Ligation of intestinal epithelial CD1d induces bioactive IL-10: Critical role of the cytoplasmic tail in autocrine signaling

The intestinal epithelium is anatomically positioned to serve as the critical interface between the lumen and the mucosal immune system. In addition to MHC class I and II antigens, intestinal epithelia constitutively express the nonclassical MHC molecule CD1d, a transmembrane molecule with a short cytoplasmic tail expressed as a β2-microglobulin-associated 48-kDa glycoprotein and novel β2-microglobulin-independent 37-kDa nonglycosylated protein on intestinal epithelia. At present, it is not known whether extracellular ligands can signal intestinal epithelial CD1d. To define signaling of CD1d cytoplasmic tail, retrovirus-mediated gene transfer was used to generate stable cell lines expressing wild-type CD1d or a chimeric molecule (extracellular CD1d and cytoplasmic CD1a), and surface CD1d was triggered by antibody crosslinking. Although wild-type CD1d was readily activated (tyrosine phosphorylation), no demonstrable signal was evident in cell lines expressing the chimeric molecule. Subsequent studies revealed that anti-CD1d crosslinking specifically induces epithelial IL-10 mRNA and protein and is blocked by the tyrosine kinase inhibitor genistein. Further studies addressing epithelial-derived IL-10 revealed that anti-CD1d crosslinking attenuates IFN-γ signaling and that such attenuation is reversed by addition of functionally inhibitory IL-10 antibodies. These results define signaling through surface CD1d, and, importantly, they demonstrate that this pathway may serve to dampen epithelial proinflammatory signals.

Neuropeptide release by efficient recruitment of diffusing cytoplasmic secretory vesicles

Neuropeptides are slowly released from a limited pool of secretory vesicles. Despite decades of research, the composition of this pool has remained unknown. Endocrine cell studies support the hypothesis that a population of docked vesicles supports the first minutes of hormone release. However, it has been proposed that mobile cytoplasmic vesicles dominate the releasable neuropeptide pool. Here, to determine the cellular basis of the releasable pool, single green fluorescent protein-labeled secretory vesicles were visualized in neuronal growth cones with the use of an inducible construct or total internal reflection fluorescence microscopy. We report that vesicle movement follows the diffusion equation. Furthermore, rapidly moving secretory vesicles are used more efficiently than stationary vesicles near the plasma membrane to support stimulated release. Thus, randomly moving cytoplasmic vesicles participate in the first minutes of neuropeptide release. Importantly, the preferential recruitment of diffusing cytoplasmic secretory vesicles contributes to the characteristic slow kinetics and limited extent of sustained neuropeptide release.

Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo

Both caspase-1- and caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. We assessed the contributions of these caspases to Fas signaling in hepatocyte cell death in vitro. Although wild-type, caspase-1−/−, and caspase-3−/− hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3−/− hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1−/− apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation were seen within 6 hr, but neither event was observed for caspase-3−/− hepatocytes. We extended these studies to thymocytes and found that apoptotic caspase-3−/− thymocytes exhibited similar “abnormal” morphological changes and delayed DNA fragmentation observed in hepatocytes. Furthermore, the cleavage of various caspase substrates implicated in mediating apoptotic events, including gelsolin, fodrin, laminB, and DFF45/ICAD, was delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.

Structure and function in rhodopsin: Topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops*

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335–T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4′-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240–V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.

KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit

ATP-sensitive potassium (“KATP”) channels are rapidly inhibited by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity to energy metabolism in a variety of cells. The KATP channel is formed from four each of a sulfonylurea receptor (SUR) regulatory subunit and an inwardly rectifying potassium (Kir6.2) pore-forming subunit. We used systematic chimeric and point mutagenesis, combined with patch-clamp recording, to investigate the molecular basis of ATP-dependent inhibition gating of mouse pancreatic β cell KATP channels expressed in Xenopus oocytes. We identified distinct functional domains of the presumed cytoplasmic C-terminal segment of the Kir6.2 subunit that play an important role in this inhibition. Our results suggest that one domain is associated with inhibitory ATP binding and another with gate closure.

Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 → Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 → Ala or Phe13 → Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.

Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum

The monolayer tapetum cells of the maturing flowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by flotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g/cm3 and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g/cm3 and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of floral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface.

Sequences within the Cytoplasmic Domain of Gp180/Carboxypeptidase D Mediate Localization to the Trans-Golgi Network

Gp180, a duck protein that was proposed to be a cell surface receptor for duck hepatitis B virus, is the homolog of metallocarboxypeptidase D, a mammalian protein thought to function in the trans-Golgi network (TGN) in the processing of proteins that transit the secretory pathway. Both gp180 and mammalian metallocarboxypeptidase D are type I integral membrane proteins that contain a 58-residue cytosolic C-terminal tail that is highly conserved between duck and rat. To investigate the regions of the gp180 tail involved with TGN retention and intracellular trafficking, gp180 and various deletion and point mutations were expressed in the AtT-20 mouse pituitary corticotroph cell line. Full length gp180 is enriched in the TGN and also cycles to the cell surface. Truncation of the C-terminal 56 residues of the cytosolic tail eliminates the enrichment in the TGN and the retrieval from the cell surface. Truncation of 12–43 residues of the tail reduced retention in the TGN and greatly accelerated the turnover of the protein. In contrast, deletion of the C-terminal 45 residues, which truncates a potential YxxL-like sequence (FxxL), reduced the protein turnover and caused accumulation of the protein on the cell surface. A point mutation of the FxxL sequence to AxxL slowed internalization, showing that this element is important for retrieval from the cell surface. Mutation of a pair of casein kinase II sites within an acidic cluster showed that they are also important for trafficking. The present study demonstrates that multiple sequence elements within the cytoplasmic tail of gp180 participate in TGN localization.

Cluster of Differentiation Antigen 4 (CD4) Endocytosis and Adaptor Complex Binding Require Activation of the CD4 Endocytosis Signal by Serine Phosphorylation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56lck, undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56lck, we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.

Cytoplasmic Dynein Heavy Chain 1b Is Required for Flagellar Assembly in Chlamydomonas

A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. The Chlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1b gene have been deleted. The two mutants assemble short flagellar stumps (<1–2 μm) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas.

Gene Knockouts Reveal Separate Functions for Two Cytoplasmic Dyneins in Tetrahymena thermophila

In many organisms, there are multiple isoforms of cytoplasmic dynein heavy chains, and division of labor among the isoforms would provide a mechanism to regulate dynein function. The targeted disruption of somatic genes in Tetrahymena thermophila presents the opportunity to determine the contributions of individual dynein isoforms in a single cell that expresses multiple dynein heavy chain genes. Substantial portions of two Tetrahymena cytoplasmic dynein heavy chain genes were cloned, and their motor domains were sequenced. Tetrahymena DYH1 encodes the ubiquitous cytoplasmic dynein Dyh1, and DYH2 encodes a second cytoplasmic dynein isoform, Dyh2. The disruption of DYH1, but not DYH2, resulted in cells with two detectable defects: 1) phagocytic activity was inhibited, and 2) the cells failed to distribute their chromosomes correctly during micronuclear mitosis. In contrast, the disruption of DYH2 resulted in a loss of regulation of cell size and cell shape and in the apparent inability of the cells to repair their cortical cytoskeletons. We conclude that the two dyneins perform separate tasks in Tetrahymena.

Specific Activation of Leukocyte β2 Integrins Lymphocyte Function–associated Antigen-1 and Mac-1 by Chemokines Mediated by Distinct Pathways via the α Subunit Cytoplasmic Domains

We show that CC chemokines induced a sustained increase in monocyte adhesion to intercellular adhesion molecule-1 that was mediated by Mac-1 (αMβ2) but not lymphocyte function–associated antigen-1 (LFA-1; αLβ2). In contrast, staining for an activation epitope revealed a rapid and transient up-regulation of LFA-1 activity by monocyte chemotactic protein-1 (MCP-1) in monocytes and Jurkat CCR2 chemokine receptor transfectants or by stromal-derived factor-1α in Jurkat cells. Differential kinetics for activation of Mac-1 (sustained) and LFA-1 (transient) avidity in response to stromal-derived factor-1α were confirmed by expression of αM or αL in αL-deficient Jurkat cells. Moreover, expression of chimeras containing αL and αM cytoplasmic domain exchanges indicated that α cytoplasmic tails conferred the specific mode of regulation. Coexpressing αM or chimeras in mutant Jurkat cells with a “gain of function” phenotype that results in constitutively active LFA-1 demonstrated that Mac-1 was not constitutively active, whereas constitutive activity was mediated via the αL cytoplasmic tail, implying the presence of distinct signaling pathways for LFA-1 and Mac-1. Transendothelial chemotaxis of monocytes in response to MCP-1 was dependent on LFA-1; however, Mac-1 was involved at MCP-1 concentrations stimulating its avidity, showing differential contributions of β2 integrins. Our data suggest that a specific regulation of β2 integrin avidity by chemokines may be important in leukocyte extravasation and may be triggered by distinct activation pathways transduced via the α subunit cytoplasmic domains.

Evidence for Four Cytoplasmic Dynein Heavy Chain Isoforms in Rat Testis

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.

β1-Integrin Cytoplasmic Subdomains Involved in Dominant Negative Function

The β1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms (“common” region) and a distal subdomain specific for each isoform (“variable” region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used β1A and β1B isoforms as well as four mutants lacking the entire cytoplasmic domain (β1TR), the variable region (β1COM), or the common region (β1ΔCOM-B and β1ΔCOM-A). By expressing these constructs in Chinese hamster ovary and β1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227–238, 1996), we show that β1B, β1COM, β1ΔCOM-B, and β1ΔCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, β1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that β1B interferes in a dominant negative manner with β1A and β3/β5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the β1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the β1B isoform.

Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity was greater when they were derived from the same protein than derived from different proteins. In fact, the chimera with a TM domain of HA and truncated CT of polyimmunoglobulin receptor did not support full fusion, indicating that the two regions are not functionally independent. Despite the fact that there is wide latitude in the sequence of the TM domain that supports fusion, a point mutation of a semiconserved residue within the TM domain of HA inhibited fusion. The ability of a foreign TM domain to support fusion contradicts the hypothesis that a pore is composed solely of fusion proteins and supports the theory that the TM domain creates fusion pores after a stage of hemifusion has been achieved.