beta -Amyloid peptide-induced apoptosis regulated by a novel protein containing a g protein activation module.

Degeneration of neurons in Alzheimer's disease is mediated by beta-amyloid peptide by diverse mechanisms, which include a putative apoptotic component stimulated by unidentified signaling events. This report describes a novel beta-amyloid peptide-binding protein (denoted BBP) containing a G protein-coupling module. BBP is one member of a family of three proteins containing this conserved structure. The BBP subtype bound human beta-amyloid peptide in vitro with high affinity and specificity. Expression of BBP in cell culture induced caspase-dependent vulnerability to beta-amyloid peptide toxicity. Expression of a signaling-deficient dominant negative BBP mutant suppressed sensitivity of human Ntera-2 neurons to beta-amyloid peptide mediated toxicity. These findings suggest that BBP is a target of neurotoxic beta-amyloid peptide and provide new insight into the molecular pathophysiology of Alzheimer's disease.

Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen.

The interaction of the N-type calcium channel beta3 subunit with the alpha1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel. The defined protein-protein interaction of the human alpha1B and beta3 subunits provides a target for small-molecule modulation of N-type channel activity. We describe a high throughput screen based on a counterselection yeast two-hybrid assay, which was used to identify small molecules that disrupt alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity. These small molecules may be a new class of calcium channel antagonists with therapeutic potential.

Investigation of growth hormone releasing hormone receptor structure and activity using yeast expression technologies.

Growth hormone releasing hormone (GHRH) is the positive regulator of growth hormone synthesis and secretion in the anterior pituitary. The peptide confers activity by binding to a seven transmembrane domain G protein-coupled receptor. Signal transduction proceeds through subsequent G alpha s stimulation of adenylyl cyclase. To investigate ligand/receptor and receptor/G protein associations, the human GHRH receptor was expressed in a modified S. cerevisiae strain which allows for facile measurement of receptor activity by cell prototrophy mediated by a reporter gene coupled to the yeast pheromone response pathway. GHRH-dependent signal activation in this system required the substitution of yeast G alpha protein with proteins containing C-terminal regions of G alpha s. A D60G variant (analogous to the little mouse mutation) of the receptor failed to respond to agonist. In parallel studies, GHRH29 and the N-terminal extracellular region of the receptor were expressed as Gal4 fusion proteins in a 2-hybrid assay. A specific interaction between these proteins was readily observed. The D60G mutation was engineered into the receptor fusion protein. This protein failed to interact with the ligand fusion, confirming the specificity of the association between unmodified proteins. These two yeast expression technologies should prove invaluable in additional structure/activity analyses of this ligand/receptor pair as well as other peptide ligands and receptors.

Functional coupling of a mammalian somatostatin receptor to the yeast pheromone response pathway.

A detailed analysis of structural and functional aspects of G-protein-coupled receptors, as well as discovery of novel pharmacophores that exert their effects on members of this class of receptors, will be facilitated by development of a yeast-based bioassay. To that end, yeast strains that functionally express the rat somatostatin receptor subtype 2 (SSTR2) were constructed. High-affinity binding sites for somatostatin ([125I-Tyr-11]S-14) comparable to those in native tissues were detected in yeast membrane extracts at levels equivalent to the alpha-mating pheromone receptor (Ste2p). Somatostatin-dependent growth of strains modified by deletion of genes encoding components of the pheromone response pathway was detected through induction of a pheromone-responsive HIS3 reporter gene, enabling cells to grow on medium lacking histidine. Dose-dependent growth responses to S-14 and related SSTR2 subtype-selective agonists that were proportional to the affinity of the ligands for SSTR2 were observed. The growth response required SSTR2, G alpha proteins, and an intact signal transduction pathway. The sensitivity of the bioassay was affected by intracellular levels of the G alpha protein. A mutation in the SST2 gene, which confers supersensitivity to pheromone, was found to significantly enhance the growth response to S-14. In sst2 delta cells, SSTR2 functionally interacted with both a chimeric yeast/mammalian G alpha protein and the yeast G alpha protein, Gpa1p; to promote growth. These yeast strains should serve as a useful in vivo reconstitution system for examination of molecular interactions of the G-protein-coupled receptors and G proteins.

Functional interaction of ligands and receptors of the hematopoietic superfamily in yeast.

Circulating peptide hormones and growth factors interact with cell surface receptors to initiate specific cellular responses. These complexes can consist of a simple association between two proteins or a more elaborate association of multiple proteins. We describe the functional expression of ligands and corresponding receptors in a microbial system useful for the rapid dissection of these important protein interactions. GH or PRL and extracellular domains of their respective receptors were functionally expressed as fusion proteins in an extended two-hybrid protein-protein interaction system. Reversible and specific ligand-receptor interactions were demonstrated by concurrent expression of free ligand peptides (GH or PRL) as binding competitors. The versatility established by expressing three heterologous proteins allowed for the investigation of higher order structures. Ligand-dependent GH receptor dimerization was demonstrated but PRL receptor dimerization was not observed in an analogous assay, suggesting that these related growth factors may not engage receptors in a similar manner. Additionally, significant association of GH receptors was observed in the absence of ligand, suggesting that there may be substantial avidity between these receptor proteins before ligand binding. Ligand-dependent and ligand-independent receptor dimerization was demonstrated by vascular endothelial growth factor and receptor proteins in similar assays. These findings indicate that extracellular protein interactions such as ligand-receptor association, as well as the formation of higher order protein structures important for the activation of hematopoietic receptors, can be rapidly investigated in this microbial expression system.

Selective uptake of estrogenic compounds by Saccharomyces cerevisiae: a mechanism for antiestrogen resistance in yeast expressing the mammalian estrogen receptor.

Estrogen antagonists such as ICI164,384 do not inhibit 17 beta-estradiol (E2)-dependent gene activity in yeast expressing the mammalian estrogen receptor although these compounds bind to receptors isolated from these cells. Various explanations have been offered for antiestrogen resistance in yeast systems including differences in cell-specific components and lack of permeability of the yeast cell wall to these compounds. We have used a strain of Saccharomyces cerevisiae transformed with the human estrogen receptor gene, and two estrogen response elements linked to a lacZ reporter gene to study the pharmacology of estrogen agonists and antagonists. The rank order of potency of estrogen agonists in this strain (CY525) is similar to that in estrogen-dependent mammalian cells: DES > or = E2 > E1 > E3 = zeranol. Competitive binding with 3H-E2 by these compounds in cell-free extracts of CY525 results in a similar order of potency with a reverse order for E1 and E3. The pure estrogen antagonist ICI164,384 also binds to the receptor from cell-free extracts of CY525 with an IC50 of approximately 14nM. As in mammalian cells ICI164,384 does not induce E2-dependent gene activity. However, unlike mammalian cells, E2-induced gene activity in CY525 is not inhibited by ICI164,384. Intact CY525 cells incubated with 3H-17 beta estradiol were found to specifically bind the labeled ligand since excess unlabeled E2 effectively competed for binding. Unlabeled DES and E1 were also found to compete, however, excess unlabeled ICI164,384, E3 and the second generation antagonist ICI182,720 were unable to displace 3H-E2 binding in intact cells. These results indicate that certain compounds enter the intact yeast cell more readily than others and offer an explanation for antagonist resistance in these organisms.

A single amino acid substitution in somatostatin receptor subtype 5 increases affinity for somatostatin-14.

Four of the five somatostatin receptor (SSTR) subtypes bind the two native forms of somatostatin, i.e., somatostatin-14 (S-14) and amino-terminally extended somatostatin-28 (S-28), with comparable affinities (approximately 0.2 nM). The SSTR5 subtype exhibits 10-50-fold higher affinity for S-28 than for S-14 (0.2 and 5 nM, respectively). To determine which domains in SSTR5 are responsible for the observed pharmacological selectivity, a series of SSTR2/SSTR5 chimeras were constructed and expressed in Chinese hamster ovary cells. Saturation and competition radioligand binding studies demonstrated that the region encompassing transmembrane domain 6 (TM6) through the carboxyl terminus plays a critical role in the lower binding affinity of S-14 for SSTR5. Substitution of this region with the corresponding region of SSTR2 produced chimeric receptors with high affinity for both S-28 and S-14. Examination of amino acid sequences revealed both a specific conserved hydrophobic residue and a conserved tyrosine in TM6 of SSTR1-4. At comparable positions in SSTR5, these residues are glycine (G258) and phenylalanine (F265), respectively. Substitution of G258 with phenylalanine did not alter the preference of SSTR5 for S-28 over S-14. However, substitution of F265 with tyrosine increased the binding affinity of S-14 by 20-fold, to an affinity comparable to that observed for the SSTR2 subtype. These data indicate that replacement of phenylalanine with tyrosine at position 265 in SSTR5 can modify ligand binding selectivity and abolish the preference for S-28 over S-14. This finding suggests that the tyrosine in the predicted TM6 may be an important contact point between somatostatin and SSTR.

cDNA cloning of an orphan opiate receptor gene family member and its splice variant.

Radioligand binding and cDNA homology studies have suggested the existence of opiate receptors distinct from the recently-cloned mu, delta and kappa receptors. XOR1S, a rat brain cDNA whose predicted translation product displays 67-72% homology with those encoded by mu 1, delta 1 and kappa 1 opiate receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XOR1L variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XOR1 mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of opiate ligands nor reproducible opiate inhibition of forskolin-stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the opiate receptor gene family, including apparent differential splicing and expression in peripheral tissues.

A unique pathway of double-strand break repair operates in tandemly repeated genes.

The RAD52 gene product of the yeast Saccharomyces cerevisiae is required for most spontaneous recombination and almost all double-strand break (DSB) repair. In contrast to recombination elsewhere in the genome, recombination in the ribosomal DNA (rDNA) array is RAD52 independent. To determine the fate of a DSB in the rDNA gene array, a cut site for the HO endonuclease was inserted into the rDNA in a strain containing an inducible HO gene. DSBs were efficiently repaired at this site, even in the absence of the RAD52 gene product. Efficient RAD52-independent DSB repair was also observed at another tandem gene array, CUP1, consisting of 18 repeat units. However, in a smaller CUP1 array, consisting of only three units, most DSBs (ca. 80%) were not repaired and resulted in cell death. All RAD52-independent DSB repair events examined resulted in the loss of one or more repeat units. We propose a model for DSB repair in repeated sequences involving the generation of single-stranded tails followed by reannealing.

Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli.

Transcriptional linkage of the enterobactin gene cluster entCEBA (P15) was confirmed by ent-lacZ gene fusion analysis. Control sequences directing iron-regulated expression of this polycistronic message were localized to the fepB-entC bidirectional promoter region. Transcriptional initiation sites defined by primer extension analysis were located 103 base-pairs apart for the divergent fepB and entC messages. Within this divergent regulatory region, strongly consensus -35 and -10 promoter determinants and potential Fur repressor-binding sequences were identified. A vector containing divergently oriented indicator gene fusions was constructed to monitor regulatory effects of mutations within this iron-responsive control region. The fepB-entC promoter-operator elements were confirmed by mutation, using the dual gene fusion system in multicopy and low copy number states. Mutations in the -35 and -10 regions of the fepB and entC promoters that decreased their similarity to consensus resulted in reduced promoter activity. Mutations in the Fur-controlled operators reduced induction ratios (iron-deficient levels/iron-rich levels) for the respective fusion gene activities by approximately sevenfold. Although operator mutants retained some degree of inducibility, complete relief of repression was observed for double operator mutants, suggesting that only minor regulatory influence is exerted by Fur occupation of the opposing operator site. DNase I footprinting experiments were performed to characterize the sequence-specific Fur interactions at the operator sequences. At the fepB operator, a 31 base-pair Fur-protected region was identified, corresponding to positions -19 to +12 with respect to the transcriptional start site. Similarly, Fur protected a 31 base-pair region in entC, corresponding to positions +1 to +31 in the message. A contiguous and sequentially occupied secondary Fur-binding site in entC was protected at higher Fur concentrations, extending the protected region to +49, and sequestering the putative Shine-Dalgarno sequence. Operator positional effects and co-operativity are discussed.

Nucleotide sequence and transcriptional organization of the Escherichia coli enterobactin biosynthesis cistrons entB and entA.

The nucleotide sequence of a 2,137-base-pair DNA fragment expressing enterobactin biosynthesis functions defined the molecular boundaries and translational products of the entB and entA genes and identified a closely linked downstream open reading frame encoding an uncharacterized protein of approximately 15,000 daltons (P15). The sequence revealed that an independent protein-coding sequence corresponding to an EntG polypeptide was not situated in the genetic region between the entB and entA cistrons, to which the EntG- phonotype had been genetically localized. As a result, the biochemical nature of the EntG function in the biosynthetic pathway requires reevaluation. The EntA polypeptide displayed significant similarities at the amino acid level to the pyridine nucleotide-binding domains of several members of a family of alcohol-polyol-sugar dehydrogenase enzymes, consistent with its function as the enzyme catalyzing the final step of dihydroxybenzoate biosynthesis. An additional role for EntA in the isochorismate synthetase activity of EntC was strongly implicated by genetic evidence. Evidence from the nucleotide sequence of this region and newly constructed ent-lacZ fusion plasmids argues strongly that these genes are linked in an iron-regulated entCEBA (P15) polycistronic operon.

Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes.

Biochemical analysis of the enzymatic activity catalyzing the conversion of chorismate to isochorismate in the enterobactin biosynthetic pathway attributed the reaction to the isochorismate synthetase enzyme, designated EntC. However, the lack of mutations defining this activity has hampered the precise identification of the entC structural gene. In this study, we engineered a stable insertion mutation into the chromosomal region between the enterobactin genes fepB and entE. This mutation disrupted the structural gene for a previously identified 44-kilodalton protein and eliminated production of 2,3-dihydroxybenzoic acid, the catechol precursor of enterobactin. The complete nucleotide sequence of this gene was determined and compared with the sequences of other genes encoding chorismate-utilizing proteins. The similarities observed in these comparisons not only indicated that the locus is entC but also supported the premise that these enzymes constitute a family of related proteins sharing a common evolutionary origin. In addition, in this and the accompanying paper (M. S. Nahlik, T. J. Brickman, B. A. Ozenberger, and M. A. McIntosh, J. Bacteriol. 171:784-790, 1989), evidence is presented indicating that the entA product is potentially a secondary factor in the chorismate-to-isochorismate conversion and that the prototypic entC lesion (entC401) resides in the structural gene for the EntA protein. Finally, polarity effects from the insertion mutation in entC on downstream biosynthetic genes indicated that this locus is the promoter-proximal cistron in an ent operon comprising at least five genes. Appropriate regulatory signals upstream of entC suggest that this operon is regulated by iron through interaction with the Fur repressor protein.

Genetic organization of multiple fep genes encoding ferric enterobactin transport functions in Escherichia coli.

Three genes were shown to provide functions specific for ferric enterobactin transport in Escherichia coli: fepA encoded the outer membrane receptor, fepB produced a periplasmic protein, and the fepC product was presumably a component of a cytoplasmic membrane permease system for this siderophore. A 10.6-kilobase-pair E. coli chromosomal EcoRI restriction fragment containing the fepB and fepC genes was isolated from a genomic library constructed in the vector pBR328. Both cistrons were localized on this clone (pITS24) by subcloning and deletion and insertion mutagenesis to positions that were separated by approximately 2.5 kilobases. Within this region, insertion mutations defining an additional ferric enterobactin transport gene (fepD) were isolated, and polarity effects from insertions into fepB suggested that fepD is encoded downstream on the same transcript. A 31,500-dalton FepC protein and a family of FepB polypeptides ranging from 34,000 to 37,000 daltons were identified in E. coli minicells, but the product of fepD was not detectable by this system. Another insertion mutation between entF and fepC was also shown to disrupt iron transport via enterobactin and thus defined the fepE locus; fepE weakly expressed a 43,000-dalton protein in minicells. It is proposed that these newly identified genes, fepD and fepE, provide functions which act in conjunction with the fepC product to form the ferric enterobactin-specific cytoplasmic membrane permease. An additional 44,000-dalton protein was identified and shown to be expressed from a gene that is situated between fepB and entE and that is transcribed in the direction opposite that of fepB. Although the function of this protein is uncharacterized, its membrane location suggests that it too may function in iron transport.