Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines

Dendritic spines receive the vast majority of excitatory synaptic contacts in the mammalian brain and are presumed to contain machinery for the integration of various signal transduction pathways. Protein phosphatase 1 (PP1) is greatly enriched in dendritic spines and has been implicated in both the regulation of ionic conductances and long-term synaptic plasticity. The molecular mechanism whereby PP1 is localized to spines is unknown. We have now characterized a novel protein that forms a complex with the catalytic subunit of PP1 and is a potent modulator of PP1 enzymatic activity in vitro. Within the brain this protein displays a remarkably distinct localization to the heads of dendritic spines and has therefore been named spinophilin. Spinophilin has the properties expected of a scaffolding protein localized to the cell membrane and contains a single consensus sequence in PSD95/DLG/zo-1, which implies cross-linking of PP1 to transmembrane protein complexes. We propose that spinophilin represents a novel targeting subunit for PP1, which directs the enzyme to those substrates in the dendritic spine compartment, e.g., neurotransmitter receptors, which mediate the regulation of synaptic function by PP1.

The small GTP-binding protein Rho links G protein-coupled receptors and Gα12 to the serum response element and to cellular transformation

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Gαs and Gαi2 families were found in human tumors; and members of the Gαq and Gα12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Gα12 family of heterotrimeric G protein α subunits strongly induced the SRE, while Gβ1γ2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Gα12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

Polyvalent binding to carbohydrates immobilized on an insoluble resin

Numerous studies have established that polyvalency is a critical feature of cell surface carbohydrate recognition. Nevertheless, carbohydrate–protein interactions are typically evaluated by using assays that focus on the behavior of monovalent carbohydrate ligands in solution. It has generally been assumed that the relative affinities of monovalent carbohydrate ligands in solution correlate with their polyvalent avidities. In this paper we show that carbohydrate ligands synthesized directly on TentaGel beads interact with carbohydrate-binding proteins in a polyvalent manner. The carbohydrate-derivatized beads can, therefore, be used as model systems for cell surfaces to evaluate polyvalent carbohydrate–protein interactions. By using a combinatorial approach to synthesize solid-phase libraries of polyvalent carbohydrates, one can rapidly address key issues in the area of cell surface carbohydrate recognition. For example, studies reported herein demonstrate that there is an unanticipated degree of specificity in recognition processes involving polyvalent carbohydrates. However, the correlation between polyvalent avidities and solution affinities is poor. Apparently, the presentation of carbohydrates on the polymer surface has a profound influence on the interaction of the ligand with the protein receptor. These findings have implications for how carbohydrates function as recognition signals in nature, as well as for how polyvalent carbohydrate–protein interactions should be studied.

Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1

The current studies explore the mechanism by which the sphingomyelin content of mammalian cells regulates transcription of genes encoding enzymes of cholesterol synthesis. Previous studies by others have shown that depletion of sphingomyelin by treatment with neutral sphingomyelinase causes a fraction of cellular cholesterol to translocate from the plasma membrane to the endoplasmic reticulum where it expands a regulatory pool that leads to down-regulation of cholesterol synthesis and up-regulation of cholesterol esterification. Here we show that sphingomyelinase treatment of cultured Chinese hamster ovary cells prevents the nuclear entry of sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound transcription factor required for transcription of several genes involved in the biosynthesis and uptake of cholesterol. Nuclear entry is blocked because sphingomyelinase treatment inhibits the proteolytic cleavage of SREBP-2 at site 1, thereby preventing release of the active NH2-terminal fragments from cell membranes. Sphingomyelinase treatment thus mimics the inhibitory effect on SREBP processing that occurs when exogenous sterols are added to cells. Sphingomyelinase treatment did not block site 1 proteolysis of SREBP-2 in 25-RA cells, a line of Chinese hamster ovary cells that is resistant to the suppressive effects of sterols, owing to an activating point mutation in the gene encoding SREBP cleavage-activating protein. In 25-RA cells, sphingomyelinase treatment also failed to down-regulate the mRNA for 3-hydroxy-3-methylglutaryl CoA synthase, a cholesterol biosynthetic enzyme whose transcription depends on the cleavage of SREBPs. Considered together with previous data, the current results indicate that cells regulate the balance between cholesterol and sphingomyelin content by regulating the proteolytic cleavage of SREBPs.

Iron regulatory protein 1 is not required for the modulation of ferritin and transferrin receptor expression by iron in a murine pro-B lymphocyte cell line

Iron regulatory proteins (IRPs) are cytoplasmic RNA binding proteins that are central components of a sensory and regulatory network that modulates vertebrate iron homeostasis. IRPs regulate iron metabolism by binding to iron responsive element(s) (IREs) in the 5′ or 3′ untranslated region of ferritin or transferrin receptor (TfR) mRNAs. Two IRPs, IRP1 and IRP2, have been identified previously. IRP1 exhibits two mutually exclusive functions as an RNA binding protein or as the cytosolic isoform of aconitase. We demonstrate that the Ba/F3 family of murine pro-B lymphocytes represents the first example of a mammalian cell line that fails to express IRP1 protein or mRNA. First, all of the IRE binding activity in Ba/F3-gp55 cells is attributable to IRP2. Second, synthesis of IRP2, but not of IRP1, is detectable in Ba/F3-gp55 cells. Third, the Ba/F3 family of cells express IRP2 mRNA at a level similar to other murine cell lines, but IRP1 mRNA is not detectable. In the Ba/F3 family of cells, alterations in iron status modulated ferritin biosynthesis and TfR mRNA level over as much as a 20- and 14-fold range, respectively. We conclude that IRP1 is not essential for regulation of ferritin or TfR expression by iron and that IRP2 can act as the sole IRE-dependent mediator of cellular iron homeostasis.

Xenopus Cdc6 confers sperm binding competence to oocytes without inducing their maturation

Amphibian eggs normally require meiotic maturation to be competent for fertilization. A necessary prerequisite for this event is sperm binding, and we show that under normal physiological conditions this property is acquired at, but not before, meiotic maturation. Immature oocytes do not bind sperm, but injection of total egg poly(A)+ mRNA into immature oocytes confers sperm binding in the absence of meiotic maturation. Using an expression cloning approach we have isolated a single cDNA from egg poly(A)+ mRNA that can induce sperm binding in immature oocytes. The cDNA was found to encode Xenopus Cdc6, a protein that previously has been shown to function in initiation of DNA replication and cell cycle control. This unanticipated finding provides evidence of a link between a regulator of the cell cycle and alterations in cell surface properties that affect gamete binding.

Role of lipid polymorphism in G protein-membrane interactions: Nonlamellar-prone phospholipids and peripheral protein binding to membranes

Heterotrimeric G proteins (peripheral proteins) conduct signals from membrane receptors (integral proteins) to regulatory proteins localized to various cellular compartments. They are in excess over any G protein-coupled receptor type on the cell membrane, which is necessary for signal amplification. These facts account for the large number of G protein molecules bound to membrane lipids. Thus, the protein-lipid interactions are crucial for their cellular localization, and consequently for signal transduction. In this work, the binding of G protein subunits to model membranes (liposomes), formed with defined membrane lipids, has been studied. It is shown that although G protein α-subunits were able to bind to lipid bilayers, the presence of nonlamellar-prone phospholipids (phosphatidylethanolamines) enhanced their binding to model membranes. This mechanism also appears to be used by other (structurally and functionally unrelated) peripheral proteins, such as protein kinase C and the insect protein apolipophorin III, indicating that it could constitute a general mode of protein-lipid interactions, relevant in the activity and translocation of some peripheral (amphitropic) proteins from soluble to particulate compartments. Other factors, such as the presence of cholesterol or the vesicle surface charge, also modulated the binding of the G protein subunits to lipid bilayers. Conversely, the binding of G protein-coupled receptor kinase 2 and the G protein β-subunit to liposomes was not increased by hexagonally prone lipids. Their distinct interactions with membrane lipids may, in part, explain the different cellular localizations of all of these proteins during the signaling process.

Cell death induction by the acute promyelocytic leukemia-specific PML/RARα fusion protein

PML/RARα is the abnormal protein product generated by the acute promyelocytic leukemia-specific t(15;17). Expression of PML/RARα in hematopoietic precursor cell lines induces block of differentiation and promotes survival. We report here that PML/RARα has a potent growth inhibitory effect on all nonhematopoietic cell lines and on the majority of the hematopoietic cell lines tested. Inducible expression of PML/RARα in fibroblasts demonstrated that the basis for the growth suppression is induction of cell death. Deletion of relevant promyelocytic leukemia (PML) and retinoic acid receptor (RARα) domains within the fusion protein revealed that its growth inhibitory effect depends on the integrity of the PML aminoterminal region (RING, B1, B2, and coiled coil regions) and the RARα DNA binding region. Analysis of the nuclear localization of the same PML/RARα deletion mutants by immunofluorescence and cell fractionation revealed that the biological activity of the fusion protein correlates with its microspeckled localization and its association to the nuclear matrix. The PML aminoterminal region, but not the RARα zinc fingers, is required for the proper nuclear localization of PML/RARα. We propose that the matrix-associated microspeckles are the active sites of PML/RARα and that targeting of RARα sequences to this specific nuclear subdomain through PML sequences is crucial to the activity of the fusion protein on survival regulation.

Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands

Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole–imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

Participation of the nuclear cap binding complex in pre-mRNA 3′ processing

Communication between the 5′ and 3′ ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5′ end is bound by the nuclear cap binding complex (CBC). This RNA–protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3′ end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5′ and 3′ ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3′ processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of “closed-loops,” due to interactions between factors bound at their 5′ and 3′ ends.

Interaction of pigeon cytochrome c-(43–58) peptide analogs with either T cell antigen receptor or I-Ab molecule

We determined that a pigeon cytochrome c-derived peptide, p43–58, possesses two anchor residues, 46 and 54, for binding with the I-Ab molecule that are compatible to the position 1 (P1) and position 9 (P9) of the core region in the major histocompatibility complex (MHC) class II binding peptides, respectively. In the present study to analyze each binding site between P1 and P9 of p43–58 to either I-Ab or T cell antigen receptor (TCR), we investigated T cell responses to a series of peptides (P2K, P3K, P4K, P5K, P6K, P7K, and P8E) that sequentially substituted charged amino acid residues for the residues at P2 to P8 of p43–58. T cells from C57BL/10 (I-Ab) mice immunized with P4K or P6K did not mount appreciable proliferative responses to the immunogens, but those primed with other peptides (P2K, P3K, P5K, P7K, and P8E) showed substantial responses in an immunogen-specific manner. It was demonstrated by binding studies that P1 and P9 functioned as main anchors and P4 and P6 functioned as secondary anchors to I-Ab. Analyses of Vβ usage of T cell lines specific for these analogs suggested that P8 interacts with the complementarity-determining region 1 (CDR1)/CDR2 of the TCR β chain. Furthermore, sequencing of the TCR on T cell hybridomas specific for these analogs indicated that P5 interacts with the CDR3 of the TCR β chain. The present findings are consistent with the three-dimensional structure of the trimolecular complex that has been reported for TCR/peptide/MHC class I molecules.

Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel, but its relationship to the primary clinical manifestation of CF, chronic Pseudomonas aeruginosa pulmonary infection, is unclear. We report that CFTR is a cellular receptor for binding, endocytosing, and clearing P. aeruginosa from the normal lung. Murine cells expressing recombinant human wild-type CFTR ingested 30–100 times as many P. aeruginosa as cells lacking CFTR or expressing mutant ΔF508 CFTR protein. Purified CFTR inhibited ingestion of P. aeruginosa by human airway epithelial cells. The first extracellular domain of CFTR specifically bound to P. aeruginosa and a synthetic peptide of this region inhibited P. aeruginosa internalization in vivo, leading to increased bacterial lung burdens. CFTR clears P. aeruginosa from the lung, indicating a direct connection between mutations in CFTR and the clinical consequences of CF.

Interaction affinity between cytokine receptor components on the cell surface

The anti-common gamma chain (γc) mAb CP.B8 is shown to inhibit interleukin 4 (IL-4)-dependent proliferation of phytohemagglutinin (PHA) activated T cells noncompetitively with respect to cytokine by blocking the IL-4-induced heterodimerization of IL-4Rα and γc receptor chains. Affinities for the binding of IL-4 to Cos-7 cells transfected with huIL-4Rα, and to PHA blasts expressing both IL-4Rα and γc, were used to estimate the affinity of the key interaction between γc and the binary IL-4Rα⋅IL-4 complex on the cell surface. This affinity was defined in terms of the dimensionless ratio [IL-4Rα⋅IL-4⋅γc]/[IL-4Rα⋅IL-4], which we designate KR. The results show that on PHA blasts this interaction is relatively weak; KR ≈ 9, implying that ≈10% of the limiting IL-4Rα chain remains free of γc even at saturating concentrations of IL-4. This quantitative treatment establishes KR as a key measure of the coupling between ligand binding and receptor activation, providing a basis for functional distinctions between different receptors that are activated by ligand-induced receptor dimerization.

Multiple loop structures critical for ligand binding of the integrin α4 subunit in the upper face of the β-propeller mode 1

A non-I-domain integrin, α4β1, recognizes vascular cell adhesion molecule 1 (VCAM-1) and the IIICS portion of fibronectin. To localize regions of α4 critical for ligand binding, we swapped several predicted loops within or near the putative ligand-binding site of α4 (which spans repeats 2–5 of the seven N-terminal repeats) with the corresponding regions of α5. Swapping residues 112–131 in repeat 2, or residues 237–247 in repeat 4, completely blocked adhesion to immobilized VCAM-1 and connecting segment 1 (CS-1) peptide. However, swapping residues 40–52 in repeat 1, residues 151–164 in repeat 3, or residues 282–288 (which contain a putative cation binding motif) in repeat 5 did not affect or only slightly reduced adhesion to these ligands. The binding of several function-blocking antibodies is blocked by swapping residues 112–131, 151–164, and 186–191 (which contain previously identified residues critical for ligand binding, Tyr-187 and Gly-190). These results are consistent with the recently published β-propeller folding model of the integrin α4 subunit [Springer, T. A. (1997) Proc. Natl. Acad. Sci. USA 94, 65–72], in which seven four-stranded β-sheets are arranged in a torus around a pseudosymmetric axis. The regions of α4 critical for ligand binding are adjacent to each other and are located in the upper face, the predicted ligand-binding site, of the β-propeller model, although they are not adjacent in the primary structure.

High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYφ motif (φ represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 ± 0.74 μM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 ± 0.10 μM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.