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At least 1,063 records · Page 59Linked to original sources
PubMed1996

Two different cytoplasmic tails direct isoforms of the membrane cofactor protein (CD46) to the basolateral surface of Madin-Darby canine kidney cells.

Membrane cofactor protein (MCP; CD46), a widely distributed regulatory protein of the complement system, was analyzed for expression in polarized epithelial cells. Both a human and a simian (Vero C1008) cell line were found to contain endogenous MCP mainly on the basolateral surface. Transfected Madin-Darby canine kidney cells stably expressing human MCP delivered this protein also predominantly to the basolateral surface. A deletion mutant lacking the cytoplasmic tail was transported in a nonpolarized fashion, indicating that the targeting signal for the basolateral transport is located in the cytoplasmic domain. A characteristic feature of MCP is the presence of various isoforms that contain either of two different cytoplasmic tails as a consequence of alternative splicing. Two isoforms differing only in the cytoplasmic tail (tail 1 or 2) were analyzed for polarized expression in Madin-Darby canine kidney cells. Surface biotinylation, as well as confocal immunofluorescence microscopy, indicated that both proteins were transported to the basolateral surface. Because no sequence similarity has been observed, the two tails contain different basolateral targeting signals. A deletion mutant lacking the only tyrosine residue in tail 1 retained the polarized expression indicating that, in contrast to most basolateral sorting signals, the transport signal of the tail 1 isoform is not dependent on tyrosine. The maintenance of a targeting motif in two distinct cytoplasmic tails suggests that the basolateral expression of MCP in polarized epithelial cells is of physiological importance.

Amino Acid Sequence↗
PubMed1996

Cytoplasmic domain of natriuretic peptide receptor-C inhibits adenylyl cyclase. Involvement of a pertussis toxin-sensitive G protein.

Natriuretic peptide receptor C (NPR-C) is a disulfide-linked homodimer with an approximately 440-amino acid extracellular domain and a 37-amino acid cytoplasmic domain; it functions in the internalization and degradation of bound ligand. The use of NPR-C-specific natriuretic peptide analogs has implicated this receptor in mediating the inhibition of adenylyl cyclase or activation of phospholipase C. In the present studies we have investigated the role of the cytoplasmic domain of NPR-C in signaling the inhibition of adenylyl cyclase. Polyclonal rabbit antisera were raised against a 37-amino acid synthetic peptide (R37A) corresponding to the cytoplasmic domain of NPR-C. Incubation of anti-R37A with rat heart particulate fractions blocked atrial natriuretic peptide-dependent inhibition of adenylyl cyclase. The cytoplasmic domain peptides R37A and TMC (10 residues of transmembrane domain appended on R37A) were equipotent in inhibiting adenylyl cyclase (Ki approximately 1 nM) in a GTP-dependent manner, whereas K37E (a scrambled peptide control for R37A) did not inhibit adenylyl cyclase activity. Prior incubation of membranes with pertussis toxin blocked R37A or TMC inhibition of cAMP production. Detergent solubilization of the rat heart particulate fraction destroyed natriuretic peptide inhibition of adenylyl cyclase, but TMC was able to inhibit cAMP production in a dose-dependent manner. Our results provide evidence that the 37-amino acid cytoplasmic domain of NPR-C is sufficient for signaling inhibition of adenylyl cyclase through a pertussis toxin-sensitive G protein.

Adenylate Cyclase Toxin↗
PubMed1996

Effect of cytoplasmic domain mutations on the agonist-stimulated ligand binding activity of the platelet integrin alphaIIbbeta3.

Function of the platelet integrin alphaIIbbeta3 is regulated by agonist-generated signals interacting with its cytoplasmic tails. When alphaIIbbeta3 is expressed in Epstein-Barr virus-transformed B lymphocytes, stimulation of the cells with phorbol 12-myristate 13-acetate results in alphaIIbbeta3-mediated lymphocyte adherence to immobilized fibrinogen, as well as soluble fibrinogen binding to alphaIIbbeta3, indicating that agonists increase the affinity of alphaIIbbeta3 for fibrinogen in these cells. To address the contribution of the alphaIIb and beta3 cytoplasmic tails to this process, we mutated each tail and expressed the mutants in B lymphocytes. Truncation of the alphaIIb tail did not impair unstimulated or stimulated lymphocyte adherence to fibrinogen, regardless whether the truncation was proximal or distal to the conserved GFFKR sequence. However, deleting GFFKR or replacing it with alanines markedly reduced alphaIIbbeta3 expression due to impaired intracellular assembly of alphaIIbbeta3 heterodimers, probably due to a mutation-induced change in the conformation of alphaIIb. Introducing beta3 mutations known to impair alphaIIbbeta3 function in platelets into the cytoplasmic tail of beta3 in lymphocytes also impaired alphaIIbbeta3 function in these cells. These studies demonstrate that the cytoplasmic tail of alphaIIb is not required for alphaIIbbeta3 function in lymphocytes, although the presence of GFFKR in the alphaIIb tail is required for alphaIIb to interact with beta3. Additionally, they indicate that signals interacting with the beta3 cytoplasmic tail are responsible for the ability of agonists to stimulate alphaIIbbeta3 function.

Amino Acid Sequence↗
PubMed1997

The role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the Escherichia coli cytoplasm.

In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each other in vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli.

Bacterial Proteins↗
PubMed1998

Function of the KKXX motif in endoplasmic reticulum retrieval of a transmembrane protein depends on the length and structure of the cytoplasmic domain.

Transmembrane glycoproteins with type 1 topology can be retrieved to the endoplasmic reticulum (ER) by a retrieval signal containing a di-lysine (KK) motif near the C terminus. To investigate the structural requirements for ER retrieval, we have constructed mutants of the simian immunodeficiency virus (SIV) envelope (Env) protein with cytoplasmic tails of different lengths and containing a KK motif at the -3 and -4 positions. Such proteins were found to be retained intracellularly when the signal was located 18 amino acids or more away from the membrane spanning domain. The retrieval signal was found to be functional even when placed at the distal end of the wild-type SIV Env protein with 164 amino acids in the cytoplasmic tail, as shown by the lack of proteolytic processing and lack of cell surface expression of the mutant proteins. However, proteins with a cytoplasmic tail length of 13 amino acids or less having the di-lysine motif at the -3 and -4 positions were not retrieved to the ER since they were found to be processed and transported to the cell surface. The surface-expressed proteins were found to be functional in inducing cell fusion, whereas the proteins retained intracellularly were defective in fusion activity. We also found that the KK motif introduced near an amphipathic helical region in the cytoplasmic tail was not functional. These results demonstrate that the ability of the KK motif to cause protein retrieval and retention in the endoplasmic reticulum depends on the length and structure of the cytoplasmic domain. The ER retrieval of the mutant proteins was found to correlate with increased intracellular binding to beta COP proteins.

Cell Line↗
PubMed1998

The cytoplasmic domains of a beta1 integrin mediate polarization in Madin-Darby canine kidney cells by selective basolateral stabilization.

In Madin-Darby canine kidney cells, newly synthesized apical and basolateral membrane proteins are generally transported directly to their respective cell surface domain due to targeting determinants that mediate sorting in the Golgi complex. In several basolateral membrane proteins, these targeting determinants reside in the cytoplasmic domains. We show here that basolateral expression of the human alpha5beta1 integrin in stably transfected Madin-Darby canine kidney cells is also mediated by the cytoplasmic domains. Distinct regions in both cytoplasmic domains were found to be sufficient to mediate basolateral expression independently from one another. Unexpectedly, newly synthesized wild-type alpha5beta1 and basolaterally expressed chimeras containing the cytoplasmic domain of either alpha5 or beta1 were integrated into both cell surface domains, preferentially apically, during biosynthesis. The apical pools of wild-type integrin and chimeric subunits were found to become quickly degraded, whereas the basolateral pools were stabilized. Thus, the cytoplasmic domains of the alpha5beta1 integrin are independently sufficient to mediate sorting by selective basolateral stabilization.

Animals↗
PubMed1998

Connexin-aequorin chimerae report cytoplasmic calcium environments along trafficking pathways leading to gap junction biogenesis in living COS-7 cells.

The cytoplasmic calcium environments along membrane trafficking pathways leading to gap junction intercellular communication channels at the plasma membrane were studied. Connexins, the constitutive proteins of gap junctions, were fused at their carboxyl terminus to the calcium-sensitive photoprotein aequorin. The cellular location of the chimeric proteins was determined by immunolocalization and subcellular fractionation. The generation of functional gap junctions by the connexin chimerae was monitored by the ability of the cells to exchange small dyes. Although aequorin fused to connexin-26 was nonfunctional, its ability to report Ca2+ and to form functional gap junctions was rescued by replacement of its cytoplasmic carboxyl tail with that of connexin-43. In COS-7 cells expressing these connexin-aequorin chimerae, calcium levels below the plasma membrane were higher (approximately 5 microM) than those in the cytoplasm (approximately 100 nM); gap junctions were able to transfer dyes under these conditions. Cytoplasmic levels of free calcium surrounding the ERGIC/Golgi reported by connexin-43 chimera (approximately 420 nM) were twice those measured by connexin-32 chimera (approximately 200 nM); both chimerae measured calcium levels substantially higher than those reported by a connexin-26 chimera (approximately 130 nM). Dispersion of the ERGIC and Golgi complex by brefeldin A led to a marked reduction in calcium levels. The results show that the various connexin chimerae were located in spatially different subcellular stores and that the ERGIC/Golgi regions of the cell maintain heterogeneous cytoplasmic domains of calcium. The implications of the subplasma-membrane Ca2+ levels on the gating of gap junctions are discussed.

Aequorin↗
PubMed1999

The Talin head domain binds to integrin beta subunit cytoplasmic tails and regulates integrin activation.

The beta subunit cytoplasmic domains of integrin adhesion receptors are necessary for the connection of these receptors to the actin cytoskeleton. The cytoplasmic protein, talin, binds to beta integrin cytoplasmic tails and actin filaments, hence forming an integrin-cytoskeletal linkage. We used recombinant structural mimics of beta(1)A, beta(1)D and beta(3) integrin cytoplasmic tails to characterize integrin-binding sites within talin. Here we report that an integrin-binding site is localized within the N-terminal talin head domain. The binding of the talin head domain to integrin beta tails is specific in that it is abrogated by a single point mutation that disrupts integrin localization to talin-rich focal adhesions. Integrin-cytoskeletal interactions regulate integrin affinity for ligands (activation). Overexpression of a fragment of talin containing the head domain led to activation of integrin alpha(IIb)beta(3); activation was dependent on the presence of both the talin head domain and the integrin beta(3) cytoplasmic tail. The head domain of talin thus binds to integrins to form a link to the actin cytoskeleton and can thus regulate integrin function.

Amino Acid Sequence↗
PubMed2000

Size-dependent DNA mobility in cytoplasm and nucleus.

The diffusion of DNA in cytoplasm is thought to be an important determinant of the efficacy of gene delivery and antisense therapy. We have measured the translational diffusion of fluorescein-labeled double-stranded DNA fragments (in base pairs (bp): 21, 100, 250, 500, 1000, 2000, 3000, 6000) after microinjection into cytoplasm and nucleus of HeLa cells. Diffusion was measured by spot photobleaching using a focused argon laser spot (488 nm). In aqueous solutions, diffusion coefficients of the DNA fragments in water (D(w)) decreased from 53 x 10(-8) to 0.81 x 10(-8) cm(2)/s for sizes of 21-6000 bp; D(w) was related empirically to DNA size: D(w) = 4.9 x 10(-6) cm(2)/s.[bp size](-0.72). DNA diffusion coefficients in cytoplasm (D(cyto)) were lower than D(w) and depended strongly on DNA size. D(cyto)/D(w) decreased from 0.19 for a 100-bp DNA fragment to 0.06 for a 250-bp DNA fragment and was <0.01 for >2000 bp. Diffusion of microinjected fluorescein isothiocyanate (FITC) dextrans was faster than that of comparably sized DNA fragments of 250 bp and greater. In nucleus, all DNA fragments were nearly immobile, whereas FITC dextrans of molecular size up to 580 kDa were fully mobile. These results suggest that the highly restricted diffusion of DNA fragments in nucleoplasm results from extensive binding to immobile obstacles and that the decreased lateral mobility of DNAs >250 bp in cytoplasm is because of molecular crowding. The diffusion of DNA in cytoplasm may thus be an important rate-limiting barrier in gene delivery utilizing non-viral vectors.

Cell Nucleus↗
PubMed2000

Cytoplasmic, nuclear, and golgi localization of RGS proteins. Evidence for N-terminal and RGS domain sequences as intracellular targeting motifs.

RGS proteins comprise a family of proteins named for their ability to negatively regulate heterotrimeric G protein signaling. Biochemical studies suggest that members of this protein family act as GTPase-activating proteins for certain Galpha subunits, thereby accelerating the turn-off mechanism of Galpha and terminating signaling by both Galpha and Gbetagamma subunits. In the present study, we used confocal microscopy to examine the intracellular distribution of several RGS proteins in COS-7 cells expressing RGS-green fluorescent protein (GFP) fusion proteins and in cells expressing RGS proteins endogenously. RGS2 and RGS10 accumulated in the nucleus of COS-7 cells transfected with GFP constructs of these proteins. In contrast, RGS4 and RGS16 accumulated in the cytoplasm of COS-7 transfectants. As observed in COS-7 cells, RGS4 exhibited cytoplasmic localization in mouse neuroblastoma cells, and RGS10 exhibited nuclear localization in human glioma cells. Deletion or alanine substitution of an N-terminal leucine repeat motif present in both RGS4 and RGS16, a domain identified as a nuclear export sequence in HIV Rev and other proteins, promoted nuclear localization of these proteins in COS-7 cells. In agreement with this observation, treatment of mouse neuroblastoma cells with leptomycin B to inhibit nuclear protein export by exportin1 resulted in accumulation of RGS4 in the nucleus of these cells. GFP fusions of RGS domains of RGS proteins localized in the nucleus, suggesting that nuclear localization of RGS proteins results from nuclear targeting via RGS domain sequences. RGSZ, which shares with RGS-GAIP a cysteine-rich string in its N-terminal region, localized to the Golgi complex in COS-7 cells. Deletion of the N-terminal domain of RGSZ that includes the cysteine motif promoted nuclear localization of RGSZ. None of the RGS proteins examined were localized at the plasma membrane. These results demonstrate that RGS proteins localize in the nucleus, the cytoplasm, or shuttle between the nucleus and cytoplasm as nucleo-cytoplasmic shuttle proteins. RGS proteins localize differentially within cells as a result of structural differences among these proteins that do not appear to be important determinants for their G protein-regulating activities. These findings suggest involvement of RGS proteins in more complex cellular functions than currently envisioned.

Amino Acid Sequence↗
PubMed2000

The rat liver Na(+)/bile acid cotransporter. Importance of the cytoplasmic tail to function and plasma membrane targeting.

To understand the potential functions of the cytoplasmic tail of Na(+)/taurocholate cotransporter (Ntcp) and to determine the basolateral sorting mechanisms for this transporter, green fluorescent protein-fused wild type and mutant rat Ntcps were constructed and the transport properties and cellular localization were assessed in transfected COS 7 and Madin-Darby canine kidney (MDCK) cells. Truncation of the 56-amino acid cytoplasmic tail demonstrates that the cytoplasmic tail of rat Ntcp is involved membrane delivery of this protein in nonpolarized and polarized cells and removal of the tail does not affect the bile acid transport function of Ntcp. Using site-directed mutagenesis, two tyrosine residues, Tyr-321 and Tyr-307, in the cytoplasmic tail of Ntcp have been identified as important for the basolateral sorting of rat Ntcp in transfected MDCK cells. Tyr-321 appears to be the major basolateral-sorting determinant, and Tyr-307 acts as a supporting determinant to ensure delivery of the transporter to the basolateral surface, especially at high levels of protein expression. When the two Tyr-based basolateral sorting motifs have been removed, the N-linked carbohydrate groups direct the tyrosine to alanine mutants to the apical surface of transfected MDCK cells. The major basolateral sorting determinant Tyr-321 is within a novel beta-turn unfavorable tetrapeptide Y(321)KAA, which has not been found in any naturally occurring basolateral sorting motifs. Two-dimensional NMR spectroscopy of a 24-mer peptide corresponding to the sequence from Tyr-307 to Thr-330 on the cytoplasmic tail of Ntcp confirms that both the Tyr-321 and Tyr-307 regions do not adopt any turn structure. Since the major motif YKAA contains a beta-turn unfavorable structure, the Ntcp basolateral sorting may not be related to the clathrin-adaptor complex pathway, as is the case for many basolateral proteins.

Amino Acid Sequence↗
PubMed2001

The cytoplasmic tail of alpha 1,2-fucosyltransferase contains a sequence for golgi localization.

The Golgi apparatus has a central role in the glycosylation of proteins and lipids. There is a sequential addition of carbohydrates by glycosyltransferases that are distributed within the Golgi in the order in which the glycosylation occurs. The mechanism of glycosyltransferase retention is considered to involve their transmembrane domains and flanking regions, although we have shown that the cytoplasmic tail of alpha1,2-fucosyltransferase is important for its Golgi localization. Here we show that the removal of the alpha1,2-fucosyltransferase cytoplasmic tail altered its function of fucosylation and its localization site. When the tail was removed, the enzyme moved from the Golgi to the trans Golgi network, suggesting that the transmembrane is responsible for retention and that the cytoplasmic tail is responsible for localization. The cytoplasmic tail of alpha1,2-fucosyltransferase contains 8 amino acids (MWVPSRRH), and mutating these to alanine indicated a role for amino acids 3 to 7 in localization with a particular role of Ser(5). Mutagenesis of Ser(5) to amino acids containing an hydroxyl (Tyr and Thr) demonstrated that the hydroxyl at position 5 is important. Thus, the cytoplasmic tail, and especially a single amino acid, has a predominant role in the localization and thus the function of alpha1,2-fucosyltransferase.

Amino Acid Sequence↗
PubMed2000

An essential cytoplasmic domain for the Golgi localization of coiled-coil proteins with a COOH-terminal membrane anchor.

Giantin is a resident Golgi protein that has an extremely long cytoplasmic domain (about 370 kDa) and is anchored to the Golgi membrane by the COOH-terminal membrane-anchoring domain (CMD) with no luminal extension. We examined the essential domain of giantin required for Golgi localization by mutational analysis. The Golgi localization of giantin was not affected by the deletion of its CMD or by substitution with the CMD of syntaxin-2, a plasma membrane protein. The giantin CMD fused to the cytoplasmic domain of syntaxin-2 could not retain the chimera in the Golgi apparatus. Sequential deletion analysis showed that the COOH-terminal sequence (positions 3059--3161) adjacent to the CMD was the essential domain required for the Golgi localization of giantin. We also examined two other Golgi-resident proteins, golgin-84 and syntaxin-5, with a similar membrane topology as giantin. It was confirmed that the cytoplasmic domain of about 100 residues adjacent to the CMD was required for their Golgi localization. Taken together, these results suggest that the COOH-terminally anchored Golgi proteins with long cytoplasmic extensions have the Golgi localization signal(s) in the cytoplasmic sequence adjacent to the CMD. This is in contrast to previous observations that a transmembrane domain is required for Golgi localization by other Golgi proteins transported from the endoplasmic reticulum.

Animals↗
PubMed2000

The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin. A possible mechanism for regulating cadherin turnover.

Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.

Amino Acid Sequence↗
PubMed2001

The proamphiregulin cytoplasmic domain is required for basolateral sorting, but is not essential for constitutive or stimulus-induced processing in polarized Madin-Darby canine kidney cells.

In this study, the role of the amphiregulin precursor (pro-AR) cytoplasmic domain in the basolateral sorting and cell-surface processing of pro-AR in polarized epithelial cells has been investigated using Madin-Darby canine kidney cells stably expressing various human pro-AR forms. Our results demonstrate that newly synthesized wild-type pro-AR (50 kDa) is delivered directly to the basolateral membrane domain with >95% efficiency, where it is sequentially cleaved within the ectodomain to release several soluble amphiregulin (AR) forms. Analyses of a pro-AR cytoplasmic domain truncation mutant (ARTL27) and two pro-AR secretory mutants (ARsec184 and ARsec190) indicated that the pro-AR cytoplasmic domain is not required for efficient delivery to the plasma membrane, but does contain essential basolateral sorting information. We show that the pro-AR cytoplasmic domain truncation mutant (ARTL27) is not sorted in polarized Madin-Darby canine kidney cells, with approximately 65% of the newly synthesized protein delivered to the apical cell surface. Under base-line conditions, ARTL27 was preferentially cleaved from the basolateral surface with 4-fold greater efficiency compared with cleavage from the apical membrane domain. However, ARTL27 ectodomain cleavage could be stimulated equivalently from either membrane domain by a variety of different stimuli. The metalloprotease inhibitor BB-94 could inhibit both base-line and stimulus-induced ectodomain cleavage of wild-type pro-AR and ARTL27. These results indicate that the pro-AR cytoplasmic domain is required for basolateral sorting, but is not essential for ectodomain processing. Preferential constitutive cleavage of ARTL27 from the basolateral cell surface also suggests that the metalloprotease activity involved in base-line and stimulus-induced ARTL27 ectodomain cleavage may be regulated differently in the apical and basolateral membrane domains of polarized epithelial cells.

Amphiregulin↗
PubMed2002

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.

Amino Acid Sequence↗
PubMed2001

The cytoplasmic tail of L-selectin interacts with members of the Ezrin-Radixin-Moesin (ERM) family of proteins: cell activation-dependent binding of Moesin but not Ezrin.

L-selectin regulates the recruitment of naive lymphocytes from the bloodstream to secondary lymphoid organs, mediating their initial capture and subsequent rolling along high endothelial cell surface-expressed ligands in peripheral lymph nodes. In vivo, distribution of L-selectin and cell surface levels determine the tethering efficiency and rolling velocity of leukocytes, respectively. Treatment of naive lymphocytes with phorbol myristate acetate (PMA) induces rapid ectodomain proteolytic down-regulation (shedding) of surface L-selectin via a protein kinase C (PKC)-dependent pathway. In an attempt to isolate proteins that are involved in regulating L-selectin expression, an affinity column was constructed using the 17-amino acid cytoplasmic tail of L-selectin. Affinity purification of extracts from lymphocytes, pre-treated with or without PMA, allowed identification of proteins that interact with the affinity column under one condition but not the other. By using this approach, members of the Ezrin-Radixin-Moesin family of proteins were found to interact specifically with the cytoplasmic tail of L-selectin. Moesin from PMA-stimulated lymphocytes, but not from unstimulated lymphocytes, bound to L-selectin tail. In contrast, ezrin from unstimulated or PMA-stimulated lymphocytes associated with L-selectin tail with equal affinity. Furthermore, the PKC inhibitor Ro 31-8220 significantly reduced the interaction of moesin, but not ezrin, with L-selectin. Alanine mutations of membrane-proximal basic amino acid residues in the cytoplasmic domain of L-selectin identified arginine 357 as a critical residue for both ezrin and moesin interaction. Finally, BIAcore affinity analysis confirmed that N-terminal moesin interacts specifically with L-selectin cytoplasmic tail, with relatively high affinity (K(d) approximately 40 nm). Based on these findings, although moesin and ezrin bind to a similar region of the cytoplasmic tail of L-selectin, moesin binding is dependent on PKC activation, which suggests that ezrin and moesin are regulated differently in lymphocytes.

Amino Acid Sequence↗
PubMed2002

Extracellular and cytoplasmic domains of endoglin interact with the transforming growth factor-beta receptors I and II.

Endoglin is an auxiliary component of the transforming growth factor-beta (TGF-beta) receptor system, able to associate with the signaling receptor types I (TbetaRI) and II (TbetaRII) in the presence of ligand and to modulate the cellular responses to TGF-beta1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and TbetaRI or TbetaRII. This study shows that full-length endoglin interacts with both TbetaRI and TbetaRII, independently of their kinase activation state or the presence of exogenous TGF-beta1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/TbetaRI interaction was different from that of endoglin/TbetaRII. TbetaRII interacts with the amino acid region 437-558 of the extracellular domain of endoglin, whereas TbetaRI interacts not only with the region 437-558 but also with the protein region located between amino acid 437 and the N terminus. Both TbetaRI and TbetaRII interact with the cytoplasmic domain of endoglin, but TbetaRI only interacts when the kinase domain is inactive, whereas TbetaRII remains associated in its active and inactive forms. Upon association, TbetaRI and TbetaRII phosphorylate the endoglin cytoplasmic domain, and then TbetaRI, but not TbetaRII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of TbetaRII, TbetaRI, and downstream Smad proteins as well as a modulation of TGF-beta signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-beta responses.

Activin Receptors, Type I↗