An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor

The polymeric immunoglobulin receptor is normally delivered from the Golgi to the basolateral surface of epithelial cells and then transports polymeric IgA and IgM to the apical surface. We now report that a 14 residue segment of the 103 amino acid cytoplasmic domain, proximal to the plasma membrane, directs the receptor to the basolateral surface. A mutant receptor lacking these 14 amino acids is sorted directly to the apical surface from the Golgi. Furthermore, this sequence is sufficient to redirect an apical membrane protein, placental alkaline phosphatase, to the basolateral plasma membrane. We conclude that this sequence contains an autonomous signal, which specifies sorting from the Golgi to the basolateral surface, a process previously postulated to occur by default.     

Identification of a novel mono-leucine basolateral sorting motif within the cytoplasmic domain of amphiregulin

Epithelial cells establish apical and basolateral (BL) membranes with distinct protein and lipid compositions. To achieve this spatial asymmetry, the cell utilizes a variety of mechanisms for differential sorting, delivery and retention of cell surface proteins. The EGF receptor (EGFR) and its ligand, amphiregulin (AREG), are transmembrane proteins delivered to the BL membrane in polarized epithelial cells. Herein, we show that the cytoplasmic domain of AREG (ACD) contains dominant BL sorting information; replacement of the cytoplasmic domain of apically targeted nerve growth factor receptor with the ACD redirects the chimera to the BL surface. Using sequential truncations and site-directed mutagenesis of the ACD, we identify a novel BL sorting motif consisting of a single leucine C-terminal to an acidic cluster (EEXXXL). In adaptor protein (AP)-1B-deficient cells, newly synthesized AREG is initially delivered to the BL surface as in AP-1B-expressing cells. However, in these AP-1B-deficient cells, recycling of AREG back to the BL surface is compromised, leading to its appearance at the apical surface. These results show that recycling, but not delivery, of AREG to the BL surface is AP-1B dependent.     

Identification of two regions within the cytoplasmic domain of the human interferon-gamma receptor required for function.

Functionally active human interferon-gamma (IFN gamma) receptors require the presence of at least two polypeptides: the IFN gamma receptor and an accessory molecule encoded by a gene on human chromosome 21. Here we have used a murine L cell line that stably contains human chromosome 21 (SCC16-5) to determine whether the receptor's cytoplasmic domain is important for receptor function. SCC16-5 stably transfected with the full-length human IFN gamma receptor cDNA bound, internalized, and responded to human IFN gamma. In contrast, SCC16-5 expressing human IFN gamma receptors lacking a cytoplasmic domain bound human IFN gamma but did not internalize or respond to it. Using a family of IFN gamma receptor deletion mutants, two functionally important regions within the intracellular domain were identified: (a) a membrane proximal region (residues 256-303) required for ligand processing and biologic responsiveness and (b) the carboxyl-terminal 39 amino acids (residues 434-472) needed exclusively for biologic responses.     

Distinct regions of the human granulocyte-colony-stimulating factor receptor cytoplasmic domain are required for proliferation and gene induction.

Using two different cell systems, we show that the cytoplasmic domain of the granulocyte-colony-stimulating factor receptor (G-CSFR) may be composed of at least two functional regions. The first, within the membrane-proximal 57 amino acids, is absolutely required to deliver a proliferative signal. This region contains two sequence motifs conserved between members of the hematopoietin receptor family. The second functional region resides between amino acids 57 and 96. This region is required for the induction of acute-phase plasma protein gene expression when the G-CSFR is transfected into human hepatoma cell lines. The G-CSFR-transfected hepatoma cells respond to G-CSF by increasing the production of the same set of plasma proteins as stimulated by interleukin-6, suggesting that the two cytokines share a common signal transduction pathway.     

Solution structure of the coxsackievirus and adenovirus receptor domain 2

The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus and adenovirus. CAR possesses an extracellular region that is comprised of 2 immunoglobulin domains termed CAR-D1 and CAR-D2. In the present work, the solution structure of CAR-D2, consisting of residues 142-235 of human CAR, has been determined by NMR spectroscopy. CAR-D2 is shown to be a beta-sandwich motif comprised of two beta-sheets, which are stabilized by two disulfide bonds. The first beta-sheet is comprised of beta-strands A, B, and E, and the second beta-sheet is comprised of beta-strands C, F, and G. A relatively hydrophobic helix is found between beta-strands C and E, which replaces beta-strand D of the classical c-type immunoglobulin fold.     

The cytoplasmic domain of the polymeric immunoglobulin receptor contains two internalization signals that are distinct from its basolateral sorting signal.

The C-terminal cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) contains two tyrosine residues, Tyr668 and Tyr734. Previous work identifying Tyr734 as a critical residue in the endocytosis of the pIgR in Madin-Darby canine kidney (MDCK) cells also suggested that a second functional internalization signal was present (Breitfeld, P. P., Casanova, J. E., McKinnon, W. C., and Mostov, K. E. (1990) J. Biol. Chem. 265, 13750-13757). To test this hypothesis, Tyr668 and Tyr734 were mutated singly or together by oligonucleotide-directed mutagenesis of pIgR cDNA, and the mutants were expressed in MDCK cells. The amount of ligand internalized within 5 min from the basolateral membrane by the pIgR in which cytoplasmic tyrosines were mutated separately to Cys668 or Ser734 or together to Cys668, Ser734 was 58, 39, and 20%, respectively, of the internalized by the wild-type pIgR. The cytoplasmic and transmembrane domains of the pIgR, when joined to the external domain of the influenza virus hemagglutinin, retained the capacity to mediate rapid internalization. As with the full-length pIgR, mutation of either tyrosine in the chimera resulted in impairment of endocytosis, with mutation of Tyr734 having a significantly greater effect than mutation on Tyr668 on the initial rate of endocytosis (3 and 44% of control values, respectively). However, unlike the full-length pIgR, mutation of both tyrosines together in the chimera did not reduce internalization further. The two tyrosines in the cytoplasmic sequence of the pIgR, although widely separated in the linear amino acid sequence, both contribute to internalization of the protein, suggesting that both can function as internalization signals. In addition, the correlation between endocytosis and basolateral targeting of the pIgR in MDCK cells was investigated. Neither tyrosine of the cytoplasmic domain was necessary for basolateral targeting of the pIgR.     

Solution structure of the coxsackievirus and adenovirus receptor domain 1

The coxsackievirus and adenovirus receptor (CAR) mediates entry of coxsackievirus B (CVB) and adenovirus (Ad). The normal cellular function of CAR, which is expressed in a wide variety of tissue types, is thought to involve homophilic cell adhesion in the developing brain. The extracellular domain of CAR consists of two immunoglobulin (Ig) domains termed CAR-D1 and CAR-D2. CAR-D1 is shown by sedimentation velocity to be monomeric at pH 3.0. The solution structure and the dynamic properties of monomeric CAR-D1 have been determined by NMR spectroscopy at pH 3.0. The determinants of the CAR-D1 monomer-dimer equilibrium, as well as the binding site of CVB and Ad on CAR, are discussed in light of the monomer structure.     

The coxsackievirus and adenovirus receptor interacts with the multi-PDZ domain protein-1 (MUPP-1) within the tight junction

The coxsackievirus and adenovirus receptor (CAR) is a component of the epithelial cell tight junction. In a yeast two-hybrid screen we identified the multi-PDZ domain protein MUPP1 as an interaction partner for the CAR cytoplasmic domain. CAR and MUPP1 were found to colocalize at the tight junction, to coprecipitate from epithelial cells, and to interact in vitro. The interaction was found to specifically involve the PDZ-binding motif within the CAR C terminus and MUPP1 PDZ domain 13. In transfected cells, CAR recruited MUPP1 to cell-cell contacts. The inhibition of CAR expression with small interfering RNA inhibited MUPP1 localization to the tight junction. The results indicated that CAR interacts with MUPP1 and is involved in MUPP1 recruitment to the tight junction.     

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.     

Structural basis for variation in adenovirus affinity for the cellular coxsackievirus and adenovirus receptor

The majority of adenovirus serotypes can bind to the coxsackievirus and adenovirus receptor (CAR) on human cells despite only limited conservation of the amino acid residues that comprise the receptor-binding sites of these viruses. Using a fluorescence anisotropy-based assay, we determined that the recombinant knob domain of the fiber protein from adenovirus serotype (Ad) 2 binds the soluble, N-terminal domain (domain 1 (D1)) of CAR with 8-fold greater affinity than does the recombinant knob domain from Ad12. Homology modeling predicted that the increased affinity of Ad2 knob for CAR D1 could result from additional contacts within the binding interface contributed by two residues, Ser408 and Tyr477, which are not conserved in the Ad12 knob. Consistent with this structural model, substitution of serine and tyrosine for the corresponding residues in the Ad12 knob (P417S and S489Y) increased the binding affinity by 4- and 8-fold, respectively, whereas the double mutation increased binding affinity 10-fold. X-ray structure analysis of Ad12 knob mutants P417S and S489Y indicated that both substituted residues potentially could form additional hydrogen bonds across the knob-CAR interface. Structural changes resulting from these mutations were highly localized, implying that the high tolerance for surface variation conferred by the stable knob scaffold can minimize the impact of antigenic drift on binding specificity and affinity during evolution of virus serotypes. Our results suggest that the interaction of knob domains from different adenovirus serotypes with CAR D1 can be accurately modeled using the Ad12 knob-CAR D1 crystal structure as a template.     

Dimeric structure of the coxsackievirus and adenovirus receptor D1 domain at 1.7 A resolution

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) comprises two extracellular immunoglobulin domains, a transmembrane helix and a C-terminal intracellular domain. The amino-terminal immunoglobulin domain (D1) of CAR is necessary and sufficient for adenovirus binding, whereas the site of coxsackievirus attachment has not yet been localized. The normal cellular role of CAR is currently unknown, although CAR was recently proposed to function as a homophilic cell adhesion molecule. RESULTS: The human CAR D1 domain was bacterially expressed and crystallized. The structure was solved by molecular replacement using the structure of CAR D1 bound to the adenovirus type 12 fiber head and refined to 1.7 A resolution, including individual anisotropic temperature factors. The two CAR D1 structures are virtually identical, apart from the BC, C"D, and FG loops that are involved both in fiber head binding and homodimerization in the crystal. Analytical equilibrium ultracentrifugation shows that a dimer also exists in solution, with a dissociation constant of 16 microM. CONCLUSIONS: The CAR D1 domain forms homodimers in the crystal using the same GFCC'C" surface that interacts with the adenovirus fiber head. The homodimer is very similar to the CD2 D1-CD58 D1 heterodimer. CAR D1 also forms dimers in solution with a dissociation constant typical of other cell adhesion complexes. These results are consistent with reports that CAR may function physiologically as a homophilic cell adhesion molecule in the developing mouse brain. Adenovirus may thus have recruited an existing and conserved interaction surface of CAR to use for its own cell attachment.     

Identification of two distinct regions within the adenovirus minimal origin of replication that are required for adenovirus type 4 DNA replication in vitro.

The adenovirus type 4 origins of replication are located at each end of the linear, protein-linked viral DNA molecule and consist of the terminal 18 bp of the viral genome. The sequence of the first 8 bp of the viral genome varies among different adenovirus serotypes, but the sequence from bp 9 to 18 is conserved in all human serotypes, suggesting that it may be of critical importance to origin function. Using an in vitro system in which purified fractions or crude extracts of adenovirus type 4-infected HeLa cells can support initiation and elongation on linearized plasmid templates containing cloned origin sequences, we examined the effect of single base changes in positions 9 to 18 of the adenovirus origin on DNA replication in vitro. Changes in positions 12 to 16 have little effect, whereas alterations at positions 9, 10, 11, 17, and 18 all reduce the efficiency of initiation of DNA replication by between 50 and 90%. Our results show that the region from bp 9 to 18 contains two sets of bases essential for DNA replication which are separated by 5 bp in which single base changes can be accommodated. The likely role of the region from bp 9 to 18 as containing the recognition sequence for a DNA-protein interaction essential for viral DNA replication is discussed.     

Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis

We deleted the cytoplasmic domain of the polymeric immunoglobulin receptor. When expressed in fibroblasts, the truncated receptor, like the wild-type, reaches the cell surface, can bind ligand, and is cleaved to secretory component. Unlike the wild-type, it is not endocytosed. When expressed in polarized Madin-Darby canine kidney cells, the mutant receptor is transported from the Golgi apparatus directly to the apical surface and cleaved to secretory component. In contrast, the wild-type receptor travels from the Golgi to the basolateral surface and is then endocytosed and sent to the apical surface. These results suggest that the cytoplasmic domain of the receptor is necessary for both basolateral localization and endocytosis.     

A role for the cytoplasmic domain in transferrin receptor sorting and coated pit formation during endocytosis

The cytoplasmic domain of transferrin receptor (TR) is essential for endocytosis of this transmembrane protein. We have investigated by electron microscopy the association of wild-type and cytoplasmic deletion mutant human TR with coated pits at the surface of transfected L cell lines. Approximately 15% of wild-type TR was concentrated in coated pits, regardless of the level of TR expression. In contrast, only 2% of deletion mutant TR was present in these structures. We also correlated the frequency of coated pits with the level of TR expression in different transfected L cell lines. Expression of more than 3 x 10(6) wild-type TR per cell was accompanied by up to a 4-fold increase in coated pits compared with nontransfected Ltk- cells. No such increase was observed in a cell line expressing a similarly high level of cytoplasmic deletion mutant TR. These results indicate that the cytoplasmic domain plays an active role in sorting and endocytosis of TR by providing an assembly site for coated pit formation.     

Functional regions of the mouse interleukin-10 receptor cytoplasmic domain.

The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.     

Kit ligand cytoplasmic domain is essential for basolateral sorting in vivo and has roles in spermatogenesis and hematopoiesis

Juxtamembrane signaling via the membrane growth factor KitL is critical for Kit mediated functions. KitL has a conserved cytoplasmic domain and has been shown to possess a monomeric leucine-dependent basolateral targeting signal. To investigate the consequences in vivo of impaired basolateral KitL targeting in polarized epithelial cells, we have mutated this critical leucine to alanine using a knock-in strategy. KitL^L263A/L263A mutant mice are pigmented normally and steady-state hematopoiesis is unaffected although peritoneal and skin mast cell numbers are significantly increased. KitL localization is affected in the Sertoli cells of the KitL^L263A/L263A testis and testis size is reduced in these mice due to aberrant spermatogonial proliferation. Furthermore, the effect of the KitL L263A mutation on the testicular phenotype is dosage dependent. The tubules of hemizygous KitL^L263A/Sl mice completely lack germ cells in contrast to the weaker testicular phenotype of KitL^L263A/L263A mice. The onset of the testis phenotype coincides with the formation of tight junctions between Sertoli cells during postnatal development. Thus, the altered sorting of KitL is dispensable for hematopoietic and melanogenic lineages, yet is crucial in the testicular environment, where the basal membranes of adjacent polarized Sertoli cells form a niche for the proliferating spermatogonia.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Fatty acid modification of the coxsackievirus and adenovirus receptor

Membrane-proximal cysteines 259 and 260 in the cytoplasmic tail of the coxsackievirus and adenovirus receptor (CAR) are known to be essential for the tumor suppression activity of CAR. We demonstrate that these residues provide an S-acylation motif for modification of CAR with the fatty acid palmitate. Substitution of alanine for cysteines 259 and 260 results in the additional localization of CAR in perinuclear compartments with no effect on the efficiency of adenovirus infection. The results indicate that palmitylation is important for stable plasma membrane expression and biological activity of CAR but is not critical for adenovirus receptor performance.