FGFR2-positive cancer indications. Further exploration for cancer therapy may thus be of interest. Citation Format: Urs B. Hagemann, Anette Sommer, Alexander Kristian, Ellen Wang, Åsmund Larsen, Uta Wirnitzer, Heidrun Ellinger-Ziegelbauer, Steffen Sandmann, Thorsten Poethko, Jenny Karlsson, Roger M. Bjerke, Lars Linden, Bertolt Kreft, Hanno Wild, Alan S. Cuthbertson. Preclinical activity of the FGFR2-targeted thorium-227 conjugate in preclinical models of colorectal, gastric and triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5199. doi:10.1158/1538-7445.AM2017-5199

Structural Basis of Pullulanase Membrane Binding and Secretion Revealed by X-Ray Crystallography, Molecular Dynamics and Biochemical Analysis

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain.

Structure, activity, and stability of metagenome‐derived glycoside hydrolase family 9 endoglucanase with an N‐terminal Ig‐like domain

A metagenome-derived glycoside hydrolase family 9 enzyme with an N-terminal immunoglobulin-like (Ig-like) domain, leaf-branch compost (LC)-CelG, was characterized and its crystal structure was determined. LC-CelG did not hydrolyze p-nitrophenyl cellobioside but hydrolyzed CM-cellulose, indicating that it is endoglucanase. LC-CelG exhibited the highest activity at 70°C and >80% of the maximal activity at a broad pH range of 5-9. Its denaturation temperature was 81.4°C, indicating that LC-CelG is a thermostable enzyme. The structure of LC-CelG resembles those of CelD from Clostridium thermocellum (CtCelD), Cel9A from Alicyclobacillus acidocaldarius (AaCel9A), and cellobiohydrolase CbhA from C. thermocellum (CtCbhA), which show relatively low (29-31%) amino acid sequence identities to LC-CelG. Three acidic active site residues are conserved as Asp194, Asp197, and Glu558 in LC-CelG. Ten of the thirteen residues that form the substrate binding pocket of AaCel9A are conserved in LC-CelG. Removal of the Ig-like domain reduced the activity and stability of LC-CelG by 100-fold and 6.3°C, respectively. Removal of the Gln40- and Asp99-mediated interactions between the Ig-like and catalytic domains destabilized LC-CelG by 5.0°C without significantly affecting its activity. These results suggest that the Ig-like domain contributes to the stabilization of LC-CelG mainly due to the Gln40- and Asp99-mediated interactions. Because the LC-CelG derivative lacking the Ig-like domain accumulated in Escherichia coli cells mostly in an insoluble form and this derivative accumulated in a soluble form exhibited very weak activity, the Ig-like domain may be required to make the conformation of the active site functional and prevent aggregation of the catalytic domain.

Crystal Structure of Glycoside Hydrolase Family 9 Endoglucanase with an N-Terminal Ig-Like Domain Isolated from Leaf-Branch Compost

Cellulase degrades glycosidic bonds of cellulose and is widely used for industrial purposes. Ten genes encoding novel cellulases, termed LC-CelA∼J, were isolated from leaf-branch compost by a metagenomic approach. Of them, LC-CelG, which is a member of glycoside hydrolase family 9 cellulases and contain an N-terminal immunoglobulin-like (Ig-like) domain, was overproduced in E. coli, purified, and characterized. LC-CelG consists of 577 amino acid residues. LC-CelG exhibited the highest activity at 70°C and pH 7.0. The crystal structure of LC-CelG was determined at 2.15A resolution. This structure resembles those of Clostridium thermocellum Cel9A (PDB: 1CLC), and contains two Ca2+ and one Zn2+ ions. Two conserved residues, Q40 and D99, in the Ig-like domain form hydrogen bonds and salt bridges respectively with the catalytic domain. To analyze the role of these interactions, two single (Q40A-CelG and D99A-CelG) and one double (Q40A/D99A-CelG) mutant proteins were constructed. The deletion mutant, ΔIg-CelG, which lacks the Ig-like domain, was also constructed. The far-UV CD spectra of these mutant proteins suggest that the structure of LC-CelG is not significantly changed by these mutations and deletion. Q40A-CelG and D99A-CelG were nearly as stable as LC-CelG, whereas Q40A/D99A-CelG and ΔIg-CelG were less stable than LC-CelG by 9.2 and 12.2°C respectively. This result suggests that Q40 and D99 cooperatively contribute to the stabilization of LC-CelG and removal of the Q40- and D99-mediated interactions is the main reason why LC-CelG is destabilized by deletion of the Ig-like domain. ΔIg-CelG was inactive at any temperature examined, whereas other mutants were as active as LC-CelG at ≤60°C, suggesting that the Ig-like domain is required for substrate binding or to make the conformation of the active site functional.

From (+)-epigallocatechin gallate to a simplified synthetic analogue as a cytoadherence inhibitor for P. falciparum

Parasite derived surface antigen PfEMP1 is a virulence factor of the human malaria parasite. PfEMP1 variants have been implicated in the cytoadherence of P. falciparum infected erythrocytes (iRBC) to several binding receptors on host vascular endothelium. Among them, binding to ICAM-1 seems to be related to severe manifestations of the disease such as cerebral malaria. The binding site for iRBC has been mapped to the BED-side of the N-terminal immunoglobulin-like domain of ICAM-1, and the DE-loop appears to be critical for binding. To date (+)-EGCG is the unique small molecule anti-cytoadherence inhibitor probably mimicking the DE-loop of ICAM-1. Here we report the discovery of a tetrahydroisoquinoline derivative, a prototype of a novel class of cytoadherence inhibitors, and an analogue of the natural compound characterized by a synthetically accessible scaffold. Molecular modeling analysis of (+)-EGCG and its synthetic tetrahydroisoquinoline analogue rationalized their binding mode to PfEMP1, confirming their ability to mimic the DE-loop.

Protein Tyrosine Phosphatase σ in Proteoglycan-Mediated Neural Regeneration Regulation

The receptor-type protein tyrosine phosphatase PTPσ mediates neural development and regeneration. Early studies on the ligands of PTPσ identified heparan sulfate proteolycan (HSPG) as a ligand. Binding of HSPG to PTPσ plays a critical role in axon guidance and synapse formation. PTPσ is also a receptor for chondroitin sulfate proteoglycan (CSPG). CSPG is deposited in high concentration at sites of neural injury. The deposited CSPG inhibits neural regeneration and axonal growth via PTPσ. The crystal structure of N-terminal immunoglobulin-like domains of PTPσ shows that the glycan binding site forms an elliptical surface patch of ∼35 by 24Å, which interacts with sulfate groups of HSPG and CSPG. In this review, we focus on the structural and functional mechanisms for the neural regeneration regulation by different types of proteoglycans. We also discuss recent results on induction of neural regeneration in the stroke model and neural transplantation. The mechanistic understanding of relationships between proteoglycans and PTPσ provides new therapeutic opportunities against diseases with impaired neural regeneration.

Homophilic interaction of the L1 family of cell adhesion molecules

Homophilic interaction of the L1 family of cell adhesion molecules plays a pivotal role in regulating neurite outgrowth and neural cell networking in vivo. Functional defects in L1 family members are associated with neurological disorders such as X-linked mental retardation, multiple sclerosis, low-IQ syndrome, developmental delay, and schizophrenia. Various human tumors with poor prognosis also implicate the role of L1, a representative member of the L1 family of cell adhesion molecules, and ectopic expression of L1 in fibroblastic cells induces metastasis-associated gene expression. Previous studies on L1 homologs indicated that four N-terminal immunoglobulin-like domains form a horseshoe-like structure that mediates homophilic interactions. Various models including the zipper, domain-swap, and symmetry-related models are proposed to be involved in structural mechanism of homophilic interaction of the L1 family members. Recently, cryo-electron tomography of L1 and crystal structure studies of neurofascin, an L1 family protein, have been performed. This review focuses on recent discoveries of different models and describes the possible structural mechanisms of homophilic interactions of L1 family members. Understanding structural mechanisms of homophilic interactions in various cell adhesion proteins should aid the development of therapeutic strategies for L1 family cell adhesion molecule-associated diseases.

Homophilic adhesion and CEACAM1-S regulate dimerization of CEACAM1-L and recruitment of SHP-2 and c-Src

Carcinoembryonic antigen (CEA)-related cell adhesion molecule 1 (CAM1 [CEACAM1]) mediates homophilic cell adhesion and regulates signaling. Although there is evidence that CEACAM1 binds and activates SHP-1, SHP-2, and c-Src, knowledge about the mechanism of transmembrane signaling is lacking. To analyze the regulation of SHP-1/SHP-2/c-Src binding, we expressed various CFP/YFP-tagged CEACAM1 isoforms in epithelial cells. The supramolecular organization of CEACAM1 was examined by cross-linking, coclustering, coimmunoprecipitation, and fluorescence resonance energy transfer. SHP-1/SHP-2/c-Src binding was monitored by coimmunoprecipitation and phosphotyrosine-induced recruitment to CEACAM1-L in cellular monolayers. We find that trans-homophilic CEACAM1 binding induces cis-dimerization by an allosteric mechanism transmitted by the N-terminal immunoglobulin-like domain. The balance of SHP-2 and c-Src binding is dependent on the monomer/dimer equilibrium of CEACAM1-L and is regulated by trans-binding, whereas SHP-1 does not bind under physiological conditions. CEACAM1-L homodimer formation is reduced by coexpression of CEACAM1-S and modulated by antibody ligation. These data suggest that transmembrane signaling by CEACAM1 operates by alteration of the monomer/dimer equilibrium, which leads to changes in the SHP-2/c-Src-binding ratio.

Structure of Reovirus σ1 in Complex with Its Receptor Junctional Adhesion Molecule-A

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. Mammalian reoviruses are highly useful experimental models for studies of viral pathogenesis and show promise as vectors for oncolytics and vaccines. Reoviruses engage cells by binding to carbohydrates and the immunoglobulin superfamily member, junctional adhesion molecule-A (JAM-A). JAM-A exists at the cell surface as a homodimer formed by extensive contacts between its N-terminal immunoglobulin-like domains. We report the crystal structure of reovirus attachment protein σ1 in complex with a soluble form of JAM-A. The σ1 protein disrupts the JAM-A dimer, engaging a single JAM-A molecule via virtually the same interface that is used for JAM-A homodimerization. Thus, reovirus takes advantage of the adhesive nature of an immunoglobulin-superfamily receptor by usurping the ligand-binding site of this molecule to attach to the cell surface. The dissociation constant (KD) of the interaction between σ1 and JAM-A is 1,000-fold lower than that of the homophilic interaction between JAM-A molecules, indicating that JAM-A strongly prefers σ1 as a ligand. Analysis of reovirus mutants engineered by plasmid-based reverse genetics revealed residues in σ1 required for binding to JAM-A and infectivity of cultured cells. These studies define biophysical mechanisms of reovirus cell attachment and provide a platform for manipulating reovirus tropism to enhance vector targeting.

Structural and Functional Analysis of Slit and Heparin Binding to Immunoglobulin-like Domains 1 and 2 of Drosophila Robo

Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.

The crystal structure of the ligand‐binding module of human TAG‐1 suggests a new mode of homophilic interaction

Human TAG-1 is a neural cell adhesion molecule that is crucial for the development of the nervous system during embryogenesis. It consists of six immunoglobulin-like and four fibronectin III-like domains and is anchored to the membrane by glycosylphosphatidylinositol. Herein we present the crystal structure of the four N-terminal immunoglobulin-like domains of TAG-1 (TAG-1(Ig1-4)), known to be important in heterophilic and homophilic macromolecular interactions. The contacts of neighboring molecules within the crystal were investigated. A comparison with the structure of the chicken ortholog resulted in an alternative mode for the molecular mechanism of homophilic TAG-1 interaction. This mode of TAG-1 homophilic interaction is based on dimer formation rather than formation of a molecular zipper as proposed for the chicken ortholog.

The solution structure of the membrane-proximal cytokine receptor domain of the human interleukin-6 receptor

The members of the interleukin-6-type family of cytokines interact with receptors that have a modular structure and are built of several immunoglobulin-like and fibronectin type III-like domains. These receptors have a characteristic cytokine receptor homology region consisting of two fibronectin type III-like domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 (IL-6) initially binds to its cognate alpha-receptor and subsequently to a homodimer of the signal transducer receptor gp130. The IL-6 receptor (IL-6R) consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane-proximal fibronectin-like domain (IL-6R-D3) accounts for more than 90% of the binding energy to IL-6. Here, we present the solution structure of the IL-6R-D3 domain solved by multidimensional heteronuclear NMR spectroscopy.

Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.

Structural similarities in the cellular receptors used by adenovirus and reovirus.

Adenovirus and reovirus are nonenveloped viruses that engage cell-surface receptors using filamentous attachment proteins with head-and-tail morphology. The coxsackievirus and adenovirus receptor (CAR) and reovirus receptor junctional adhesion molecule 1 (JAM1) are immunoglobulin superfamily members that form homodimers stabilized by ionic and hydrophobic contacts between their N-terminal immunoglobulin-like domains. Both proteins are expressed at regions of cell-cell contact and contain sequences in their cytoplasmic tails that anchor the proteins to the actin cytoskeleton. Like CAR and JAM1, the attachment proteins of adenovirus and reovirus, fiber and sigma1, respectively, also share key structural features. Both fiber and sigma1 have defined regions of flexibility within the tail, which is constructed in part using an unusual triple beta-spiral motif. The head domains of both proteins are formed by an 8-stranded beta-barrel with identical beta-strand connectivity. Strikingly, both adenovirus fiber and reovirus 1 engage their receptors by interacting with sequences that also mediate formation of receptor homodimers. Therefore, while adenovirus and reovirus belong to different virus families and have few overall properties in common, the observed similarities between the receptors and attachment proteins of these viruses suggest a conserved mechanism of attachment and an evolutionary relationship.

The CD200 and CD200 receptor cell surface proteins interact through their N-terminal immunoglobulin-like domains.

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) involved in regulation of macrophage function. Both CD200 and CD200R contain two Ig superfamily domains like many other leukocyte membrane proteins. Site-directed mutagenesis of CD200R showed that, like CD200, it interacted through its N-terminal domain. This indicated that the cell-cell interaction spans four Ig superfamily domains and this distance is similar to many interactions found between T cells and antigen-presenting cells. This suggests that this topology is also important in interactions of CD200 on a variety of cells with CD200R on myeloid cells, and comparable contact sites may be important mediating regulation in other cell-cell interactions. The mutagenesis showed that the binding involved the predicted GFCC' face of its N-terminal domain, like that of CD200, suggesting that the interaction evolved from a homotypic interaction.

Direct Determination of the Interleukin-6 Binding Epitope of the Interleukin-6 Receptor by NMR Spectroscopy

All cytokines belonging to the interleukin-6 (IL-6)-type family of cytokines utilize receptors that have a modular build of several immunoglobulin-like and fibronectin type III-like domains. Characteristic of these receptors is a cytokine receptor homology region consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 first binds to its specific receptor and subsequently to a homodimer of the signal transducer protein gp130. The interleukin-6 receptor consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane proximal fibronectin-like domain accounts for more than 90% of the binding energy to IL-6. Here, the key residues of this fibronectin-like domain involved in the interaction with IL-6 are described. Chemical shift mapping data with 15N-labeled IL-6R-D3 and unlabeled IL-6 coupled with recent structural data clearly reveal the epitope within the IL-6R-D3 responsible for mediating the high affinity interaction with its cognate cytokine.

Structure-Guided Design of Sialic Acid-Based Siglec Inhibitors and Crystallographic Analysis in Complex with Sialoadhesin

The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC 50 values (relative to methyl-α-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-α-9- N-benzoyl-amino-9-deoxy-Neu5Ac (BENZ), Me-α-9- N-(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-α-9- N-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.

Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy.

Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play an important role in angiogenesis associated with tumor growth and metastasis. VEGF exerts its biologic activities through 2 transmembrane tyrosine kinase receptors: the fms-like tyrosine kinase receptor (Flt-1, or VEGFR1) and kinase insert domain-containing receptor (KDR or VEGFR2). We have previously produced a panel of antibodies directed against KDR from mice immunized with the recombinant form receptor. These antibodies efficiently neutralized VEGF-induced KDR activation and mitogenesis of human umbilical vascular endothelial cells (HUVEC). Murine antibodies, however, may not be suitable candidates for human therapy because of their propensity to elicit human anti-mouse antibody response. Here we isolated several high-affinity human Fab antibody fragments directed against KDR from an antibody phage display library constructed from the pooled B lymphocytes of nonimmunized healthy human donors. These human Fab fragments bind specifically to KDR with nanomolar affinity and block KDR/VEGF interaction with IC(50) of approximately 2-20 nM. Further, they effectively inhibit VEGF-stimulated mitogenesis of HUVEC and migration of human leukemia cells. Epitope mapping studies demonstrated that all neutralizing human antibodies bound the epitope(s) located within the first 3 N-terminal immunoglobulin-like domains of KDR, the same region that encompasses the binding site of VEGF. Our results suggest that these human anti-KDR antibodies may have potential application in the treatment of cancer and other diseases in which pathologic angiogenesis occurs.

Identification of the domain in the human interleukin-11 receptor that mediates ligand binding

The interleukin-11 receptor (IL-11R) belongs to the hematopoietic receptor superfamily. The functional receptor complex comprises IL-11, IL-11R and the signal-transducing subunit gp130. The extracellular part of the IL-11R consists of three domains: an N-terminal immunoglobulin-like domain, D1, and two fibronectin-type III-like (FNIII) domains and D2 and D3. The two FNIII domains comprise the cytokine receptor-homology region defined by a set of four conserved cysteine residues in the N-terminal domain (D2) and a WSXWS sequence motif in the C-terminal domain (D3). We investigated the structural and functional role of the third extracellular receptor domain of IL-11R. A molecular model of the human IL-11/IL-11R complex allowed the identification of amino acid residues in IL-11R to be involved in ligand binding. Most of them were located in the third extracellular domain, which therefore should be able to bind with high affinity to IL-11. To prove this prediction, domain D3 of the IL-11R was expressed in Escherichia coli, refolded and purified. For structural characterization, circular dichroism, fluorescence and NMR spectroscopy were used. By plasmon resonance experiments, we show that the ligand-binding capacity of this domain is as high as that one for the whole receptor. These results provide a basis for further structural investigations that could be used for the rational design of potential agonists and antagonists essential in human therapy.

Multiple Regions within the Coxsackievirus and Adenovirus Receptor Cytoplasmic Domain Are Required for Basolateral Sorting

The coxsackievirus and adenovirus receptor (CAR) mediates attachment and infection by coxsackie B viruses and many adenoviruses. In human airway epithelia, as well as in transfected Madin-Darby canine kidney cells, CAR is expressed exclusively on the basolateral surface. Variants of CAR that lack the cytoplasmic domain or are attached to the cell membrane by a glycosylphosphatidylinositol anchor are expressed on both the apical and basolateral surfaces. We have examined the localization of CAR variants with progressive truncations of the cytoplasmic domain, as well as with mutations that ablate a potential PDZ (PSD95/dlg/ZO-1) interaction motif and a putative tyrosine-based sorting signal. In addition, we have examined the targeting of two murine CAR isoforms, with different C-terminal sequences. The results suggest that multiple regions within the CAR cytoplasmic domain contain information that is necessary for basolateral targeting.