The ANK repeat: a ubiquitous motif involved in macromolecular recognition.

Many proteins rely on stable, noncovalent interactions with other macromolecules to perform their function. The identification of a repeated sequence motif, the ANK repeat, in diverse proteins whose common function involves binding to other proteins indicates one way nature may achieve a wide range of protein-protein interactions. In this article, we describe evidence that these ANK repeats are involved in the specific recognition of proteins and possibly DNA, and present a model for the folding of the motif.

The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats.

Ankyrin repeats are a 33-amino acid motif present in a number of proteins of diverse functions including transcription factors, cell differentiation molecules, and structural proteins. This motif has been shown to mediate protein interactions in the case of ankyrin as well as several other repeat-bearing proteins. In ankyrin, 24 tandemly arrayed repeats are arranged to form a globular, membrane-binding domain. This report provides evidence that the repeats in this domain fold into four independently folded subdomains of six repeats each. Limited proteolytic digestions of defined regions of the membrane-binding domain identified protease-sensitive sites, which divided this domain into subdomains of approximately six repeats each. Hydrodynamic measurements and circular dichroism spectroscopy of expressed subdomains confirmed that these six-repeat regions exist as folded, globular structures. The requirement of a complete set of six repeats for proper folding was determined using a series of protein constructs, which sequentially deleted repeats from the last subdomain. Deletion of even one repeat resulted in a 40% loss of alpha-helicity. Deletions removing three or more repeats abolished the helical signal completely. The spherical shapes of the intact domain and of the subdomains (inferred from hydrodynamic values) suggest that the four subdomains are organized in either a tetrahedral or square planar configuration. Two six-repeat subdomains were found to be required for high affinity association with the anion exchanger, suggesting that at least some of the protein interactions mediated by ankyrin repeats involve multiple subdomains.

The ANK repeats of erythrocyte ankyrin form two distinct but cooperative binding sites for the erythrocyte anion exchanger.

The 24 ANK repeats of the membrane-binding domain of ankyrin form four folded subdomains of six ANK repeats each. These four repeat subdomains mediate interactions with at least seven different families of membrane proteins. In the erythrocyte, the main membrane target of ankyrin is the Cl-/HCO3- anion exchanger. This report presents the first evidence that ankyrin contains two separate binding sites for anion exchanger dimers. One site utilizes repeat subdomain two (repeats 7-12) while the other requires both repeat subdomains three and four (repeats 13-24). The two sites are positively coupled with a Hill coefficient of 1.4. Since the anion exchanger exists as a dimer in the membrane, the presence of two binding sites on ankyrin allows ankyrin to interact with four anion exchangers simultaneously. These findings provide a direct demonstration of the versatility of ANK repeats in protein recognition, and have important implications for the organization of ankyrin-linked integral membrane proteins in erythrocytes as well as other cells.

Mechanism for binding site diversity on ankyrin. Comparison of binding sites on ankyrin for neurofascin and the Cl-/HCO3- anion exchanger.

Ankyrins are a family of spectrin-binding proteins that associate with at least seven distinct membrane proteins, including ion transporters and cell adhesion molecules. The membrane-binding domain of ankyrin is comprised of a tandem array of 24 ANK repeats organized into four 6-repeat folding domains. Tandem arrays of ANK repeats have been proposed to mediate protein interactions in a variety of proteins including factors involved in the regulation of transcription and the cell cycle. This report provides several new insights into the versatility of ANK repeats of ankyrin in protein recognition, using neurofascin and the Cl-/HCO3- anion exchanger as model ligands and ankyrinR as the prototypic ankyrin. Different combinations of ANK repeat domains from this ankyrin form two distinct, high affinity binding sites for neurofascin. One site requires both repeat domains 3 and 4. The other site involves both repeat domains 2 and 3, although domain 2 has significant activity alone. The sites appear to be independent with Kd values of 3 and 14 nM, respectively. Both the Cl-/HCO3- anion exchanger and neurofascin can interact simultaneously with repeat domains 3 and 4, because neurofascin is unable to displace binding of the anion exchanger cytoplasmic domain to domains 3 and 4, despite having a 3-5-fold higher affinity. These results demonstrate two levels of diversity in the binding sites on ankyrin: one resulting from different combinations of ANK repeat domains and another from different determinants within the same combination of repeat domains. One consequence of this diversity is that ankyrin can accommodate two neurofascin molecules as well as the anion exchanger through interactions mediated by ANK repeats. The ability of ankyrin to simultaneously associate with multiple types of membrane proteins is an unanticipated finding with implications for the assembly of integral membrane proteins into specialized regions of the plasma membrane.

A requirement for ankyrin binding to clathrin during coated pit budding.

Recent studies suggest that the mobility of clathrin-coated pits at the cell surface are restricted by an actin cytoskeleton and that there is an obligate reduction in the amount of spectrin on membranes during coated pit budding. The spectrin-actin cytoskeleton associates with membranes primarily through ankyrins, which interact with the cytoplasmic region of numerous integral membrane proteins. We now report that the fourth repeat domain (D4) of ankyrin(R) binds to the N-terminal domain of clathrin heavy chain with high affinity. Addition of peptides containing the D4 region inhibited clathrin-coated pit budding in vitro. In addition, microinjection of D4 containing peptides blocked the endocytosis of fluorescent low density lipoprotein (LDL). Ankyrin(R) peptides that contained repeat domains other than D4 had no effect on either in vitro budding or internalization of LDL. Finally, immunofluorescence shows that ankyrin is uniformly associated with endosomes that contain fluorescent LDL. These results suggest that ankyrin plays a role in the budding of clathrin-coated pits during endocytosis.

Presence of oxidized cholesterol in caveolae uncouples active platelet-derived growth factor receptors from tyrosine kinase substrates.

Platelet-derived growth factor receptor beta (PDGFRbeta) in fibroblasts is concentrated in caveolae where it controls the tyrosine phosphorylation of multiple proteins. Caveolae are enriched in cholesterol and sphingolipids, but the role of these lipids in PDGFR signal transduction is unknown. We report that introduction of cholest-4-en-3-one into caveolae membranes uncouples PDGFR autophosphorylation from tyrosine phosphorylation of neighboring proteins. Cholest-4-en-3-one appears to interfere with the normal interaction between PDGFR and its partners. The results suggest that tightly packed caveolae lipids form a membrane platform that functions as a lipid scaffold for organizing the molecular interactions of multiple signaling pathways.

Polarized distribution of endogenous Rac1 and RhoA at the cell surface.

Rac1 and RhoA regulate membrane ruffling and stress fiber formation. Both molecules appear to exert their control from the plasma membrane. In fibroblasts stimulated with platelet-derived growth factor or lysophosphatidic acid, the reorganization of the cytoskeleton begins at specific sites on the cell surface. We now report that endogenous Rac1 and RhoA also have a polarized distribution at the cell surface. Cell fractionation and immunogold labeling show that in quiescent fibroblasts both of these molecules are concentrated in caveolae, which are plasma membrane domains that are associated with actin-rich regions of the cell. Treatment of these cells with platelet-derived growth factor stimulated the recruitment of additional Rac1 and RhoA to caveolae fractions, while lysophosphatidic acid only caused the recruitment of RhoA. We could reconstitute the recruitment of RhoA using either whole cell lysates or purified caveolae. Surprisingly, pretreatment of the lysates with exoenzyme C3 shifted both resident and recruited RhoA from caveolae to noncaveolae membranes. The shift in location was not caused by inactivation of the RhoA effector domain. Moreover, chimeric proteins containing the C-terminal consensus site for Rac1 and RhoA prenylation were constitutively targeted to caveolae fractions. These results suggest that the polarized distribution of Rho family proteins at the cell surface involves an initial targeting of the protein to caveolae and a mechanism for retaining it at this site.

[The sleep apnea syndrome]. / Le syndrome d'apnée pendant le sommeil.

The authors presents an pathologic entity whose importance has been outlined in the last few years: sleep apnea syndromes. They insisted on the level of study of this syndrome in western countries. They presents the etiology, the pathophysiology and the main complication and they emphasizes the risks of traffic accidents due to the fall asleep and the cardiovascular complication. The therapeutical possibilities depending on the evolutive stage has been surveyed.

Feline cytotoxic large granular lymphocytes induced by recombinant human IL-2.

Large granular lymphocytes (LGL) have been characterized phenotypically and functionally as cytotoxic T lymphocytes, NK cells or lymphokine-activated killer cells. The most prominent morphologic feature of LGL is large cytoplasmic granules that are thought to contain the molecules responsible for cell lysis. In this study, we describe the morphologic and functional characteristics of IL-2-dependent cytotoxic lymphocytes derived from feline PBL. Stimulation of feline PBL with Con A followed by culturing in 50 U of gibbon monkey IL-2 human rIL-2 induced long term lymphocyte cultures. These lymphocytes are cytotoxic for the feline leukemia virus-induced T cell lymphoma (FL74), in a 4-h 51Cr release assay. All cell lines are either constitutively cytotoxic for FL74 cells, or cytotoxic in a lectin-dependent cell cytotoxic assay, the latter being a characteristic of low passage cultures. In contrast, no cell lines express self lysis or lysis for other lines. [3H]TdR uptake showed that 1 U of human rIL-2 produces a 50% maximal proliferative response by feline lymphocytes suggesting a high degree of homology between the ligand binding sites of feline and human IL-2R. Feline cytotoxic lymphocytes possess abundant cytoplasm containing large azurophilic granules characteristic of LGL. These granules are bound by a bilipid membrane and contain numerous smaller membrane-bound vesicles 50 to 60 nm in diameter. A model is proposed, whereby subsequent to binding of LGL to target cell the large granules fuse to the LGL plasma membrane and release the small vesicles into the binding pocket. The vesicles then transport the lytic molecules directly and selectively to the target cell membrane.