Beta Heterodimer Research Articles

Identification of Healthy Donor Phenotypic and Functional Signatures That Predict Allogeneic, Hypoimmune CD19-Directed CAR T Cell Potency

Allogeneic, hypoimmune (HIP) CAR T cells may provide off-the-shelf CAR T cells that allow for expanded access for patients, overcome manufacturing failures, and provide an efficacious alternative to CAR T cells manufactured from heavily pre-treated T cells. However, donor T cells are the primary source of variability during production of allogeneic HIP CAR T cells, and may influence clinical efficacy. Previously, we reported a series of T cell functional assays that differentiate T cell potency as a means to select high-quality CAR T cell donors for clinical trials. Here, we explore donor phenotypic and functional signatures that are associated with desirable pre-clinical attributes for the generation of allogeneic HIP CD19-directed CAR T cells. Human T cells from healthy donors were obtained by leukapheresis and used to generate allogeneic HIP CD19-directed CAR T cells by disrupting B2M, CIITA, and TRAC genes using Cas12b nuclease, and lentiviral transduction to overexpress CD47 and to express the CD19 CAR.Phenotype profiling (PBMC subsets, memory subsets, viability, exhaustion, and activation markers) was assessed on apheresis, CD4 or CD8 enriched subsets and post-CAR manufacturing. Generated CAR T cells were evaluated in vitro by repetitive tumor challenge (3x) performed over a 14-day period using the IncuCyte platform. Bulk cytokine analysis was measured by MesoScale Discovery (MSD). In vivo efficacy was evaluated using a systemic NALM-6 challenge in immune-deficient NSG mice at HIP CD19 CAR T cell doses of 5e5 and 5e6 cells per animal. Gene expression profiling was assessed on pre-production, post-CAR T cell manufacturing, and post-serial tumor challenge CD4+ and CD8+ T cells using the Nanostring nCounter CAR T panel. Functional and phenotypic attributes were used for weighted donor performance scoring. To do this, cell functional assays were defined to produce single, quantitative indices to enable the classification of CAR T cells based on functional performance. We assessed differences in CAR T cell performance and rank scored donor performance. Associations between donor attributes, phenotype/ functional T cell attributes and ranked donor performance were evaluated using Spearman's correlations. Correlative comparison of phenotype and donor attributes in relation to weighted donor performance scores indicated that cellular subtypes at leukapheresis correlate with CAR T performance. Gene expression profiling shows that outperforming donors upregulate expression of mucosal associated invariant T (MAIT) cell canonical TCRb chain TRBV28. Outperforming donors have increased expression of Tc1/Th1 and Tc17/Th17 associated cytokines: IFNg, Il-17A, Il-17F, Il23, Il22, and have reduced expression of Tc2/Th2 associated cytokine Il5. Outperforming donors also showed reduced expression of MHC class II of alpha and beta heterodimers (HLA-DQA1 and HLA-DQB1). The information collected across phenotypic and functional comparisons will be compared with clinical performance.

Structural insight to human Retinoid X receptor alpha-Thyroid hormone receptor beta heterodimer by molecular modelling and MD-simulation studies: role of conserved water molecules

The Retinoid X receptor alpha–Thyroid hormone receptor beta (RXRα-THRβ) heterodimer plays an important role in physiological function of humans specially in the growth and development. Extensive MD-simulation studies on the aquated complexes of modelled RXRα-THRβ heterodimer with DNA-duplex have indicated the role of some conserved/semiconserved water molecules in the complexation process in presence or absence of Triiodothyronine (T3) and 9-cis retinoic acid (9CR) in the respective Ligand Binding Domain (LBD) domain. Among the seventeen conserved/semi-conserved water molecules, the W1-W4 water centers have been observed to mediate the interaction between the residues of A-chain (DBD of RXR) to consensus sequence (C-chain) of DNA. The W5-W8 water centers involve in recognition of the residues of B-chain (DBD of THR) to C-chain of DNA. The W9-W13 centers have connected the different residues of B-chain (THR) to D-chain of DNA through H-bonds, whereas W14-W17 water molecules were involved in the interaction of A-chain′s (RXR) residues to D-chain of DNA. In our previous study with homodimeric THRβ from Rattus norvegicus we have identified fifteen conserved water molecules at the DNA-DBD interface. Moreover, the conformational flexibility of Met313 (in the LBD of THR) from open to close form in presence or absence of T3 molecule in the holo and Apo-protein may provide a plausible rational on the possible role of that residue to acts as gate which could restrict the solvent molecules to enter into the hydrophobic T3-binding pocket of LBD during the absence of ligand molecule and thus could help the stabilization of that domain in THRβ structure. Communicated by Ramaswamy H. Sarma

T Cell Receptor Engineered Lymphocytes for Cancer Therapy.

T lymphocytes are capable of specific recognition and elimination of target cells. Physiological antigen recognition is mediated by the T cell receptor (TCR), which is an alpha beta heterodimer comprising the products of randomly rearranged V, D, and J genes. The exquisite specificity and functionality of T cells can be leveraged for cancer therapy: specifically, the adoptive transfer of T cells that express tumor-reactive TCRs can induce regression of solid tumors in patients with advanced cancer. However, the isolation and expression of a tumor antigen-specific TCRs is a highly involved process that requires identifying an immunogenic epitope, ensuring human cells are of the correct haplotype, performing a laborious T cell expansion process, and carrying out downstream TCR sequencing and cloning. Recent advances in single-cell sequencing have begun to streamline this process. This protocol synthesizes and expands upon methodologies to generate, isolate, and engineer human T cells with tumor-reactive TCRs for adoptive cell therapy. Though this process is perhaps more arduous than the alternative strategy of using chimeric antigen receptors (CARs) for engineering, the ability to target intracellular proteins using TCRs substantially increases the types of antigens that can be safely targeted. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of human autologous dendritic cells from monocytes Basic Protocol 2: In vitro priming and expansion of human antigen-specific T cells Basic Protocol 3: Cloning of antigen-specific T cell receptors based on single-cell VDJ sequencing data Basic Protocol 4: Validation of T cell receptor expression and functionality in vitro Basic Protocol 5: Rapid expansion of T cell receptor-transduced T cells and human T cell clones.

Urine reference intervals for human chorionic gonadotropin (hCG) isoforms by immunoextraction-tandem mass spectrometry to detect hCG use.

Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles and can normalize suppressed testosterone concentrations in males following prolonged anabolic steroid use. Because of the potential for abuse by males, hCG is on the World Anti-Doping Agency (WADA) list of prohibited substances. The majority of WADA-accredited laboratories measure urinary hCG using an automated immunoassay. Only immunoassays that recognize the intact alpha and beta heterodimer of hCG (intact hCG) should be used to measure urinary hCG for doping control purposes since intact hCG is the only biologically active molecule. WADA further requires that confirmation testing is performed using an intact hCG immunoassay that is different from the one used in the initial testing procedure or by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study we measured the concentration of intact hCG, free β-subunit (hCGβ) and β-subunit core fragment (hCGβcf) in 570, 275, and 256 male urine samples, respectively, by an immunoextraction LC-MS/MS method. Mean concentrations of intact hCG, hCGβ and hCGβcf were 0.04IU/L, 0.47pmol/L and 0.16pmol/L, respectively. The upper reference limits (97.5th percentile) for intact hCG, hCGβ and hCGβcf were 0.21IU/L, 0.40pmol/L, and 1.86pmol/L, respectively. Based on these data, we recommend a threshold of 1.0IU/L for intact hCG (false positive rate of <1 in 10 000) for detecting male athletes that dope with hCG.

The Impact of Perturbing Dynamic Amino Acid Networks in a (β/α)8 Barrel Enzyme

Amino acid networks describe the web of noncovalent interactions between residues spanning an enzyme. These networks may be responsible for the propagation of regulatory signals across the protein that influence conformation, binding of substrate(s), and catalysis. Tryptophan synthase (TS), the final enzyme in the tryptophan biosynthetic pathway, is a tetramer consisting of a pair of alpha and beta heterodimers arranged in a linear conformation. The alpha and beta subunits are connected by a 25Å intramolecular tunnel that channels indole, a product from the alpha reaction, to the active site in the beta subunit. In addition to this tunnel, the conformational states of these subunits are highly coordinated making TS an ideal and heavily studied model for substrate channeling and enzyme-enzyme interactions. We used nuclear magnetic resonance chemical shift covariance analysis to delineate amino acid networks in the alpha subunit, a (β/α)8 barrel enzyme. We have shown that these observed networks change between the resting state (in the absence of substrates) and the working state (under active catalytic turnover). Furthermore, the loss of a hydrogen bond between the dynamic β2α2 and β6α6 loops in the T183V variant significantly changes these networks and the catalytic rate seen in the wild-type. These networks were perturbed by making small Ala to Gly substitutions of surface residues correlated to the catalytic Glu49. These modifications resulted in modest decreases in the catalytic rate, although they are 25 Å away from the active site. Amino acid networks are important for the function of an enzyme and may be manipulated to tune its function.

The novel peptide F29 facilitates the DNA-binding ability of hypoxia-inducible factor-1α

Hypoxia-inducible factor-1alpha/beta (HIF-1alpha/beta) is a heterodimeric transcriptional activator that mediates gene expression in response to hypoxia. HIF-1alpha has been noted as an effective therapeutic target for ischemic diseases such as myocardiac infarction, stroke and cancer. By using a yeast two-hybrid system and a random peptide library, we found a 16-mer peptide named F29 that directly interacts with the bHLH-PAS domain of HIF-1alpha. We found that F29 facilitates the interaction of the HIF-1alpha/beta heterodimer with its target DNA sequence, hypoxia-responsive element (HRE). The transient transfection of an F29-expressing plasmid increases the expression of both an HRE-driven luciferase gene and the endogenous HIF-1 target gene, vascular endothelial growth factor (VEGF). Taken together, we conclude that F29 increases the DNA-binding ability of HIF-1alpha, leading to increased expression of its target gene VEGF. Our results suggest that F29 can be a lead compound that directly targets HIF-1alpha and increases its activity.

Contributions of the individual hydrophobic clefts of the Escherichia coli sliding clamp to clamp loading, DNA replication and clamp recycling

The homodimeric Escherichia coli β sliding clamp contains two hydrophobic clefts with which proteins involved in DNA replication, repair and damage tolerance interact. Deletion of the C-terminal five residues of β (βC) disrupted both clefts, severely impairing interactions of the clamp with the DnaX clamp loader, as well as the replicative DNA polymerase, Pol III. In order to determine whether both clefts were required for loading clamp onto DNA, stimulation of Pol III replication and removal of clamp from DNA after replication was complete, we developed a method for purification of heterodimeric clamp proteins comprised of one wild-type subunit (β+), and one βC subunit (β+/βC). The β+/βC heterodimer interacted normally with the DnaX clamp loader, and was loaded onto DNA slightly more efficiently than was β+. Moreover, β+/βC interacted normally with Pol III, and stimulated replication to the same extent as did β+. Finally, β+/βC was severely impaired for unloading from DNA using either DnaX or the δ subunit of DnaX. Taken together, these findings indicate that a single cleft in the β clamp is sufficient for both loading and stimulation of Pol III replication, but both clefts are required for unloading clamp from DNA after replication is completed.

Luteinizing hormone promotes gonadal tumorigenesis in inhibin-deficient mice

The inhibins are secreted alpha:beta heterodimers of the TGF-beta superfamily that are mainly synthesized in Sertoli cells and granulosa cells, and are critical regulators of testicular and ovarian development and function. Mice homozygous for a targeted deletion of the inhibin alpha subunit gene (Inha(-/-)) develop sex cord-stromal tumors in a gonadotropin-dependent manner. Here, we determine the contribution of LH to gonadal tumorigenesis by generating mice deficient in both inhibins and LH. Inha(-/-)Lhb(-/-) mice have increased survival and delayed tumor progression, and these observations correlate with lower serum FSH and estradiol levels compared to Inha(-/-) controls. Double mutant testicular tumors demonstrate decreased expression of cyclin D2, while double mutant ovarian tumors have elevated expression of p15(INK4b) and trend toward higher levels of p27(Kip1). We conclude that LH is not required for tumor formation in the absence of inhibins but promotes tumor progression, likely through alterations in serum hormone levels and cell cycle regulators.

INVESTIGATING THE ROLE OF INHIBINS IN OVARIAN FOLLICLE DEVELOPMENT

The inhibins are alpha:beta heterodimers of the TGF-beta superfamily that are essential for balancing growth and differentiation within the ovary and testis. Inhibindeficient mice (Inha−/−) develop sex cord-stromal tumors at an early age, indicating that inhibins function as tumor suppressors with gonadal specificity. Inha−/− females are infertile at 6 weeks of age, however, pharmacologic superovulation before advanced tumor growth (i.e., 3–4 weeks old) allows for fertilization, but with substantially reduced efficiency compared to wild-type controls. Combined with the observation that ovaries from adult mutants do not contain corpora lutea, these results point to defects in folliculogenesis that may be independent of tumor development. To pursue these preliminary findings, we treated 3-week-old wild-type and Inha−/− female mice with eCG (44–46 hours), or with eCG (44–46 hours) followed by hCG (6 hours), and collected ovaries to analyze follicular architecture. Stimulated wild-type ovaries contain numerous large antral follicles, each with an oocyte surrounded by two to three layers of cumulus cells that begin to expand in response to hCG. In contrast, treated mutant ovaries demonstrate an increase in the overall number of follicles, however with fewer antral follicles. The antral follicles that are present appear small and have cumulus defects. These observations suggest that inhibins are essential for the proper regulation of gonadotropin-dependent follicular development. To identify key differences in inhibin-deficient granulosa cells that may in part be responsible for the defects in folliculogenesis, we isolated total RNA from wild-type and Inha−/− granulosa cells collected from eCG- and eCG/hCGtreated mice. Gene expression profiling was performed using the Affymetrix Mouse Genome 430 2.0 microarray (n = 3 chips for each of 4 groups). Our analyses reveal that eCG-stimulated Inha−/− granulosa cells have decreased expression of cell adhesion-related genes that may correlate with the observed abnormalities in follicular organization. Moreover, these cells have increased expression of multiple cell cycle genes and decreased expression of genes involved in terminal differentiation. Together, these findings indicate that the loss of inhibins disrupts the balance of growth and differentiation within ovarian follicles, ultimately driving them toward a proliferative state. This work was supported by National Institutes of Health Grant CA60651 (to M.M.M.) and the Joseph and Matilda Melnick Endowed Fund (to A.K.N.). (poster)

Ubiquitination of red blood cell α‐spectrin does not affect heterodimer formation

Erythrocyte alpha-spectrin is ubiquitinated in repeats alpha20/alpha21, which also represents the nucleation site for contact with the beta subunit which leads to heterodimer formation by a zippering mechanism. In this study we have determined the second-order rate constant for association of ubiquitinated alpha'-spectrin, nonubiquitinated alpha-spectrin, and beta-spectrin into the alpha'beta or alphabeta heterodimer. The rate constant for incorporation of monomers into heterodimers at 37 degrees C were (5.181 +/- 0.001) x 10(5) M(-1) sec(-1) for total alpha-spectrin (alpha + alpha'), (5.121 +/- 0.001) x 10(5) M(-1) sec(-1) for alpha'-spectrin, and (5.178 +/- 0.003) x 10(5) M(-1) sec(-1) for beta-spectrin. We conclude that ubiquitination of alpha-spectrin does not regulate heterodimer formation.

Comparison of the Function of the .BETA.C and .BETA.E Subunits of Activin in AML12 Hepatocytes

To investigate the function of the beta(C) and beta(E) subunits of activin, we overexpressed these subunits in AML12 cells, a normal hepatocyte cell line, using adenovirus vector. Overexpression of the beta(C) subunit increased [3H]thymidine incorporation and the cell number. In contrast, both [3H]thymidine incorporation and the cell number were reduced in the beta(E) overexpressing cells. When AML cells overexpressing the beta(E) subunit were cultured in medium containing 1% serum for 48 h, many of the cells died by apoptosis, whereas cells overexpressing the beta(C) subunit or beta-galactosidase survived in the same condition. To examine dimer formation, the beta(C) and beta(E) subunits were expressed in AML12 cells. In these cells, the beta(C) homodimer, the beta(E) homodimer and the beta(C)-beta(E) heterodimer were detected. When the expression level of the beta(E) subunit was increased, formation of the beta(E) homodimer was increased, while formation of the beta(C)-beta(E) heterodimer was slightly reduced. Overexpression of the beta(E) subunit did not significantly affect the formation of the beta(C) homodimer. These results indicate that the beta(C) and beta(E) subunits form homo- and heterodimers, and that the functions of the two subunits are quite different.

Masking of the CD3 gamma di-leucine-based motif by zeta is required for efficient T-cell receptor expression.

The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.

Structural and Functional Analysis of Integrin α2I Domain Interaction with Echovirus 1

Integrins are cell surface receptors for several microbial pathogens including echovirus 1 (EV1), a picornavirus. Cryo-electron microscopy revealed that the functional domain (alpha(2)I) of human alpha(2)beta(1) integrin binds to a surface depression on the EV1 capsid. This three-dimensional structure of EV1 bound to alpha(2)I domain provides the first structural details of an integrin interacting with a picornavirus. The model indicates that alpha(2)beta(1) integrin cannot simultaneously bind both EV1 and the physiological ligand collagen. Compared with collagen binding to the alpha(2)I domain, the virus binds with a 10-fold higher affinity but in vitro uncoating of EV1 was not observed as a result of attachment of alpha(2)I. A molecular model, constructed on the basis of the EV1-integrin complex, shows that multiple alpha(2)beta(1) heterodimers can bind at adjacent sites around the virus 5-fold symmetry axes without steric hindrance. In agreement with this, virus attachment to alpha(2)beta(1) integrin on the cell surface was found to result in integrin clustering, which can give rise to signaling and facilitate the initiation of the viral entry process that takes place via caveolae-mediated endocytosis.

Quantitation of HLA class II protein incorporated into human immunodeficiency type 1 virions purified by anti-CD45 immunoaffinity depletion of microvesicles.

Among the many host cell-derived proteins found in human immunodeficiency virus type 1 (HIV-1), HLA class II (HLA-II) appears to be selectively incorporated onto virions and may contribute to mechanisms of indirect imunopathogenesis in HIV infection and AIDS. However, the amount of HLA-II on the surface of HIV-1 particles has not been reliably determined due to contamination of virus preparations by microvesicles containing host cell proteins, including HLA-II. Even rigorous sucrose density centrifugation is unable to completely separate HIV-1 from microvesicles. CD45, a leukocyte integral membrane protein, is found on microvesicles, yet appears to be excluded from HIV-1 particles. Exploiting this observation, we have developed a CD45-based immunoaffinity depletion method for removing CD45-containing microvesicles that yields highly purified preparations of virions. Examination of CD45-depleted HIV-1(MN) by high-pressure liquid chromatography, protein sequencing, and amino acid analyses determined a molar ratio of HLA-II to Gag of 0.04 to 0.05 in the purified virions, corresponding to an estimated average of 50 to 63 native HLA-II complexes (i.e., a dimer of alpha and beta heterodimers) per virion. These values are approximately 5- to 10-fold lower than those previously determined for other virion preparations that contained microvesicles. Our observations demonstrate the utility of CD45 immunoaffinity-based approaches for producing highly purified retrovirus preparations for applications that would benefit from the use of virus that is essentially free of microvesicles.

Estrogen signaling: a subtle balance between ER alpha and ER beta.

The biological actions of estrogens are mediated by estrogen binding to one of two specific estrogen receptors (ERs) ERalpha and ERbeta, which belong to the nuclear receptor superfamily, a family of ligand-regulated transcription factors. ERalpha and ERbeta are products of different genes and exhibit tissue- and cell-type specific expression. The characterization of mice lacking ERalpha, or ERbeta, or both has revealed that both receptor subtypes have overlapping but also unique roles in estrogen-dependent action in vivo. Additionally, ERalpha and ERbeta have different transcriptional activities in certain ligand, cell-type, and promoter contexts. Both receptors, however, are coexpressed in a number of tissues and form functional heterodimers. The biological roles of ERalpha /beta heterodimers in the presence of each respective homodimer are unknown. When coexpressed, ERbeta exhibits an inhibitory action on ERalpha -mediated gene expression and in many instances opposes the actions of ERalpha. A number of ERalpha and ERbeta isoforms have also been described, many of which alter estrogen-mediated gene expression. Uncovering the molecular mechanisms regulating the expression of both ERs, and how ERalpha and ERbeta directly or indirectly affect each other's function are paramount to understanding the cellular and biological events of estrogen-mediated gene regulation in normal and diseased tissues.

Analysis of the cruciform binding activity of recombinant 14-3-3zeta-MBP fusion protein, its heterodimerization profile with endogenous 14-3-3 isoforms, and effect on mammalian DNA replication in vitro.

The human cruciform binding protein (CBP), a member of the 14-3-3 protein family, has been recently identified as an origin of DNA replication binding protein and involved in DNA replication. Here, pure recombinant 14-3-3zeta tagged with maltose binding protein (r14-3-3zeta-MBP) at its N-terminus was tested for binding to cruciform DNA either in the absence or presence of F(TH), a CBP-enriched fraction, by electromobility shift assay (EMSA), followed by Western blot analysis of the electroeluted CBP-cruciform DNA complex. The r14-3-3zeta-MBP was found to have cruciform binding activity only after preincubation with F(TH). Anti-MBP antibody immunoprecipitation of F(TH) preincubated with r14-3-3zeta-MBP, followed by Western blot analysis with antibodies specific to the beta, gamma, epsilon, zeta, and sigma 14-3-3 isoforms showed that r14-3-3zeta-MBP heterodimerized with the endogenous beta, epsilon, and zeta isoforms present in the F(TH) but not with the gamma or sigma isoforms. Immunoprecipitation of endogenous 14-3-3zeta from nuclear extracts (NE) of HeLa cells that were either serum-starved (s-s) or blocked at the G(1)/S or G(2)/M phases of the cell cycle revealed that at G(1)/S and G(2)/M, the zeta isoform heterodimerized only with the beta and epsilon isoforms, while in s-s extracts, the 14-3-3zeta/epsilon heterodimer was never detected, and the 14-3-3zeta/beta heterodimer was seldom detected. Furthermore, addition of r14-3-3zeta-MBP to HeLa cell extracts used in a mammalian in vitro replication system increased the replication level of p186, a plasmid bearing the minimal 186-bp origin of the monkey origin of DNA replication ors8, by approximately 3.5-fold. The data suggest that specific dimeric combinations of the 14-3-3 isoforms have CBP activity and that upregulation of this activity leads to an increase in DNA replication.

Detection of Integrin αIIbβ3Clustering in Living Cells

In platelets, bidirectional signaling across integrin alpha(IIb)beta(3) regulates fibrinogen binding, cytoskeletal reorganization, cell aggregation, and spreading. Because these responses may be influenced by the clustering of alpha(IIb)beta(3) heterodimers into larger oligomers, we established two independent methods to detect integrin clustering and evaluate factors that regulate this process. In the first, weakly complementing beta-galactosidase mutants were fused to the C terminus of individual alpha(IIb) subunits, and the chimeras were stably expressed with beta(3) in Chinese hamster ovary cells. Clustering of alpha(IIb)beta(3) should bring the mutants into proximity and reconstitute beta-galactosidase activity. In the second method, alpha(IIb) was fused to either a green fluorescent protein (GFP) or Renilla luciferase and transiently expressed with beta(3). Here, integrin clustering should stimulate bioluminescence resonance energy transfer between a cell-permeable luciferase substrate and GFP. These methods successfully detected integrin clustering induced by anti-alpha(IIb)beta(3) antibodies. Significantly, they also detected clustering upon soluble fibrinogen binding to alpha(IIb)beta(3). In contrast, no clustering was observed following direct activation of alpha(IIb)beta(3) by MnCl(2) or an anti-alpha(IIb)beta(3)-activating antibody Fab in the absence of fibrinogen. Intracellular events also influenced alpha(IIb)beta(3) clustering. For example, a cell-permeable, bivalent FK506-binding protein (FKBP) ligand stimulated clustering when added to cells expressing an alpha(IIb)(FKBP)(2) chimera complexed with beta(3). Furthermore, alpha(IIb)beta(3) clustering occurred in the presence of latrunculin A or cytochalasin D, inhibitors of actin polymerization. These effects were enhanced by fibrinogen, suggesting that actin-regulated clustering modulates alpha(IIb)beta(3) interaction with ligands. These studies in living cells establish that alpha(IIb)beta(3) clustering is modulated by fibrinogen and actin dynamics. More broadly, they should facilitate investigations of the mechanisms and consequences of integrin clustering.

Expression of membrane-bound and soluble guanylyl cyclase mRNAs in embryonic and adult retina of the medaka fish Oryzias latipes.

Localization of mRNAs for four membrane-bound guanylyl cyclases (membrane GCs; OlGC3, OlGC4, OlGC5, and OlGC-R2), three soluble guanylyl cyclase subunits (soluble GC; OlGCS-alpha(1), OlGCS-alpha(2), and OlGCS-beta(1)), neuronal nitric oxide synthase (nNOS), and cGMP-dependent protein kinase I (cGK I) was examined in the embryonic and adult retinas of the medaka fish Oryzias latipes by in situ hybridization. All of the membrane GC mRNAs were detected in the photoreceptor cells of the adult and embryonic retinas, but in different parts; the OlGC3 and OlGC5 mRNAs were expressed in the proximal part and the OlGC4 and OlGC-R2 mRNAs were expressed in the outer nuclear layer. The mRNA for nNOS was expressed in a scattered fashion on the inner side of the inner nuclear layer in the adult and embryonic retinas. The mRNAs (OlGCS-alpha(2) and OlGCS- beta(1)) of two soluble GC subunits (alpha(2) and beta(1)) were expressed mainly in the inner nuclear layer and the ganglion cell layer of the embryonic retina while the mRNAs of the soluble GC alpha(1) subunit and cGK I were not detected in either the adult or embryonic retina. These results suggest that NO itself and/or the cGMP generated by soluble GC (alpha(2)/beta(1) heterodimer) play a novel role in the neuronal signaling and neuronal development in the medaka fish embryonic retina in addition to the role played by phototransduction through membrane GCs in the adult and embryonic retinas.

Transition From Rolling to Firm Adhesion Is Regulated by the Conformation of the I Domain of the Integrin Lymphocyte Function-associated Antigen-1

The integrin lymphocyte function-associated antigen-1 (alpha(L)beta(2)), which is known for its ability to mediate firm adhesion and migration, can also contribute to tethering and rolling in shear flow. The alpha(L) I domain can be mutationally locked with disulfide bonds into two distinct conformations, open and closed, which have high and low affinity for the ligand intercellular adhesion molecule 1 (ICAM-1), respectively. The wild type I domain exists primarily in the lower energy closed conformation. We have measured for the first time the effect of conformational change on adhesive behavior in shear flow. We show that wild type and locked open I domains, expressed in alpha(L)beta(2) heterodimers or as isolated domains on the cell surface, mediate rolling adhesion and firm adhesion, respectively. alpha(L)beta(2) is thus poised for the conversion of rolling to firm adhesion upon integrin activation in vivo. Isolated I domains are surprisingly more effective than alpha(L)beta(2) in interactions in shear flow, which may in part be a consequence of the presence of alpha(L)beta(2) in a bent conformation. Furthermore, the force exerted on the C-terminal alpha-helix appears to stabilize the open conformation of the wild type isolated I domain and contribute to its robustness in supporting rolling. An allosteric small molecule antagonist of alpha(L)beta(2) inhibits both rolling adhesion and firm adhesion, which has important implications for its mode of action in vivo.

Characterization of integrin expression in the mouse ovary.

Integrin alpha:beta heterodimers mediate cell contacts to the extracellular matrix and initiate intracellular signaling cascades in response to a variety of factors. Integrins interact with many determinants of cellular phenotypes and play roles in controlling the development, structural integrity, and function of every type of tissue. Despite their importance, little is known about the regulation of integrin subunits in the mammalian ovary and how they function in folliculogenesis. To determine their relevance to ovarian physiology, we have studied the expression of integrin subunit mRNAs by Northern blot analysis and in situ hybridization in ovaries of wild-type, growth differentiation factor 9 (Gdf 9) knockout, FSHbeta (Fshb) knockout, and inhibin alpha (Inha) knockout mice. Integrin alpha6 mRNA is expressed in oocytes and granulosa cells of single-layer follicles and in oocytes and theca cells of multilayer follicles. Integrin alpha6 is highly expressed in Gdf 9 knockout ovaries, which are enriched in oocytes and primary (single layer) follicles because of a block at this stage of follicular development. Integrin alpha(v) mRNA is most highly expressed in the granulosa cells of multilayer growing follicles, and therefore only low levels of expression are detectable in the Gdf 9 knockout ovaries. Integrin beta1 mRNA exhibits a broad expression pattern in ovaries, including oocytes, granulosa cells, theca cells, and corpora lutea. Integrin beta3 mRNA is expressed in theca and interstitial cells and is upregulated in corpora lutea. It is nearly undetectable in ovaries of Fshb knockout mice, which develop preantral follicles but have no luteal cells. Integrin beta5 mRNA is predominantly expressed in granulosa cells of multilayer follicles. It is expressed at high levels in the Fshb knockout mice and in a compartmentalized manner in the granulosa cell/Sertoli cell tumors that develop in the Inha knockout mice. Specific integrins are associated with ovarian cellular phenotypes in mice, which raises intriguing possibilities as to integrin functions in oocyte competence, follicular development, luteinization, and granulosa cell proliferation.