Ikuko F. Mizukami

Function of the lectin domain of mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by β-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcγRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) β2 integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by β-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by β-glucan. A single CD11b lectin site for β-glucan and uPAR was suggested because the binding of either β-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of 125I-labeled β-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of 125I-labeled β-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.

Huntingtin Interacting Protein 1 Is a Clathrin Coat Binding Protein Required for Differentiation of late Spermatogenic Progenitors

ABSTRACT Huntingtin-interacting protein 1 (HIP1) interacts with huntingtin, the protein whose gene is mutated in Huntingtons disease. In addition, a fusion between HIP1 and platelet-derived growth factor β receptor causes chronic myelomonocytic leukemia. The HIP1 proteins, including HIP1 and HIP1-related (HIP1r), have an N-terminal polyphosphoinositide-interacting epsin N-terminal homology, domain, which is found in proteins involved in clathrin-mediated endocytosis. HIP1 and HIP1r also share a central leucine zipper and an actin binding TALIN homology domain. Here we show that HIP1, like HIP1r, colocalizes with clathrin coat components. We also show that HIP1 physically associates with clathrin and AP-2, the major components of the clathrin coat. To further understand the putative biological role(s) ofHIP1, we have generated a targeted deletion of murineHIP1. HIP1−/− mice developed into adulthood, did not develop overt neurologic symptoms in the first year of life, and had normal peripheral blood counts. However, HIP1-deficient mice exhibited testicular degeneration with increased apoptosis of postmeiotic spermatids. Postmeiotic spermatids are the only cells of the seminiferous tubules that express HIP1. These findings indicate that HIP1 is required for differentiation, proliferation, and/or survival of spermatogenic progenitors. The association of HIP1 with clathrin coats and the requirement of HIP1 for progenitor survival suggest a role for HIP1 in the regulation of endocytosis.

Serum antibodies to huntingtin interacting protein-1: a new blood test for prostate cancer.

Huntingtin-interacting protein 1 (HIP1) is frequently overexpressed in prostate cancer. HIP1 is a clathrin-binding protein involved in growth factor receptor trafficking that transforms fibroblasts by prolonging the half-life of growth factor receptors. In addition to human cancers, HIP1 is also overexpressed in prostate tumors from the transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse model. Here we provide evidence that HIP1 plays an important role in mouse tumor development, as tumor formation in the TRAMP mice was impaired in the Hip1null/null background. In addition, we report that autoantibodies to HIP1 developed in the sera of TRAMP mice with prostate cancer as well as in the sera from human prostate cancer patients. This led to the development of an anti-HIP1 serum test in humans that had a similar sensitivity and specificity to the anti-alpha-methylacyl CoA racemase (AMACR) and prostate-specific antigen tests for prostate cancer and when combined with the anti-AMACR test yielded a specificity of 97%. These data suggest that HIP1 plays a functional role in tumorigenesis and that a positive HIP1 autoantibody test may be an important serum marker of prostate cancer.

A soluble form of the urokinase plasminogen activator receptor (suPAR) can bind to hematopoietic cells.

The receptor for urokinase plasminogen activator (uPAR; CD87) is a 50‐ to 65‐kDa glycosylphosphatidylinositol (GPI)‐anchored glycoprotein expressed by leukocytes and tumor cells where it facilitates uPA‐dependent, plasmin‐mediated pericellular proteolysis during cellular invasion. Because uPAR is inducibly shed into culture supernatants and human body fluids, we tested the hypothesis that soluble uPAR (suPAR) can bind to the plasma membrane of hematopoietic cells where it might modulate their invasive phenotype. As measured by flow cytometry, recombinant biotinylated‐suPAR (B‐suPAR) bound in a specific fashion to THP‐1 leukemia cells and blood PMNs and monocytes (but not to lymphocytes). B‐suPAR also demonstrated specific binding to a variety of leukemic lines, including cells that are positive or negative for membrane uPAR expression. Binding of B‐suPAR to THP‐1 cells was enhanced four‐ to sevenfold by 24‐h exposure of cells to PMA or by coincubation with uPA ligand (but not its isolated catalytic and binding fragments). Conversely, binding of B‐suPAR to PMNs was unaffected by brief exposure to fMLP, and was inhibited by coincubation with uPA. B‐suPAR binding to PMA‐differentiated THP‐1 cells in the presence of uPA was further enhanced by acid washing (removing endogenous uPA) but was partially inhibited by treatment of cells with trypsin. Pretreatment of PMA‐differentiated THP‐1 cells and unstimulated PMNs with soluble sugars, calcium chelators, and antibodies specific for integrins or extracellular matrix proteins failed to consistently block the binding of B‐suPAR. Whereas the binding of suPAR did not measurably affect cell‐associated plasmin activation, suPAR did competitively inhibit the binding of exogenous uPA to membrane‐associated uPAR. These observations support the hypothesis that suPAR can bind specifically to trypsin‐sensitive receptors expressed by certain normal and neoplastic hematopoietic cells where its binding is variably influenced by uPA ligand. J. Leukoc. Biol. 64: 203–213; 1998.

Association of calmodulin inhibition, erythrocyte membrane stabilization and pharmacological effects of drugs

The present study was designed to determine whether there is an association of drug-induced inhibition of calmodulin functions, drug-induced membrane stabilization (protection against osmotic lysis), and pharmacological effects of drugs. First, data on drugs which have been studied for both calmodulin inhibition and membrane antihemolysis were collected from the literature and an association of the two properties was established. Second, ten additional drugs were selected for study of all three properties. Four drugs, with known antihemolytic effects, were studied for calmodulin inhibition. One drug, which was a known calmodulin inhibitor, was studied for antihemolysis. Our results show that membrane-stabilizing drugs are usually calmodulin inhibitors, and vice versa; that drugs in certain therapeutic classes inhibit calmodulin-activated functions and protect against osmotic lysis; and finally, that there is a significant correlation (P less than 0.01) in terms of potency between these two actions of drugs. Data from the literature which bear on these mechanisms of drug actions suggest that the interactions between drugs and calmodulin, and drugs and the membrane, appear to be hydrophobic in nature. At this point, we do not know whether there is some causal relationship between calmodulin inhibition and the antihemolytic effect of drugs, or whether the two are simply a result of hydrophobic properties of drugs. Similarly, the roles of calmodulin inhibition and/or membrane antihemolysis in producing therapeutic efficacy are unknown.

Hip1-related Mutant Mice Grow and Develop Normally but Have Accelerated Spinal Abnormalities and Dwarfism in the Absence of HIP1

ABSTRACT In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo.

A component in mammalian muscle synaptic basal lamina induces clustering of acetylcholine receptors.

Publisher Summary This chapter discusses the clustering of acetylcholine receptors (AChR). It presents a study in which an AChR clustering factor or factors from mammalian (rat) diaphragm muscle endplate extracellular matrix (SBL) was isolated. The purified factor, which can be extracted from the matrix by high salt (2 M MgC1 2 ) or low pH (5.4), appeared to be a glycoprotein with an apparent molecular mass of about 75,000 daltons; however, there might be two proteins with close molecular masses. The addition of the crude extracellular matrix fraction from the regions of muscle enriched in endplates (EP) or of purified factor(s) from the extracellular matrix of endplate, but not nonendplate (NEP) regions, produced a significant increase, between 4- and 15-fold, in the numbers of the clusters of α-bungarotoxin binding sites on both the rat and chick embryonic myotubes. The phenomenon is both dose and time dependent. NEP and EP matrix preparations in a modified in vitro hybridoma protocol are used to generate a monoclonal antibody designated synaptic basal lamina antibody-1 that blocked the induction of AChR aggregation by the factor(s); the antibody had no effect on the myotubes. The antibody can also be used to identify an antigen in the basal lamina of the rat skeletal muscle in vivo and in vitro that is concentrated at synapses and that colocalizes with α-bungarotoxin binding sites at the synapse.