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.

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.

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.

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.

An Atypical Sorting Determinant in the Cytoplasmic Domain of P-Selectin Mediates Endosomal Sorting

We previously identified the 11 amino acid C1 region of the cytoplasmic domain of P-selectin as essential for an endosomal sorting event that confers rapid turnover on P-selectin. The amino acid sequence of this region has no obvious similarity to other known sorting motifs. We have analyzed the sequence requirements for endosomal sorting by measuring the effects of site-specific mutations on the turnover of P-selectin and of the chimeric protein LLP, containing the lumenal and transmembrane domains of the low density lipoprotein receptor and the cytoplasmic domain of P-selectin. Endosomal sorting activity was remarkably tolerant of alanine substitutions within the C1 region. The activity was eliminated by alanine substitution of only one amino acid residue, leucine 768, where substitution with several other large side chains, hydrophobic and polar, maintained the sorting activity. The results indicate that the endosomal sorting determinant is not structurally related to previously reported sorting determinants. Rather, the results suggest that the structure of the sorting determinant is dependent on the tertiary structure of the cytoplasmic domain.

The Kinesin-related Proteins, Kip2p and Kip3p, Function Differently in Nuclear Migration in Yeast

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δ kar9Δ double mutants were viable. Conversely, kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δ dhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.

Regulation of Adherence and Virulence by the Entamoeba histolytica Lectin Cytoplasmic Domain, Which Contains a β2 Integrin Motif

Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the β2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin’s role in virulence.

Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules in Paramecium

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the α- and β- tubulin subunits are modified by only 1–5 and 2–9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.

Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.

The Cytoplasmic Zinc Finger Protein ZPR1 Accumulates in the Nucleolus of Proliferating Cells

The zinc finger protein ZPR1 translocates from the cytoplasm to the nucleus after treatment of cells with mitogens. The function of nuclear ZPR1 has not been defined. Here we demonstrate that ZPR1 accumulates in the nucleolus of proliferating cells. The role of ZPR1 was examined using a gene disruption strategy. Cells lacking ZPR1 are not viable. Biochemical analysis demonstrated that the loss of ZPR1 caused disruption of nucleolar function, including preribosomal RNA expression. These data establish ZPR1 as an essential protein that is required for normal nucleolar function in proliferating cells.

An Intronic Enhancer Is Required for Deflagellation-induced Transcriptional Regulation of a Chlamydomonas reinhardtii Dynein Gene

Chlamydomonas reinhardtii flagellar regeneration is accompanied by rapid induction of genes encoding a large set of flagellar structural components and provides a model system to study coordinate gene regulation and organelle assembly. After deflagellation, the abundance of a 70-kDa flagellar dynein intermediate chain (IC70, encoded by ODA6) mRNA increases approximately fourfold within 40 min and returns to predeflagellation levels by ∼90 min. We show by nuclear run-on that this increase results, in part, from increased rates of transcription. To localize cis induction elements, we created an IC70 minigene and measured accumulation, in C. reinhardtii, of transcripts from the endogenous gene and from introduced promoter deletion constructs. Clones containing 416 base pairs (bp) of 5′- and 2 kilobases (kb) of 3′-flanking region retained all sequences necessary for a normal pattern of mRNA abundance change after deflagellation. Extensive 5′- and 3′- flanking region deletions, which removed multiple copies of a proposed deflagellation-response element (the tub box), did not eliminate induction, and the IC70 5′-flanking region alone did not confer deflagellation responsiveness to a promoterless arylsulfatase (ARS) gene. Instead, an intron in the IC70 gene 5′-untranslated region was found to contain the deflagellation response element. These results suggest that the tub box does not play an essential role in deflagellation-induced transcriptional regulation of this dynein gene.

LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.