Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo.

Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene product) contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (furin) shared many properties with Kex2p including activity at pH 7.3 and a requirement for calcium. By using antifurin antibodies, immunoblot analysis detected two furin translation products (90 and 96 kD), while immunofluorescence indicated localization to the Golgi apparatus. Coexpression of either Kex2p or furin with the mouse beta-nerve growth factor precursor (pro-beta-NGF) resulted in greatly enhanced conversion of the precursor to mature nerve growth factor. Thus, the sequence homology shared by furin and the yeast KEX2 prohormone processing enzyme is reflected by significant functional homology both in vitro and in vivo.

Intracellular targeting and structural conservation of a prohormone-processing endoprotease.

The prohormone-processing endoprotease (KEX2 gene product) of the yeast Saccharomyces cerevisiae is a membrane-bound, 135,000-dalton glycoprotein, which contains both asparagine-linked and serine- and threonine-linked oligosaccharide and resides in a secretory compartment. Analysis of mutant kex2 genes truncated at their 3' end indicates that carboxyl terminal domains of the enzyme are required for its proper localization within the cell. A human gene product, "furin," shares 50% identity with the catalytic domain of Kex2 protease and is, therefore, a candidate for a human prohormone-processing enzyme.

Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel.

The neurotransmitter serotonin (5HT) activates a variety of second messenger signaling systems and through them indirectly regulates the function of ion channels. Serotonin also activates ion channels directly, suggesting that it may also mediate rapid, excitatory responses. A complementary DNA clone containing the coding sequence of one of these rapidly responding channels, a 5HT3 subtype of the serotonin receptor, has been isolated by screening a neuroblastoma expression library for functional expression of serotonin-gated currents in Xenopus oocytes. The predicted protein product has many of the features shared by other members of the ligand-gated ion channel family. The pharmacological and electrophysiological characteristics of the cloned receptor are largely consistent with the properties of native 5HT3 receptors. Messenger RNA encoding this receptor is found in the brain, spinal cord, and heart. This receptor defines a new class of excitatory ligand-gated channels.

Yeast KEX2 protease has the properties of a human proalbumin converting enzyme.

Several classes of proteolytic enzymes have been proposed to have a role in the processing of precursor forms of proproteins at paired basic amino acid residues. In higher eukaryotes, a single endopeptidase has yet to fulfill the necessary criteria as the physiologically relevant convertase. The observation of proalbumin circulating in a child with a bleeding disorder caused by an unusual alpha 1-antitrypsin mutation led to speculation that the presence of this alpha 1-antitrypsin mutant was inhibitory to the convertase. This provided an additional means of characterizing the processing enzyme. In this study the yeast KEX2 enzyme, a calcium-dependent thiol protease, was found to have all the properties expected for this processing enzyme. KEX2 correctly recognized and cleaved the prosequence in proalbumin. In addition, KEX2 was specifically inhibited by the mutant alpha 1-antitrypsin but not by other serine protease inhibitors.

A functional prepro-alpha-factor gene in Saccharomyces yeasts can contain three, four, or five repeats of the mature pheromone sequence.

The chromosomal region containing a structural gene for the mating pheromone precursor prepro-alpha-factor was examined in a variety of Saccharomyces yeasts by using a cloned putative prepro-alpha-factor gene of Saccharomyces cerevisiae as the probe. Analysis by restriction endonuclease digestion and Southern blot hybridization indicated that the physical arrangement of this region is highly conserved in all the Saccharomyces species analyzed, but displays length polymorphisms of limited size (50 to 60 base pairs). The observed polymorphisms were shown to be due solely to differences in the number of tandemly arranged spacer peptide/pheromone units within the coding sequence of these genes. Analysis of polyadenylated RNA indicated that these genes specified RNA transcripts and that these RNA molecules could be translated in vitro into prepro-alpha-factor polypeptides immunoprecipitable with anti-alpha-factor antibodies. The sizes of both the mRNAs and the proteins synthesized from them reflected exactly the differences observed in the lengths of the genes. These findings demonstrate conclusively that the putative prepro-alpha-factor DNA cloned from S. cerevisiae, as well as the sequences detected in the other Saccharomyces species, are indeed expressed and functional genes, and suggest that proper proteolytic processing of prepro-alpha-factor is unaffected by the number of pheromone repeats encoded within this precursor protein.

The MF alpha 1 gene of Saccharomyces cerevisiae: genetic mapping and mutational analysis of promoter elements.

The activity and cell-type specificity of the promoter of the MF alpha 1 gene of Saccharomyces cerevisiae were examined by measuring expression of an MF alpha 1-SUC2 gene fusion in MATa, MAT alpha, and MATa/MAT alpha cells. A high level of invertase activity was observed only in MAT alpha cells. Weak expression occurred in MATa cells when the hybrid gene was carried on a multicopy plasmid or on a centromere-containing plasmid, but not when the hybrid gene was integrated at the normal MF alpha 1 locus. Analysis of a set of 5'-deletions of the promoter region of the MF alpha 1-SUC2 gene on the multicopy plasmid indicated that sequences from -354 to -274 upstream of the translational start site were required for high level expression in MAT alpha cells. Smaller internal deletions and insertions within the promoter region of the MF alpha 1-SUC2 gene were inserted into the genome at the normal MF alpha 1 locus. These mutations further delineated four promoter domains important for expression: (1) two 26 bp elements (-365 to -340 and -312 to -287) with imperfect dyad symmetry; (2) a 40 bp segment (-264 to -226) that lies about 120 bp upstream of the TATA box; and (3) the TATA box itself (-128 to -122). The transcriptional start sites of the normal MF alpha 1 promoter and of a mutant lacking the TATA box were determined. The MF alpha 1 locus was mapped to the left arm of chromosome XVI, about 22 cM centromere-proximal to the PEP4 gene.

cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues.

A cDNA encoding the human fur gene product was isolated from a human hepatoma cell line. The cDNA encodes a protein with significant amino acid sequence identity to the prokaryotic subtilisin family of serine proteases. More extensive sequence identity was found when the protein was compared with eukaryotic proteases such as PRB1 of Saccharomyces cerevisiae, and with PC2 and PC3, the only other known mammalian subtilisin-like proteases. In contrast to these proteins, however, the fur gene product shares a more extensive topographic and functional homology with the KEX2 endoprotease of S. cerevisiae. Each protease contains a signal peptide, a glycosylated extra cytoplasmic domain, a hydrophobic membrane-spanning region, and a short, hydrophilic "tail" sequence. As with KEX2, the expressed human protease was shown to cleave mammalian proproteins at their paired basic amino acid processing sites. We have, therefore, proposed the function-based acronym PACE (paired basic amino acid cleaving enzyme) for this prototypic mammalian proprotein processing enzyme.

Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin.

We have developed the yeast Kluyveromyces lactis as a host organism for the production of the milk-clotting enzyme chymosin. In contrast to Saccharomyces cerevisiae, we found that this yeast is capable of the synthesis and secretion of fully active prochymosin. Various signal sequences could be used to efficiently direct the secretion of prochymosin in Kluyveromyces, but not in S. cerevisiae. We conclude that the efficient synthetic and secretory capacity of this heterologous protein is a property of the yeast Kluyveromyces. These results have led to the development of a large scale production process for chymosin.

Signaling by extracellular nucleotides.

ATP and other nucleotides can be released from cells through regulated pathways, or following the loss of plasma membrane integrity. Once outside the cell, these compounds take on new roles as intercellular signaling molecules that elicit a broad spectrum of physiological responses through the activation of numerous cell surface receptor subtypes. This review summarizes recent advances in the molecular characterization of ATP receptors and discusses roles for cloned receptors in established and novel physiological processes.

New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor.

The adenosine-5'-triphosphate (ATP) molecule is an extracellular messenger in neural and non-neural tissues, where it activates several cell-surface-receptor subtypes, including G-protein-coupled receptors and ligand-gated ion channels. ATP-gated channels (termed P2x receptors) have been characterized on smooth muscle cells and autonomic and sensory neurons, where they mediate membrane depolarization and, in some cases, Ca2+ entry. P2x receptors are functionally heterogeneous, but resemble acetylcholine- and serotonin-gated channels with respect to ion selectivity and kinetic parameters of channel gating. We report here that despite such close functional similarities, the deduced sequence of a cloned P2x receptor predicts an unusual subunit structure resembling voltage-insensitive cation channels. Thus, the P2x receptor provides a striking example of convergent evolution, whereby proteins have been fashioned with similar functional properties from subunits having very different structural characteristics. There is sequence similarity between the ATP receptor and RP-2, a gene activated in thymocytes undergoing programmed cell death. RP-2 may encode a receptor for ATP or another metabolite released during apoptosis.

Amino acid sequence of beta-galactosidase. VI. Limited tryptic digestion of the citraconylated protein and sequences of tryptic peptides.

Hydrolysis with trypsin of citraconyl-carboxy-methyl-beta-galactosidase was carried out under limiting conditions. No Asp-Arg-X sequences were cleaved and many large peptides were produced. Butanol extraction from dilute acid proved very useful for separating the more hydrophobic fragments. Peptides were purified and sequenced. From this digest and two earlier preparations, all 80 theoretically possible tryptic fragments have been isolated and their structures determined.

Amino acid sequence of beta-galactosidase. X. Sequence of the COOH-terminal segment, CNBr peptides 18 to 24, residues 654 to 1021.

The sequence of the COOH-terminal third (omega) of beta-galactosidase is presented. The size of the 7 cyanogen bromide peptides of this segment is larger on the average, about 52 amino acid residues as compared to an average size of 42 for cyanogen bromide peptides in the whole molecule. Tyrosine, phenylalanine, and valine are low in this segment whereas alanine and lysine are high. This region has a slight excess of basic groups.

Expression cloning of an ATP receptor from mouse neuroblastoma cells.

Extracellular ATP activates cell-surface metabotropic and ionotropic nucleotide (P2) receptors in vascular, neural, connective, and immune tissues. These P2 receptors mediate a wealth of physiological processes, including nitric oxide-dependent vasodilation of vascular smooth muscle and fast excitatory neurotransmission in sensory afferents. Although ATP is now recognized as a signaling molecule, the cellular and molecular mechanisms underlying its actions have been difficult to study due to the absence of selective P2 receptor antagonists and cloned receptor genes. Nonetheless, five mammalian P2 receptor subtypes have been tentatively assigned based solely on agonist specificity and signaling properties. Here we report the cloning of a mouse cDNA encoding a P2 receptor that shares striking homology with several G protein-coupled peptide receptors. When expressed in Xenopus laevis oocytes, the cloned receptor resembles a metabotropic P2U receptor; activation by either ATP or UTP elicits the mobilization of intracellular calcium. mRNA encoding the P2U purinergic receptor is found in neural and nonneural tissues.

A capsaicin-receptor homologue with a high threshold for noxious heat.

Pain-producing heat is detected by several classes of nociceptive sensory neuron that differ in their thermal response thresholds. The cloned capsaicin receptor, also known as the vanilloid receptor subtype 1 (VR1), is a heat-gated ion channel that has been proposed to mediate responses of small-diameter sensory neurons to moderate (43 degrees C) thermal stimuli. VR1 is also activated by protons, indicating that it may participate in the detection of noxious thermal and chemical stimuli in vivo. Here we identify a structurally related receptor, VRL-1, that does not respond to capsaicin, acid or moderate heat. Instead, VRL-1 is activated by high temperatures, with a threshold of approximately 52 degrees C. Within sensory ganglia, VRL-1 is most prominently expressed by a subset of medium- to large-diameter neurons, making it a candidate receptor for transducing high-threshold heat responses in this class of cells. VRL-1 transcripts are not restricted to the sensory nervous system, indicating that this channel may be activated by stimuli other than heat. We propose that responses to noxious heat involve these related, but distinct, ion-channel subtypes that together detect a range of stimulus intensities.

Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations.

Functional complementation of mutations in the yeast Saccharomyces cerevisiae has been used to clone three multifunctional human genes involved in de novo purine biosynthesis. A HepG2 cDNA library constructed in a yeast expression vector was used to transform yeast strains with mutations in adenine biosynthetic genes. Clones were isolated that complement mutations in the yeast ADE2, ADE3, and ADE8 genes. The cDNA that complemented the ade8 (phosphoribosylglycinamide formyltransferase, GART) mutation, also complemented the ade5 (phosphoribosylglycinamide synthetase) and ade7 [phosphoribosylaminoimidazole synthetase (AIRS; also known as PAIS)] mutations, indicating that it is the human trifunctional GART gene. Supporting data include homology between the AIRS and GART domains of this gene and the published sequence of these domains from other organisms, and localization of the cloned gene to human chromosome 21, where the GART gene has been shown to map. The cDNA that complemented ade2 (phosphoribosylaminoimidazole carboxylase) also complemented ade1 (phosphoribosylaminoimidazole succinocarboxamide synthetase), supporting earlier data suggesting that in some organisms these functions are part of a bifunctional protein. The cDNA that complemented ade3 (formyltetrahydrofolate synthetase) is different from the recently isolated human cDNA encoding this enzyme and instead appears to encode a related mitochondrial enzyme.

beta-Galactosidase chimeras: primary structure of a lac repressor-beta-galactosidase protein.

A protein possessing both lac repressor and beta-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Z genes are fused. A 77-residue cyanogen bromide peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of beta-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of beta-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of beta-galactosidase are not essential for the activities of these two proteins. The exact site of the fusion is not known because lac repressor residue 356 and beta-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the two end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.

Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site.

Intracellular proteolytic processing of precursor polypeptides is an essential step in the maturation of many proteins, including plasma proteins, hormones, neuropeptides, and growth factors. Most frequently, propeptide cleavage occurs after paired basic amino acid residues. To date, no mammalian propeptide processing enzyme with such specificity has been purified or cloned and functionally characterized. We report the isolation and functional expression of a cDNA encoding a propeptide-cleaving enzyme from a human liver cell line. The encoded protein, called PACE (paired basic amino acid cleaving enzyme), has structural homology to the well-characterized subtilisin-like protease Kex2 from yeast. The functional specificity of PACE for mediating propeptide cleavage at paired basic amino acid residues was demonstrated by the enhancement of propeptide processing of human von Willebrand factor when coexpressed with PACE in COS-1 cells.

Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone alpha-factor. These cells accurately process the hybrid protein and efficiently secrete authentic biologically active human epidermal growth factor into the medium.