Membrane-proximal Extracellular Domain Research Articles

A novel IgG-based FLT3xCD3 bispecific antibody for the treatment of AML and B-ALL

BackgroundIn lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for...

Daratumumab Activity Is Agnostic to Fc Gamma Receptor 3A (FcγR3A) 158 Genotype in Patients with Primary Refractory or Relapsed Systemic AL Amyloidosis

Introduction: Treatment targeted at the clonal plasma cells that produce the pathologic immunoglobulin light chain in patients with systemic AL amyloidosis, if effective and timely, can normalize serum free light chains resulting in organ responses and significantly improved overall survival. A third of patients with AL have no response to initial therapy, and over half of those who respond to initial therapy will relapse in 4 years; non-responders and relapsed patients with cardiac involvement are at especially high risk of shortened survival. Single agent daratumumab has been shown to have a response rate of 36% in relapsed refractory multiple myeloma patients (Lokhorst, Plesner et al. 2015). Daratumumab is an option for patients with AL who have marrow plasma cell burden > 10%, consistent with myeloma. Antibody- dependent cellular cytotoxicity is a mechanism of action of daratumumab, and this implicates recognition of the FC portion of the therapeutic antibody by FC receptors on effector cells such as macrophages and NK cells. A single nucleotide polymorphism at codon 158 of the FcγR3A receptor has been shown to affect the therapeutic efficacy of monoclonal antibodies(Mellor, Brown et al. 2013). The aim of this study was to assess hematologic and organ responses to daratumumab in a cohort of AL patients with primary refractory or relapsed clonal plasma cell disease and to test whether FcγR3A genotype influenced response to daratumumab.Materials and Methods: Patients with biopsy proven AL amyloidosis and organ involvement evaluated at Tufts Medical Center who received daratumumab were identified and evaluated for hematologic and organ responses according to consensus criteria (Comenzo, Reece et al. 2012, Palladini, Hegenbart et al. 2014). FcγR3a genotype for each patient was identified by PCR amplification and sequencing using genomic DNA from blood or marrow mononuclear cells. The translation of 158F or 158V in the alleles of the membrane-proximal extracellular domain was determined by T/G zygosity at nt559 in the gene sequence.Results: Fifteen patients with AL amyloidosis (λ=10, κ=5) who had primary refractory (no response after at least 2 cycles of initial therapy) (n = 5) or relapsed clonal plasma cell disease (n = 10) received daratumumab at standard doses at a median of 29 months from diagnosis (range, 2-107). Median age at diagnosis was 62 years (47-73) and 60% were male. The median lines of prior therapy were 3 (1-5). Eighty seven percent (n=13) had received bortezomib-based therapy and 53% (n=8) had undergone high-dose melphalan with autologous stem cell transplant. Nine patients had renal involvement (6 with stage 3), and 9 had cardiac involvement with a median NT-proBNP of 2373pg/ml (553-18021). All patients are alive with a median follow-up of 7 months (1-16). Hematologic responses were seen in 87% (CR=5, VPGR=8) with a median time to best response of 71 days. Forty four percent (n=4) had a renal response within an average of 107 days while 55% (n=5) achieved a cardiac response within an average of 149 days. Responses did not correlate with FcγR3A genotype (see figure below). Those with 158V/V or V/F had 3 CR, 3 VGPR and 1 no response (NR) while those with 158F/F had 2 CR, 5 VGPR and 1 NR. The ratio of FcγR3A 158 genotypes in patients with systemic AL amyloidosis has not previously been studied.Conclusions: Daratumumab achieved rapid hematologic responses in over 85% of patients with primary refractory or relapsed systemic AL amyloidosis with significant organ responses. Daratumumab activity was agnostic to FcγR3A 158 genotypes in this cohort of patients, in whom, of note, the distribution of genotypes is of interest. The basis for daratumumab activity may be linked to other mechanisms such as direct cytotoxicity or to the low fraction of cells in S-phase in AL plasma cell clones (Gertz, Kyle et al. 1989). Daratumumab is a very active agent in AL and its rapid clinical evaluation in patients with newly diagnosed AL is anticipated. [Display omitted] DisclosuresComenzo:Prothena: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Karyopharm: Research Funding; Takeda: Research Funding.

Cadherin Organization in Desmosomes Probed using Fluorescence Polarization Microscopy

Desmosomes are intercellular junctions found in epithelia and cardiac muscle that resist mechanical stress by linking the intermediate filaments of neighboring cells. Disruption of the desmosome-intermediate filament complex (DIFC) can cause a loss of tissue integrity as well as abnormalities in tissue differentiation, leading to a variety of diseases. Understanding these diseases will require expanding our knowledge of the relationship between structure and function in desmosomes. Of particular interest is the arrangement, or order, of the desmosomal cadherins. Cadherins are calcium-dependent, transmembrane adhesion proteins. In a single desmosome, many copies of these proteins engage in trans-binding with the cadherins of neighboring cells to form the adhesive interface. Large macromolecular complexes and membrane-associated proteins are difficult to study using traditional structural techniques. To overcome this, we pioneered excitation-resolved fluorescence polarization microscopy (FPM) to study the organization of desmosomes. After placing fluorescent tags in proteins of interest, FPM allows for the quantification of their order within a macromolecular assembly. Unlike methods that require sample fixation, FPM can be performed on living cells, providing insights into DIFC organization under physiologically relevant conditions, while preserving dynamic information. Through this methodology, our lab has shown that the most membrane-proximal extracellular domain of desmoglein 3 is ordered in desmosomes and that this order is rapidly lost after calcium removal. Here, we show that this behavior is conserved in another cadherin isoform, desmolgein 2. We also designed constructs to measure the order of the cadherin cytoplasmic domains, allowing correlation of order across the membrane. Finally, we use FPM to examine the effect of mutations that abrogate binding between intracellular DIFC components. This approach provides a unique tool to study desmosomal architecture and elucidate the roles of specific protein-protein interactions as they relate to desmosome structure and function.

Protocadherin cis-dimer architecture and recognition unit diversity

Clustered protocadherins (Pcdhs) mediate numerous neural patterning functions, including neuronal self-recognition and non-self-discrimination to direct self-avoidance among vertebrate neurons. Individual neurons stochastically express a subset of Pcdh isoforms, which assemble to form a stochastic repertoire of cis-dimers. We describe the structure of a PcdhγB7 cis-homodimer, which includes the membrane-proximal extracellular cadherin domains EC5 and EC6. The structure is asymmetric with one molecule contributing interface surface from both EC5 and EC6, and the other only from EC6. Structural and sequence analyses suggest that all Pcdh isoforms will dimerize through this interface. Site-directed mutants at this interface interfere with both Pcdh cis-dimerization and cell surface transport. The structure explains the known restrictions of cis-interactions of some Pcdh isoforms, including α-Pcdhs, which cannot form homodimers. The asymmetry of the interface approximately doubles the size of the recognition repertoire, and restrictions on cis-interactions among Pcdh isoforms define the limits of the Pcdh recognition unit repertoire.

A Novel CSF3R Mutation Uncovers the Importance of Membrane-Proximal N-Glycosylation for Receptor Regulation

Mutations in CSF3R (aka GCSFR) occur in the majority of patients with Chronic Neutrophilic Leukemia (CNL) and also are rarely found in adult and pediatric Acute Myeloid Leukemia. The most common CSF3R mutation in CNL is T618I (aka T595I), a point mutation in the membrane-proximal extracellular domain that causes ligand independence. Mutations that lead to a premature stop in the cytoplasmic domain are also found in CNL and result in increased expression of CSF3R on the cell surface. Understanding the biology of other novel mutations in CSF3R may lead to insight into both receptor biology and oncogenesis.The CSF3R N610H mutation was identified in a patient with a myeloproliferative neoplasm, which was most consistent with a JAK2, CALR, MPL mutation negative primary myelofibrosis. This patient had a history of mild leukocytosis for several years with most recent white blood cell counts between 13.3 and 15.3 x 103/uL. A bone marrow biopsy revealed 90% cellularity with mildly increased reticulin fibrosis, increased myeloid to erythroid ratio, no overt dysplasia, and less than 5% blasts. The cells were karotypically normal with a micro deletion of the 3' end of PDGFRB (5q) identified by FISH at 59%. The patient has had minimal symptoms with no anemia or thrombocytopenia and is currently being monitored but not receiving any intervention. The patient's bone marrow was next-generation sequenced using a 42-gene myeloid malignancy targeted mutation hotspot panel which revealed a mutation at N610H in CSF3R at a 50% mutant allele frequency.Given its proximity to the most common CSF3R mutation found in Chronic Neutrophilic Leukemia (T618I, aka T595I), we were interested in understanding whether the N610H mutation might contribute to disease biology. N610 (also known as N586 in the traditional numbering system that does not include the signal peptide) is part of an N-X-T motif, which is a consensus sequence for N-linked glycosylation. Haniu et al (Biochemistry 1996) demonstrated that N610 is one of 8 sites that are N-glycosylated on CSF3R. We confirm by mass spectrometry (MS) based analysis that N610 is occupied with a bisecting complex N-glycan (in vitro). We further found that the N610H mutation and a more conservative N610Q substitution are highly activating in CSF3R, leading to cytokine-independent growth in the murine Ba/F3 cell line. Furthermore, like the T618I mutation, these mutations render the receptor ligand-independent. N610H and N610Q lead to a robust increase in downstream signaling through the JAK/STAT pathway as demonstrated by an increase in the levels of phospho-STAT3. The loss of N-glycosylation in the membrane-proximal region of CSF3R may therefore increase ligand-independent receptor activation and promote oncogenesis. This study highlights the insight that rare human mutations can provide into the relationship between receptor structure and function. DisclosuresRadich:Ariad: Consultancy; Novartis: Consultancy, Research Funding; Gilliad: Consultancy; Incyte: Consultancy. Bertozzi:GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Catalent Biologics: Membership on an entity's Board of Directors or advisory committees; Verily: Membership on an entity's Board of Directors or advisory committees; Enable Bioscience: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Oh:CTI: Research Funding; Janssen: Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Cadherin is involved in the action of Bacillus thuringiensis toxins Cry1Ac and Cry2Aa in the beet armyworm, Spodoptera exigua

Bacillus thuringiensis (Bt) insecticidal crystal (Cry) proteins are effective against some insect pests in sprays and transgenic crops, although the evolution of resistance could threaten the long-term efficacy of such Bt use. One strategy to delay resistance to Bt crops is to “pyramid” two or more Bt proteins that bind to distinct receptor proteins within the insect midgut. The most common Bt pyramid in cotton (Gossypium hirsutum L.) employs Cry1Ac with Cry2Ab to target several key lepidopteran pests, including the beet armyworm, Spodoptera exigua (Hübner), which is a serious migratory pest of many vegetable crops and is increasingly important in cotton in China. While cadherin and aminopeptidase-N are key receptors of Cry1 toxins in many lepidopterans including S. exigua, the receptor for Cry2A toxins remains poorly characterized. Here, we show that a heterologous expressed peptide corresponding to cadherin repeat 7 to the membrane proximal extracellular domain (CR7-MPED) in the S. exigua cadherin 1b (SeCad1b) binds Cry1Ac and Cry2Aa. Moreover, SeCad1b transcription was suppressed in S. exigua larvae by oral RNA interference and susceptibility to Cry1Ac and Cry2Aa was significantly reduced. These results indicate that SeCad1b plays important functional roles of both Cry1Ac and Cry2Aa, having major implications for resistance management for S. exigua in Bt crops.

Cadherin AdCad1 in Alphitobius diaperinus larvae is a receptor of Cry3Bb toxin from Bacillus thuringiensis

Bacillus thuringiensis (Bt) Cry proteins are used as components of biopesticides or expressed in transgenic crops to control diverse insect pests worldwide. These Cry toxins bind to receptors on the midgut brush border membrane and kill enterocytes culminating in larval mortality. Cadherin proteins have been identified as Cry toxin receptors in diverse lepidopteran, coleopteran, and dipteran species. In the present work we report a 185 kDa cadherin (AdCad1) from larvae of the lesser mealworm (Alphitobius diaperinus) larvae as the first identified receptor for Cry3Bb toxin. The AdCad1 protein contains typical structural components for Cry toxin receptor cadherins, including nine cadherin repeats (CR9), a membrane-proximal extracellular domain (MPED) and a cytosolic region. Peptides corresponding to the CR9 and MPED regions bound Cry3Bb toxin with high affinities (23 nM and 40 nM) and significantly synergized Cry3Bb toxicity against A. diperinus larvae. Silencing of AdCad1 expression through RNA interference resulted in highly reduced susceptibility to Cry3Bb in A. diperinus larvae. The CR9 peptide fed with toxin to RNAi-treated larvae restored Cry3Bb toxicity. These results are evidences that AdCad1 is a functional receptor of Cry3Bb toxin and that exogenously fed CR9 peptide can overcome the effect of reduced AdCad1expression on Cry3Bb toxicity to larvae.

Increased survival and reduced renal injury in MRL/lpr mice treated with a human Fcγ receptor II (CD32) peptide

Systemic lupus erythematosus (SLE) is a multisystem chronic inflammatory disease affecting many organs. The deposition in kidney tissue of immune complexes and their interaction with macrophages is thought to trigger the inflammatory response leading to glomerulonephritis. It has been demonstrated that inhibition of this interaction in murine models can alleviate the disease. Six synthetic peptides were derived from the membrane-proximal extracellular domain (EC2) of human Fcγ receptor II (huFcγRII). Of these, one peptide, huRII6, was shown to be a potent competitive inhibitor of IgG binding to recombinant FcγRII in vitro. To examine the possible therapeutic impact of huRII6 in vivo, this peptide, or a control, was given by subcutaneous injection to female MRL/lpr mice from weeks 7 to 36, resulting in an enhanced survival rate compared with control-treated animals and a reduction of proteinuria. Histopathological examination of the kidneys showed a reduction in deposition of immune complexes and preservation of structure. Such a functional peptide should prove useful for examining the role of IgG-FcγR interactions in experimental models of disease and may provide for the development of FcR-targeting drugs to treat autoimmune disorders.

The cytoplasmic domains of TNFα-converting enzyme (TACE/ADAM17) and L-selectin are regulated differently by p38 MAPK and PKC to promote ectodomain shedding

L-selectin mediates the initial tethering and subsequent rolling of leucocytes along luminal walls of inflamed venules. TACE [TNFalpha (tumour necrosis factor alpha)-converting enzyme] is responsible for cleaving the membrane-proximal extracellular domain of L-selectin (also known as shedding), which reduces the efficiency of leucocyte recruitment to sites of inflammation. Many reports have highlighted roles for PKC (protein kinase C) and p38 MAPK (mitogen-activated protein kinase) in promoting L-selectin shedding with little insight into the mechanism involved. By using PMA and the phosphatase inhibitors cantharidin and calyculin A, we could selectively activate PKC or p38 MAPK respectively to promote TACE-dependent shedding of L-selectin. Interestingly, the intracellular mechanisms leading to the shedding event differed dramatically. For example, regulatory elements within the L-selectin cytoplasmic tail, such as ERM (ezrin/radixin/moesin)-binding and serine residues, were important for PKC- but not p38 MAPK-dependent shedding. Also, increased and sustained cell surface levels of TACE, and phosphorylation of its cytoplasmic tail (a hallmark of TACE activation), occurred in lymphocytes and monocytes following p38 MAPK activation. Finally, we showed that TNFalpha-induced shedding of L-selectin in monocytes was strikingly similar to cantharidin-induced shedding and suggest that this newly characterized mechanism could be physiologically relevant in inflammatory cells.

Cadherin fragments from Anopheles gambiae synergize Bacillus thuringiensis Cry4Ba's toxicity against Aedes aegypti larvae.

A peptide from cadherin AgCad1 of Anopheles gambiae larvae was reported as a synergist of Bacillus thuringiensis subsp. israelensis Cry4Ba's toxicity to the Anopheles mosquito (G. Hua, R. Zhang, M. A. Abdullah, and M. J. Adang, Biochemistry 47:5101-5110, 2008). We report that CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) and a longer peptide, CR9 to CR11 (CR9-11), from AgCad1 act as synergists of Cry4Ba's toxicity to Aedes aegypti larvae, but a Diabrotica virgifera virgifera cadherin-based synergist of Cry3 (Y. Park, M. A. F. Abdullah, M. D. Taylor, K. Rahman, and M. J. Adang, Appl. Environ. Microbiol. 75:3086-3092, 2009) did not affect Cry4Ba's toxicity. Peptides CR9-11 and CR11-MPED bound Cry4Ba with high affinity (13 nM and 23 nM, respectively) and inhibited Cry4Ba binding to the larval A. aegypti brush border membrane. The longer CR9-11 fragment was more potent than CR11-MPED in enhancing Cry4Ba against A. aegypti.

The Membrane Proximal Extracellular Domain of Human hGC-B Folds Independently

Human Guanylyl Cyclase B (hGC-B) is a single-transmembrane receptor protein which upon binding C-type natriuretic peptide (CNP) to its extracellular domain catalyzes the intracellular conversion of GTP to the second messenger cGMP. cGMP in turn affects various physiological processes such as smooth muscle contraction, cell proliferation, phototransduction, and salt as well as fluid homeostasis. The 3-dimensional binding site of the peptide hormone is unknown, and the binding mechanism is not yet understood. Therefore, a model of the C-terminal moiety of the extracellular domain of human GC-B containing the potential binding site was derived from the crystal structure of (GC-A). The selected protein sequence was provided with an N-terminal TEV-cleavage site and fused with a 109 aa thioredoxin-tag and a hexahistidine-tag. The identity of the purified 25 kDa protein was confirmed by protein mass fingerprint and its secondary structure was determined by CD- and NMR-spectroscopy. The protein proved to be properly folded with the observed secondary structure matching the predicted secondary structure and the homologous structure in the extracellular domain of GC-A. Size exclusion chromatography confirmed the monomeric state of P-hGC-B.

Soluble lectin-like oxidized low density lipoprotein receptor-1 in type 2 diabetes mellitus

The lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) can be proteolytically cleaved and released as soluble forms (sLOX-1). We have determined serums LOX-1 in type 2 diabetes and evaluated the effect of glucose and advanced glycation end products (AGEs) on sLOX-1 in vitro and in vivo. Endothelial cells were incubated with glucose or AGEs, and sLOX-1 in cell medium was measured. Serum sLOX-1 was measured in 219 diabetic patients and 187 controls by ELISA. The effect of lowering glucose and AGEs on sLOX-1 was determined in 38 poorly controlled diabetic patients after improvement in glycemic control. Incubation of endothelial cells with AGE-BSA led to a dose-dependent increase in sLOX-1, whereas the effect of glucose on sLOX-1 was less marked. Serum sLOX-1 was 9% higher in diabetic patients compared with controls (P<0.01). In the poorly controlled patients, serum sLOX-1 decreased by 12.5% after improvement in glycemic control (P<0.05). The magnitude of reduction in sLOX-1 correlated with the improvement in hemoglobin A1c and AGEs but not with the reduction in oxidized LDL. sLOX-1 level is increased in type 2 diabetes. Both glucose and AGEs are important determinants of LOX-1 expression, and lowering glucose and AGEs leads to a reduction in sLOX-1.

Anopheles gambiae Cadherin AgCad1 Binds the Cry4Ba Toxin of Bacillus thuringiensis israelensis and a Fragment of AgCad1 Synergizes Toxicity

A midgut cadherin AgCad1 cDNA was cloned from Anopheles gambiae larvae and analyzed for its possible role as a receptor for the Cry4Ba toxin of Bacillus thuringiensis strain israelensis. The AgCad1 cadherin encodes a putative 1735-residue protein organized into an extracellular region of 11 cadherin repeats (CR) and a membrane-proximal extracellular domain (MPED). AgCad1 mRNA was detected in midgut of larvae by polymerase chain reaction (PCR). The AgCad1 protein was localized, by immunochemistry of sectioned larvae, predominately to the microvilli in posterior midgut. The localization of Cry4Ba binding was determined by the same technique, and toxin bound microvilli in posterior midgut. The AgCad1 protein was present in brush border membrane fractions prepared from larvae, and Cry4Ba toxin bound the same-sized protein on blots of those fractions. The AgCad1 protein was expressed transiently in Drosophila melanogaster Schneider 2 (S2) cells. 125I-Cry4Ba toxin bound AgCad1 from S2 cells in a competitive manner. Cry4Ba bound to beads extracted 200 kDa AgCad1 and a 29 kDa fragment of AgCad1 from S2 cells. A peptide containing the AgCad1 region proximal to the cell (CR11-MPED) was expressed in Escherichia coli. Although Cry4Ba showed limited binding to CR11-MPED, the peptide synergized the toxicity of Cry4Ba to larvae. AgCad1 in the larval brush border is a binding protein for Cry4Ba toxin. On the basis of binding results and CR11-MPED synergism of Cry4Ba toxicity, AgCad1 is probably a Cry4Ba receptor.

Univalent Binding of the Cry1Ab Toxin of Bacillus thuringiensis to a Conserved Structural Motif in the Cadherin Receptor BT-R1

The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding to BT-R1, a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta [Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S., Midboe, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R1 in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis, Insect Biochem. Mol. Biol. 32, 1025-1036]. BT-R1 represents a family of invertebrate cadherins whose ectodomains (ECs) are composed of multiple cadherin repeats (EC1 through EC12). In the present work, we determined the Cry1Ab toxin binding site in BT-R1 in the context of cadherin structural determinants. Our studies revealed a conserved structural motif for toxin binding that includes two distinct regions within the N- and C-termini of EC12. These regions are characterized by unique sequence signatures that mark the toxin-binding function in BT-R1 as well as in homologous lepidopteran cadherins. Structure modeling of EC12 discloses the conserved motif as a single broad interface that holds the N- and C-termini in close proximity. Binding of toxin to BT-R1, which is univalent, and the subsequent downstream molecular events responsible for cell death depend on the conserved motif in EC12.

ELISA for Soluble Form of Lectin-Like Oxidized LDL Receptor-1, A Novel Marker of Acute Coronary Syndrome

Lectin-like oxidized LDL receptor-1 (LOX-1) is prominently expressed in atherosclerotic plaques (1)(2). LOX-1 expressed on the cell surface is proteolytically cleaved at its membrane-proximal extracellular domain (ECD) and released in soluble form (sLOX-1) (2)(3). Serum sLOX-1 is increased at an earlier stage of acute coronary syndrome (ACS) than are other cardiac makers, such as creatine kinase-MB fraction, troponin T, and high-sensitivity C-reactive protein (4). We describe the development of an ELISA for sLOX-1. The cleavage sites on human LOX-1 (hLOX-1) remain unknown. We tried to obtain human sLOX-1 (hsLOX-1) from cultures of CHO-K1 cells stably expressing hLOX-1 (3)(5), but the amount of hsLOX-1 obtained was too small for ELISA development. hsLOX-1 should be similar to the ECD of hLOX-1 (hLOX-1-ECD), considering a structure of bovine sLOX-1 (3). We produced recombinant hLOX-1-ECD [rhLOX-1-ECD; hLOX-1 (84–273)] in Escherichia coli and purified it as described previously (5); we then used the soluble fraction of rhLOX-1-ECD instead of hsLOX-1 as the assay calibrator and immunogen. We estimated the amount of rhLOX-1-ECD by ultraviolet spectrophotometry, using the …

Binding a bent integrin

A bent integrin still has room for its ligand, according to Adair et al. on page 1109. Integrin activation by internal signals may therefore not depend on the protein assuming a more extended conformation. Figure Fibronectin (green and yellow) can bind to a bent integrin (blue and red). To prevent unregulated adhesion, most integrins are expressed in an inactive form. EM and crystal structures showed that the extracellular domain of the integrin αVβ3 without its ligand can assume a bent conformation. 2D EM reconstructions of the same domain in the presence of a small peptide suggested that the bound form, in contrast, was extended. These results supported a switchblade model, in which the binding of cytoplasmic signals initiates the movement of several intra- and extracellular domains that opens and activates the integrin by exposing its ligand-binding site. But Adair et al. suggest that such large-scale changes may not be necessary. The authors generated 3D EM reconstructions using a larger, physiological ligand (in this case, a piece of fibronectin). Particles to be analyzed were also selected automatically to minimize bias. The 3D maps showed that most of the fibronectin-bound integrin complexes were in a bent conformation. The maps with and without the ligand looked generally similar. But in the ligand's presence, additional densities consistent with the size of two fibronectin domains were found at the ligand-binding sites. If, as the findings indicate, no large-scale opening is needed to make room for ligand, then the mechanism of activation by intracellular signals should be revisited. For instance, a slight sideways movement of a membrane-proximal extracellular domain of the β-subunit away from the ligand-binding site may be sufficient. This model involves much smaller conformational changes than does the switchblade model. It is possible that the switchblade movement has quite a different function. For example, it may be part of the conformational changes induced by ligand binding that culminate in intracellular responses.

Involvement of CD4 D3–D4 membrane proximal extracellular domain for the inhibitory effect of oxidative stress on activation-induced CD4 down-regulation and its possible role for T cell activation

During antigen presentation, CD4 functions to stabilize T cell receptor (TCR)–class II MHC interactions and coordinate Ag-induced T cell activation signals. These activation signals cause CD4 down-regulation, presumably acting to optimize T cell activation. We previously reported that oxidative stress interferes with activation-induced CD4 down-regulation in T cells. In this study, we have further investigated inhibition of CD4 down-regulation by oxidative stress and its role for T cell activation. A construct comprised of the mouse FcγRIIB extracellular domain and the transmembrane/cytoplasmic domains of human CD4 (FcγR/CD4) was expressed in a human T cell line. Oxidant actually potentiated down-regulation of the FcγR/CD4 chimera and induced Lck dissociation from both CD4 and FcγR/CD4, which is a crucial intracellular process for activation-induced CD4 down-regulation, suggesting a critical role of CD4 ectodomain in the inhibition of CD4 down-regulation by oxidative stress. Furthermore, insertion of CD4 D3–D4 membrane proximal extracellular region between FcγR extracellular domain and CD4 transmembrane/cytoplasmic domains in FcγR/CD4 chimera made this molecule behave like native CD4 molecule under oxidative stress condition. These data imply that the inhibitory effect of oxidative stress on CD4 down-regulation is executed via D3–D4 domain of CD4 ectodomain. As to its role for T cell activation, CD4 coaggregation with CD3 under the oxidative conditions enhanced activation signal induced by CD3 aggregation. Our results demonstrate that Ag-induced T cell activation which is normally concomitant with CD4 down-regulation may be disturbed through the aberrant regulation of CD4 expression by oxidative stress.

Human Herpesvirus 6 and Measles Virus Employ Distinct CD46 Domains for Receptor Function

We employed a quantitative cell fusion assay to identify structural domains of CD46 required for its function as a receptor for human herpesvirus 6 (HHV-6). We examined the activities of recombinant variants of CD46, including different isoforms as well as engineered truncations and molecular chimeras with decay-accelerating factor, a related protein in the family of regulators of complement activation (RCA). We observed strong receptor activity for all four CD46 isoforms, which differ in the membrane-proximal extracellular and cytoplasmic domains, indicating that the critical determinants for HHV-6 receptor activity reside outside the C-terminal portion of CD46. Analysis of the short consensus repeat (SCR) regions that comprise most of the extracellular portion of CD46 indicated a strong dependence on SCRs 2 and 3 and no requirement for SCRs 1 or 4. Fusion-inhibition studies with SCR-specific monoclonal antibodies supported the essential role of SCRs 2 and 3 in HHV-6 receptor activity. These findings contrast markedly with fusion mediated by measles virus glycoproteins for which we observed a strict dependence on SCRs 1 and 2, consistent with previous reports. These results expand the emerging notion that CD46 and other members of the RCA family are co-opted in distinct manners by different infectious pathogens.

VE-PTP and VE-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts.

VE-cadherin is the essential adhesion molecule in endothelial adherens junctions, and the regulation of protein tyrosine phosphorylation is thought to be important for the control of adherens junction integrity. We show here that VE-PTP (vascular endothelial protein tyrosine phosphatase), an endothelial receptor-type phosphatase, co-precipitates with VE-cadherin, but not with beta-catenin, from cell lysates of transfected COS-7 cells and of endothelial cells. Co-precipitation of VE-cadherin and VE-PTP required the most membrane-proximal extracellular domains of each protein. Expression of VE-PTP in triple-transfected COS-7 cells and in CHO cells reversed the tyrosine phosphorylation of VE-cadherin elicited by vascular endothelial growth factor receptor 2 (VEGFR-2). Expression of VE-PTP under an inducible promotor in CHO cells transfected with VE-cadherin and VEGFR-2 increased the VE-cadherin-mediated barrier integrity of a cellular monolayer. Surprisingly, a catalytically inactive mutant form of VE-PTP had the same effect on VE-cadherin phosphorylation and cell layer permeability. Thus, VE-PTP is a transmembrane binding partner of VE-cadherin that associates through an extracellular domain and reduces the tyrosine phosphorylation of VE-cadherin and cell layer permeability independently of its enzymatic activity.

Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R 1 in Manduca sexta: : involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis

Many subspecies of the soil bacterium Bacillus thuringiensis produce various parasporal crystal proteins, also known as Cry toxins, that exhibit insecticidal activity upon binding to specific receptors in the midgut of susceptible insects. One such receptor, BT-R 1 (210 kDa), is a cadherin located in the midgut epithelium of the tobacco hornworm, Manduca sexta. It has a high binding affinity (K d ∼ 1 nM) for the Cry1A toxins of B. thuringiensis. Truncation analysis of BT-R 1 revealed that the only fragment capable of binding the Cry1A toxins of B. thuringiensis was a contiguous 169-amino acid sequence adjacent to the membrane-proximal extracellular domain. The purified toxin-binding fragment acted as an antagonist to Cry1Ab toxin by blocking the binding of toxin to the tobacco hornworm midgut and inhibiting insecticidal action. Exogenous Cry1Ab toxin bound to intact COS-7 cells expressing BT-R 1 cDNA, subsequently killing the cells. Recruitment of BT-R 1 by B. thuringiensis indicates that the bacterium interacts with a specific cell adhesion molecule during its pathogenesis. Apparently, Cry toxins, like other bacterial toxins, attack epithelial barriers by targeting cell adhesion molecules within susceptible insect hosts.