γ Subunit of the AP-1 Adaptor Complex Binds Clathrin: Implications for Cooperative Binding in Coated Vesicle Assembly

The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from the trans-Golgi network (TGN). The beta 1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the beta chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the gamma subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the gamma hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the gamma subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-gamma hinge, but not by GST-gamma appendage. However, the hinge and appendage domains of gamma function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the gamma and beta1 subunits of AP-1.     

Microbial Conversion of α-Ionone, α-Methylionone, and α-Isomethylionone

α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion.     

Cross Talk between β1 and αV Integrins: β1 Affects β3 mRNA Stability

There is increasing evidence that a fine-tuned integrin cross talk can generate a high degree of specificity in cell adhesion, suggesting that spatially and temporally coordinated expression and activation of integrins are more important for regulated cell adhesive functions than the intrinsic specificity of individual receptors. However, little is known concerning the molecular mechanisms of integrin cross talk. With the use of beta(1)-null GD25 cells ectopically expressing the beta(1)A integrin subunit, we provide evidence for the existence of a cross talk between beta(1) and alpha(V) integrins that affects the ratio of alpha(V)beta(3) and alpha(V)beta(5) integrin cell surface levels. In particular, we demonstrate that a down-regulation of alpha(V)beta(3) and an up-regulation of alpha(V)beta(5) occur as a consequence of beta(1)A expression. Moreover, with the use of GD25 cells expressing the integrin isoforms beta(1)B and beta(1)D, as well as two beta(1) cytoplasmic domain deletion mutants lacking either the entire cytoplasmic domain (beta(1)TR) or only its "variable" region (beta(1)COM), we show that the effects of beta(1) over alpha(V) integrins take place irrespective of the type of beta(1) isoform, but require the presence of the "common" region of the beta(1) cytoplasmic domain. In an attempt to establish the regulatory mechanism(s) whereby beta(1) integrins exert their trans-acting functions, we have found that the down-regulation of alpha(V)beta(3) is due to a decreased beta(3) subunit mRNA stability, whereas the up-regulation of alpha(V)beta(5) is mainly due to translational or posttranslational events. These findings provide the first evidence for an integrin cross talk based on the regulation of mRNA stability.     

Assembly and Intracellular Targeting of the βγ Subunits of Heterotrimeric G Proteins

The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent α, β, and γ subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified βγ complexes, recognize β subunits when not associated with γ and can thus be used to monitor assembly of βγ complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of βγ complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with γ subunits, their localization, and their detergent solubility. Association of β and α subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of βγ subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

Activation of Distinct α5β1-mediated Signaling Pathways by Fibronectin's Cell Adhesion and Matrix Assembly Domains

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α₅β₁ integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α₅β₁ integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α₅β₁ integrin, suggesting that these two domains of fibronectin cannot bind to the α₅β₁ integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α₅β₁ integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.     

The Laminin α Chains: Expression, Developmental Transitions, and Chromosomal Locations of α1-5, Identification of Heterotrimeric Laminins 8–11, and Cloning of a Novel α3 Isoform

Laminin trimers composed of α, β, and γ chains are major components of basal laminae (BLs) throughout the body. To date, three α chains (α1–3) have been shown to assemble into at least seven heterotrimers (called laminins 1–7). Genes encoding two additional α chains (α4 and α5) have been cloned, but little is known about their expression, and their protein products have not been identified. Here we generated antisera to recombinant α4 and α5 and used them to identify authentic proteins in tissue extracts. Immunoprecipitation and immunoblotting showed that α4 and α5 assemble into four novel laminin heterotrimers (laminins 8–11: α4β1γ1, α4β2γ1, α5β1γ1, and α5β2γ1, respectively). Using a panel of nucleotide and antibody probes, we surveyed the expression of α1-5 in murine tissues. All five chains were expressed in both embryos and adults, but each was distributed in a distinct pattern at both RNA and protein levels. Overall, α4 and α5 exhibited the broadest patterns of expression, while expression of α1 was the most restricted. Immunohistochemical analysis of kidney, lung, and heart showed that the α chains were confined to extracellular matrix and, with few exceptions, to BLs. All developing and adult BLs examined contained at least one α chain, all α chains were present in multiple BLs, and some BLs contained two or three α chains. Detailed analysis of developing kidney revealed that some individual BLs, including those of the tubule and glomerulus, changed in laminin chain composition as they matured, expressing up to three different α chains and two different β chains in an elaborate and dynamic progression. Interspecific backcross mapping of the five α chain genes revealed that they are distributed on four mouse chromosomes. Finally, we identified a novel full-length α3 isoform encoded by the Lama3 gene, which was previously believed to encode only truncated chains. Together, these results reveal remarkable diversity in BL composition and complexity in BL development.     

Differential Subcellular Localization of Protein Phosphatase-1 α, γ1, and δ Isoforms during Both Interphase and Mitosis in Mammalian Cells

Protein phosphatase-1 (PP-1) is involved in the regulation of numerous metabolic processes in mammalian cells. The major isoforms of PP-1, α, γ1, and δ, have nearly identical catalytic domains, but they vary in sequence at their extreme NH₂ and COOH termini. With specific antibodies raised against the unique COOH-terminal sequence of each isoform, we find that the three PP-1 isoforms are each expressed in all mammalian cells tested, but that they localize within these cells in a strikingly distinct and characteristic manner. Each isoform is present both within the cytoplasm and in the nucleus during interphase. Within the nucleus, PP-1 α associates with the nuclear matrix, PP-1 γ1 concentrates in nucleoli in association with RNA, and PP-1 δ localizes to nonnucleolar whole chromatin. During mitosis, PP-1 α is localized to the centrosome, PP-1 γ1 is associated with microtubules of the mitotic spindle, and PP-1 δ strongly associates with chromosomes. We conclude that PP-1 isoforms are targeted to strikingly distinct and independent sites in the cell, permitting unique and independent roles for each of the isoforms in regulating discrete cellular processes.     

Complementary Roles for Receptor Clustering and Conformational Change in the Adhesive and Signaling Functions of Integrin αIIbβ3

Integrin α_IIbβ₃ mediates platelet aggregation and “outside-in” signaling. It is regulated by changes in receptor conformation and affinity and/or by lateral diffusion and receptor clustering. To document the relative contributions of conformation and clustering to α_IIbβ₃ function, α_IIb was fused at its cytoplasmic tail to one or two FKBP12 repeats (FKBP). These modified α_IIb subunits were expressed with β₃ in CHO cells, and the heterodimers could be clustered into morphologically detectable oligomers upon addition of AP1510, a membrane-permeable, bivalent FKBP ligand. Integrin clustering by AP1510 caused binding of fibrinogen and a multivalent (but not monovalent) fibrinogen-mimetic antibody. However, ligand binding due to clustering was only 25–50% of that observed when α_IIbβ₃ affinity was increased by an activating antibody or an activating mutation. The effects of integrin clustering and affinity modulation were additive, and clustering promoted irreversible ligand binding. Clustering of α_IIbβ₃ also promoted cell adhesion to fibrinogen or von Willebrand factor, but not as effectively as affinity modulation. However, clustering was sufficient to trigger fibrinogen-independent tyrosine phosphorylation of pp72^Syk and fibrinogen-dependent phosphorylation of pp125^FAK, even in non-adherent cells. Thus, receptor clustering and affinity modulation play complementary roles in α_IIbβ₃ function. Affinity modulation is the predominant regulator of ligand binding and cell adhesion, but clustering increases these responses further and triggers protein tyrosine phosphorylation, even in the absence of affinity modulation. Both affinity modulation and clustering may be needed for optimal function of α_IIbβ₃ in platelets.     

The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.     

Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets

It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

An α-Tubulin Mutant Destabilizes the Heterodimer: Phenotypic Consequences and Interactions with Tubulin-binding Proteins

Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant α-tubulin to probe cellular regulation of microtubule assembly. tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and β-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724 haploid cells when the β-tubulin–binding protein Rbl2p is either overexpressed or absent. It also explains why the TUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind α-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components.     

Structural Requirements for Function of Yeast GGAs in Vacuolar Protein Sorting, α-Factor Maturation, and Interactions with Clathrin

The GGAs (Golgi-localized, gamma-ear-containing, ARF-binding proteins) are a family of multidomain adaptor proteins involved in protein sorting at the trans-Golgi network of eukaryotic cells. Here we present results from a functional characterization of the two Saccharomyces cerevisiae GGAs, Gga1p and Gga2p. We show that deletion of both GGA genes causes defects in sorting of carboxypeptidase Y (CPY) and proteinase A to the vacuole, vacuolar morphology, and maturation of alpha-factor. A structure-function analysis reveals a requirement of the VHS, GAT, and hinge for function, while the GAE domain is less important. We identify putative clathrin-binding motifs in the hinge domain of both yeast GGAs. These motifs are shown to mediate clathrin binding in vitro. While mutation of these motifs alone does not block function of the GGAs in vivo, combining these mutations with truncations of the hinge and GAE domains diminishes function, suggesting functional cooperation between different clathrin-binding elements. Thus, these observations demonstrate that the yeast GGAs play important roles in the CPY pathway, vacuole biogenesis, and alpha-factor maturation and identify structural determinants that are critical for these functions.     

α3β1 Integrin Is Required for Normal Development of the Epidermal Basement Membrane

Integrins α3β1 and α6β4 are abundant receptors on keratinocytes for laminin-5, a major component of the basement membrane between the epidermis and the dermis in skin. These integrins are recruited to distinct adhesion structures within keratinocytes; α6β4 is present in hemidesmosomes, while α3β1 is recruited into focal contacts in cultured cells. To determine whether differences in localization reflect distinct functions of these integrins in the epidermis, we studied skin development in α3β1-deficient mice. Examination of extracellular matrix by immunofluorescence microscopy and electron microscopy revealed regions of disorganized basement membrane in α3β1-deficient skin. Disorganized matrix was first detected by day 15.5 of embryonic development and became progressively more extensive as development proceeded. In neonatal skin, matrix disorganization was frequently accompanied by blistering at the dermal-epidermal junction. Laminin-5 and other matrix proteins remained associated with both the dermal and epidermal sides of blisters, suggesting rupture of the basement membrane itself, rather than detachment of the epidermis from the basement membrane as occurs in some blistering disorders such as epidermolysis bullosa. Consistent with this notion, primary keratinocytes from α3β1-deficient skin adhered to laminin-5 through α6 integrins. However, α3β1-deficient keratinocytes spread poorly compared with wild-type cells on laminin-5, demonstrating a postattachment requirement for α3β1 and indicating distinct roles for α3β1 and α6β4. Our findings support a novel role for α3β1 in establishment and/or maintenance of basement membrane integrity, while α6β4 is required for stable adhesion of the epidermis to the basement membrane through hemidesmosomes.     

A model for how Gβγ couples Gα to GPCR

Representing ∼5% of the human genome, G-protein-coupled receptors (GPCRs) are a primary target for drug discovery; however, the molecular details of how they couple to heterotrimeric G protein subunits are incompletely understood. Here, I propose a hypothetical initial docking model for the encounter between GPCR and Gβγ that is defined by transient interactions between the cytosolic surface of the GPCR and the prenyl moiety and the tripeptide motif, asparagine–proline–phenylalanine (NPF), in the C-terminus of the Gγ subunit. Analysis of class A GPCRs reveals a conserved NPF binding site formed by the interaction of the TM1 and H8. Functional studies using differentially prenylated proteins and peptides further suggest that the intracellular hydrophobic core of the GPCR is a prenyl binding site. Upon binding TM1 and H8 of GPCRs, the propensity of the C-terminal region of Gγ to convert into an α helix allows it to extend into the hydrophobic core of the GPCR, facilitating the GPCR active state. Conservation of the NPF motif in Gγ isoforms and interacting residues in TM1 and H8 suggest that this is a general mechanism of GPCR–G protein signaling. Analysis of the rhodopsin dimer also suggests that Gγ–rhodopsin interactions may facilitate GPCR dimer transactivation.