Role of the regulatory domain of the EGF-receptor cytoplasmic tail in selective binding of the clathrin-associated complex AP-2.

BACKGROUND: After stimulation of a cell by the mitogenic epidermal growth factor (EGF), the EGF receptor (EGF-R) is cleared from the cell surface in order to turn off receptor signaling. This internalization is mediated via clathrin-coated pits and coated vesicles, and ultimately the receptors are delivered to the lysosome and destroyed. It is believed that clathrin-associated protein complexes or adaptors (APs) link the entrapment of EGF-R and other nutrient and growth-factor receptors to the formation of the clathrin-coated pit. Two classes of APs are known--AP-2, found at the plasma membrane, and AP-1, found in the trans-Golgi network. Activated EGF-R associates with AP-2s at the plasma membrane, but the mechanism responsible for this association is not known. Here, we investigate, in vivo and in vitro, three aspects of the interaction between APs and EGF-R: firstly, we ask whether EGF-R at the plasma membrane distinguishes between AP-1 and AP-2; secondly, we ask which part of the receptor's cytoplasmic tail is responsible for binding; finally, we ask whether autophosphorylation by EGF-R is essential for the interaction. RESULTS: We demonstrate that EGF-R displays a selective association for AP-2 over AP-1 in vivo, and that this preferential interaction can also be detected using surface plasmon resonance in vitro. Using a truncated mutant and a kinase-dead mutant of EGF-R, we show that the regulatory domain of the cytoplasmic tail is essential for the recruitment of AP-2 in vivo and that this domain is required for association between purified AP-2 and EGF-R in vitro. Finally, we demonstrate, in vivo and in vitro, that tyrosine auto-phosphorylation by the receptor is not an essential pre-condition for the recruitment of AP-2. CONCLUSIONS: EGF-R binds selectively to AP-2s, and the regulatory domain of its cytoplasmic tail is required for this interaction. The lack of correlation between receptor autophosphorylation and AP-2 recruitment suggests that activation of the EGF-R kinase stimulates endocytosis by the phosphorylation of a factor distinct from EGF-R itself, as also proposed by others based on experiments measuring receptor traffic and entrapment.

Regulation of intestinal lactase in adult hypolactasia.

Relative deficiency of intestinal lactase activity during adulthood, adult hypolactasia, is a common condition worldwide. We studied the regulation of lactase-phlorizin hydrolase in normal and adult hypolactasic subjects by correlating transcript abundance in intestinal biopsies with relative synthetic rates for the protein in cultured intestinal explants. After metabolic labelling studies in six subjects, precursor lactase-phlorizin hydrolase was identified in amounts directly proportional to the enzyme-specific activity suggesting that levels of intestinal lactase are regulated by synthetic rate. Total intestinal RNA was extracted from biopsies of these subjects and three hypolactasic adults who had participated in previous biosynthesis studies. Transcript levels were markedly reduced in deficient subjects who demonstrated diminished lactase-phlorizin hydrolase synthesis. The sequence of 1 kb of 5'-flanking region of the lactase-phlorizin hydrolase gene was determined in two hypolactasic subjects and two controls. No sequence variability was identified to account for differences in mRNA levels or biosynthetic rates between the two groups. A single hypolactasic subject previously characterized as demonstrating delayed posttranslational processing, showed message levels intermediate between other deficients and controls. These results suggest that in the majority of our subjects, pretranslational mechanisms account for the predominate regulatory control of lactase-phlorizin hydrolase expression in the proximal intestine.

Wortmannin alters the transferrin receptor endocytic pathway in vivo and in vitro.

Treatment with the phosphatidylinositol 3-kinase inhibitor wortmannin promotes approximately 30% decrease in the steady-state number of cell-surface transferrin receptors. This effect is rapid and dose dependent, with maximal down-regulation elicited with 30 min of treatment and with an IC50 approximately 25 nM wortmannin. Wortmannin-treated cells display an increased endocytic rate constant for transferrin internalization and decreased exocytic rate constants for transferrin recycling. In addition to these effects in vivo, wortmannin is a potent inhibitor (IC50 approximately 15 nM) of a cell-free assay that detects the delivery of endocytosed probes into a common compartment. Inhibition of the in vitro assay involves the inactivation of a membrane-associated factor that can be recruited onto the surface of vesicles from the cytosol. Its effects on the cell-free assay suggest that wortmannin inhibits receptor sorting and/or vesicle budding required for delivery of endocytosed material to "mixing" endosomes. This idea is consistent with morphological changes induced by wortmannin, which include the formation of enlarged transferrin-containing structures and the disruption of the perinuclear endosomal compartment. However, the differential effects of wortmannin, specifically increased transferrin receptor internalization and inhibition of receptor recycling, implicate a role for phosphatidylinositol 3-kinase activity in multiple sorting events in the transferrin receptor's membrane traffic pathway.

A new approach to high sensitivity differential hybridization.

We describe a new approach to differential hybridization, designed to identify cDNA clones representing rare mRNA species. Duplicate filters carrying a library of cDNA from phorbolmyristate acetate (PMA)-induced EL-4 cells in lambda gt11 were hybridized with high concentrations of unlabeled, cloned, single-stranded cDNA from induced and control EL-4 cells, respectively. Plaques binding single-stranded cDNA were revealed by a second round of hybridization with 35S-labeled DNA complementary to the vector moiety of the single-stranded cDNA. Plaques corresponding to PMA-induced mRNAs occurring at a level of about 1 part in 15,000 were isolated. We believe the method is at least ten times more sensitive than conventional differential hybridization.

Region-specific expression of multiple lactase-phlorizin hydrolase genes in intestine of rabbit.

We have identified a total of 4 sequences coding for lactase-phlorizin hydrolase (LPH) in the rabbit. Individual rabbits each yielded three different LPH cDNA sequences, or three chromosomal segments containing exon 1 of the LPH gene, representing either three genes or allelic variants of two genes. The three sequences were found in differing proportions in intestinal mRNA depending on the position along the small intestine from which the RNA was isolated. If all these mRNAs are translated, posttranslational mechanisms previously implicated in the regulation of LPH in the rabbit will be acting on different enzyme species in different parts of the intestine. However, we find no evidence for more than one LPH gene in the rat, and have previously shown that humans have only one LPH gene.

Comparison of intestinal phospholipase A/lysophospholipase and sucrase-isomaltase genes suggest a common structure for enterocyte-specific promoters.

Intestinal phospholipase A/lysophospholipase (IPAL) is an intestine-specific brush-border enzyme expressed during development and along the intestinal crypt-villus axis in a pattern similar to another well characterized brush-border enzyme, sucrase-isomaltase (SI). A tissue-specific DNase I hypersensitive site was identified in chromatin from intestinal nuclei immediately upstream from the transcriptional start site of the IPAL gene. Footprinting analysis showed that two DNA elements within the IPAL promoter were protected by intestinal nuclear proteins. The IPAL-FP1 element was shown to be a monomer binding site for Cdx1 and Cdx2, intestine-specific homeobox proteins. Moreover, this site was important for transcriptional activation of the promoter in intestinal cell lines via interaction with Cdx proteins. Nuclear proteins from both liver and intestine interacted with the IPAL-FP2 element, forming a complex consistent with binding to HNF1. Cdx and HNF1 binding sites have also been shown to be the two major regulatory elements responsible for transcriptional activation of the SI gene promoter, which directs intestine-specific transcription in transgenic mice. These findings suggest that enterocyte genes that are expressed in similar developmental patterns may be regulated by the interaction of common DNA elements and their associated transcription factors.

Action of organic antagonists on neuronal calcium currents.

The low-voltage activated, fast inactivating calcium (Ca) conductance recently found in dorsal root ganglion cells is largely inert to the Ca agonist and antagonists (BAY K 8644, nifedipine and verapamil). The ubiquitous, slowly inactivating Ca conductance is strongly depressed by verapamil (20 to 100 X 10(-6) M) and with [Ca]o greater than or equal to 2 mM also by BAY K 8644, while nifedipine was weakly agonistic or ineffective in a comparable range of drug concentrations (10(-9) to 5 X 10(-7 M). The difference in affinity to verapamil renders possible a pharmacological separation of the two channels.

Characterisation of the enzyme intermediates of the sarcoplasmic transport ATPase by the use of inhibitors.

1. Based on a detailed reaction scheme of the phosphorylation process of the sarcoplasmic transport ATPase the inhibition mechanisms of benzoctamine, DIO 9, AMP-PNP and of Ca2+-ions at relatively high concentrations (1 approximately 100 microM) were determined. 2. The inhibition mechanisms were analyzed by measuring the gamma-phosphate exchange between ATP and ADP and evaluated by applying conventional and an extended Dixon plot procedures. 3. The kinetic patterns of the inhibition were shown to be compatible with the assumed reaction scheme. 4. Each inhibitor combines with definite intermediates: Benzoctamine with the intermediate species Ca2MgE and Ca2Mg2E-ATP; AMP-PNP with Ca2Mg2E approximately P; DIO 9 with E and MgE and Ca2+ at relatively high concentrations with E. 5. The central intermediate blocked by benzoctamine can partially exist as Ca2Mg2E ADPP-benzoctamine which is detected as phosphoprotein after acid denaturation.

Structure of the chromosomal gene and cDNAs coding for lactase-phlorizin hydrolase in humans with adult-type hypolactasia or persistence of lactase.

Lactase-phlorizin hydrolase (LPH) splits lactose in the small intestine. LPH activity is high in the suckling; in many human populations the activity declines in adults, leading to adult-type hypolactasia, whereas in other populations the high LPH activity persists in adults. In the present work, we compared LPH sequences at the gene and cDNA level among adult subjects with high and low LPH activity. The complete intron-exon organization, including the sequences of all 17 exons and of the borders of all introns (as well as about 1,000 bp of 5' flanking region), was established for the cloned chromosomal LPH gene of a subject with persistence of lactase. Using PCR, we directly sequenced the exons of a hypolactasic subject. Except for silent mutations and the unknown linkage phase at two heterozygous positions, both coding sequences were identical. We further examined the LPH mRNA of a hypolactasic subject by S1 mapping and by sequencing a set of overlapping PCR products produced from cDNA templates. Except for allelic differences, the LPH sequence of the hypolactasic subject was identical to that of the LPH cDNAs of three subjects with persistence of lactase (one cDNA isolated previously by cloning and two characterized in the present work by PCR). No allele was peculiar to the hypolactasic subject. We conclude that humans with high or low levels of lactase can code for identical LPH enzymes.

Rabbit beta-globin mRNA production in mouse L cells transformed with cloned rabbit beta-globin chromosomal DNA.

Mouse thymidine kinase-negative L cells were transformed with a cloned rabbit chromosomal beta-globin gene linked to the clone thymidine kinase gene of herpes simplex virus type 1. Most thymidine kinase-positive cell lines contained one or more copies of rabbit beta-globin DNA and produced up to 2,000 copies of rabbit beta-globin RNA per cell indistinguishable from its authentic counterpart. No mouse beta-globin mRNA was detected.

Parallel dimers and anti-parallel tetramers formed by epidermal growth factor receptor pathway substrate clone 15.

The recently discovered localization of epidermal growth factor receptor pathway substrate clone 15 (Eps15) to plasma membrane clathrin-coated pits and its constitutive association with the endocytic clathrin adaptor protein complex, AP-2, strongly suggest that Eps15 has an important role in the pathway of clathrin-dependent endocytic traffic. We report here that Eps15 forms dimers and tetramers of distinct shape. The Eps15 dimer is an elongated molecule, 32 nm in length. There is a globular "head" at one end of the molecule and an extended "stalk" of 25 nm which is kinked at about 17 nm away from the head. In the Eps15 dimer, two subunits are arranged parallel to each other, so that the head corresponds to two side by side copies of the N-terminal region I, which contains the three Eps15 homology domains. The proximal part of the stalk is the coiled-coil central region II containing 20 heptad repeats. The kink is at the boundary between region II and the C-terminal region III, which contains the AP-2 binding site, 15 aspartic-proline-phenylalanine repeats, and proline-rich Src homology domain ligand sites. The Eps15 tetramer has a "dumbbell" shape, approximately 31 nm in length; it is formed by the anti-parallel association of two Eps15 dimers. Formation of these Eps15 tetramers appears to require contacts between regions I of one dimer and regions III of a second apposing dimer. The extended shapes of the Eps15 dimers and tetramers suggest how Eps15 oligomers are located in the clathrin coat. We discuss the implications for accessibility to partners and for proposed functions of Eps15.

A role for the hinge/ear domain of the beta chains in the incorporation of AP complexes into clathrin-coated pits and coated vesicles.

The clathrin-associated adaptor protein (AP) complexes drive the polymerization of clathrin in coated pits to form coated vesicles. It has previously been shown that the carboxyl-terminal hinge/ear domain of the beta 2 chain contains a binding site for clathrin and that removal of this domain from APs or from isolated beta 2 chains abrogates their ability to form clathrin coats in vitro. We show here that the hinge/ear domain is necessary for efficient incorporation of AP complexes into coated pits and coated vesicles in cells, a result that is consistent with the view that the beta chains indeed provide an important interaction between the AP complexes and clathrin.

Interaction of the bovine papillomavirus E6 protein with the clathrin adaptor complex AP-1.

The E6 gene of the bovine papillomavirus type 1 (BPV-1) is expressed in fibropapillomas caused by BPV-1 and in tissue culture cells transformed by BPV-1. It encodes one of the two major oncoproteins of BPV-1. In this study, we demonstrate an interaction between the BPV-1 E6 protein and AP-1, the TGN (trans-Golgi network)-specific clathrin adaptor complex. AP-1 is a four-subunit protein complex required for clathrin-mediated cellular transport from the TGN. The AP-1/E6 interaction was observed in vitro and in cells. The E6 binding site on AP-1 was mapped to the N-terminal trunk domain of the gamma subunit. BPV-1 E6 preferentially associated with membrane-bound AP-1 in cells but not with free cytosolic AP-1. BPV-1 E6 was further shown to be recruited to isolated Golgi membranes and to copurify with clathrin-coated vesicles. The recruitment of BPV-1 E6 to Golgi membranes was AP-1 independent, but the E6 interaction with AP-1 was required for its association with clathrin-coated vesicles. Furthermore, AP-1 proteins could compete with BPV-1 E6 for binding to Golgi membranes, suggesting that the recruitment of BPV-1 E6 and AP-1 to Golgi membranes involves a common factor. Taken together, our results suggest that cytosolic BPV-1 E6 is first recruited to the TGN, where it is then recognized by membrane-bound AP-1 and subsequently recruited into TGN-derived clathrin-coated vesicles. We propose that BPV-1 E6, through its interaction with AP-1, can affect cellular processes involving clathrin-mediated trafficking pathway.

Messenger RNAs expressed in intestine of adult but not baby rabbits. Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity.

Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits. Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S-transferase, a cytochrome P450, and a long form of ferritin mRNA), while six code for previously unknown proteins. One clone, AdRab-B, codes for a protein of 1458 amino acids, including (i) a putative signal sequence at the NH2 terminus, (ii) four internal repeats, 308-346 amino acids in length, (iii) a hydrophobic stretch near the COOH terminus, which represents a potential membrane anchor, and (iv) a short hydrophilic stretch at the very COOH terminus. The corresponding protein was studied with the aid of antibodies prepared against polypeptides expressed from segments of the cDNA in Escherichia coli. The protein was shown to be proteolytically processed in the intestine (but not when expressed in COS cells) and to be targeted to the brush border membrane of the enterocytes. Finally, the protein was found to have esterase and phospholipase A/lysophospholipase activity.

Determination of respiratory resistance by an oscillation method. Studies of long-term and short-term variability and dependence upon lung volume and compliance.

The respiratory resistance (Ros) as measured by an oscillation method, and the airway resistance (Raw) as measured by body plethysmography were determined in 10 healthy volunteers thrice daily every week over a period of 22 weeks. Ros in 9 of this group was measured at different breathing levels at constant respiratory rate and volume. Additionally, the phase angle between oscillatory pressure and flow (phi), the thoracic gas volume, the end-tidal volume, the functional residual capacity, Raw and the lung compliance were determined. Furthermore Ros of 116 patients was measured together with the lung and thorax compliance. Compared to Raw, the variability of Ros is smaller and the sensitivity is higher. There is a reciprocal relationship between Ros and lung volume, lung compliance, and compliance of the lung and thorax system. Therefore, measurements of Ros should be performed at a normal, stabilized mean breathing level.

Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus.

Mx+ mice are much more resistant to influenza virus than Mx- strains. The resistance is mediated by interferon (IFN) alpha/beta. After IFN treatment, Mx+ but not Mx- cells accumulate Mx protein and become specifically resistant to orthomyxoviruses. cDNA encoding Mx protein was cloned and sequenced. Southern analyses indicate that Mx- alleles derive from their Mx+ counterpart by deletions. IFN-treated Mx+ cells contained a 3.5 kb Mx mRNA, while Mx- cells showed only traces of shorter Mx RNA. Mx- cells transformed with Mx cDNA expressed Mx protein constitutively to varying extents; resistance of individual cells to influenza virus correlated with Mx protein expression. Thus, specific resistance to influenza virus in vivo may be attributed to Mx protein expression and is independent of other IFN-mediated effects.

The in-vitro synthesized tomato shikimate kinase precursor is enzymatically active and is imported and processed to the mature enzyme by chloroplasts.

Full-length cDNA clones encoding shikimate kinase (EC 2.7.1.71), an enzyme of the central section of the shikimate pathway, have been isolated from tomato (Lycopersicon esculentum L., cv. UC82b). The open reading frame has the capacity to encode a peptide of 300 amino acids. The in-vitro synthesized peptide catalysed the phosphorylation of shikimate thus confirming the identity of the isolated cDNA clones. The N-terminal portion of the deduced amino acid sequence resembles known chloroplast-specific transit peptides. The existence of such a transit peptide was proven by the uptake of the in-vitro synthesized peptide as well as its processing by isolated chloroplasts. Multiple sites of polyadenylation were observed in shikimate kinase mRNAs. The results of Northern and Southern blot analyses are consistent with the existence of only one shikimate kinase gene per haploid genome in tomato. These results are discussed with respect to the dual pathway hypothesis of the shikimate pathway in higher plants.

Distinct pathways for basolateral targeting of membrane and secretory proteins in polarized epithelial cells.

Polarized epithelial cells target distinct sets of membrane and secretory proteins to their apical and basolateral domains. Here we examine whether constitutively secreted and membrane proteins that are bound for the same domain share the same carrier vesicles. To address the issue, differential effects of microtubule depolymerization on basolateral protein targeting in the polarized Madin-Darby canine kidney II cell line were studied. We find that the basolateral insertion of the active, ouabain-binding Na+,K(+)-ATPase and of a set of very late antigen integrins is little affected by microtubule disruption. Under equivalent conditions, the basolateral secretion of the basement membrane protein laminin is strongly suppressed. More specifically, it is demonstrated that microtubules are involved in targeting laminin, but not integrins, from the compartment related to the accumulation of newly synthesized proteins at 20 degrees C (trans-Golgi network) to the basolateral domain. Our study also reveals that laminin associated with basolateral binding sites interacts with those sites only secondarily to secretion. The data provide evidence for a branch in the basolateral targeting pathway, with secreted and membrane proteins loaded into distinct carrier vesicles.

Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1.

The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).