Dependence of agonist activation on a conserved apolar residue in the third intracellular loop of the AT1 angiotensin receptor.

The coupling of agonist-activated seven transmembrane domain receptors to G proteins is known to involve the amino-terminal region of their third cytoplasmic loop. Analysis of the amino acids in this region of the rat type in angiotensin (AT1a) receptor identified Leu-222 as an essential residue in receptor activation by the physiological agonist, angiotensin II (Ang II). Nonpolar replacements for Leu-222 yielded functionally intact AT1 receptors, while polar or charged residues caused progressive impairment of Ang II-induced inositol phosphate generation. The decrease in agonist-induced signal generation was associated with a parallel reduction of receptor internalization, and was most pronounced for the Lys-222 mutant receptor. Although this mutant showed normal binding of the peptide antagonist, [Sar1,Ile6]Ang II, its affinity for Ang II was markedly reduced, consistent with its inability to adopt the high-affinity conformation. A search revealed that many Gq-coupled receptors contain an apolar amino acid (frequently leucine) in the position corresponding to Leu-222 of the AT1 receptor. These findings suggest that such a conserved apolar residue in the third intracellular loop is a crucial element in the agonist-induced activation of the AT1 and possibly many other G protein-coupled receptors.

The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain.

Mutations in the whn gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/ winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene revealed the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. As the whnrnu mutant protein lacks this domain, our results indicate that a transactivation function is essential for the activity of the whn transcription factor.

Mutations affecting conserved cysteine residues within the extracellular domain of Neu promote receptor dimerization and activation.

Overexpression of the Neu/ErbB-2 receptor tyrosine kinase has been implicated in the genesis of human breast cancer. Indeed, expression of either activated or wild-type neu in the mammary epithelium of transgenic mice results in the induction of mammary tumors. Previously, we have shown that many of the mammary tumors arising in transgenic mice expressing wild-type neu occur through somatic activating mutations within the neu transgene itself. Here we demonstrate that these mutations promote dimerization of the Neu receptor through the formation of disulfide bonds, resulting in its constitutive activation. To explore the role of conserved cysteine residues within the region deleted in these altered Neu proteins, we examined the transforming potential of a series of Neu receptors in which the individual cysteine residues were mutated. These analyses indicated that mutation of certain cysteine residues resulted in the oncogenic activation of Neu. The increased transforming activity displayed by the altered receptors correlated with constitutive dimerization that occurred in a disulfide bond-dependent manner. We further demonstrate that addition of 2-mercaptoethanol to the culture medium interfered with the specific transforming activity of the mutant Neu receptors. These observations suggest that oncogenic activation of Neu results from constitutive disulfide bond-dependent dimerization.

Creation of a novel protein-coding region at the RNA level in black pine chloroplasts: the pattern of RNA editing in the gymnosperm chloroplast is different from that in angiosperms.

The phenomenon of RNA editing has been found to occur in chloroplasts of several angiosperm plants. Comparative analysis of the entire nucleotide sequence of a gymnosperm [Pinus thunbergii (black pine)] chloroplast genome allowed us to predict several potential editing sites in its transcripts. Forty-nine such sites from 14 genes/ORFs were analyzed by sequencing both cDNAs from the transcripts and the corresponding chloroplast DNA regions, and 26 RNA editing sites were identified in the transcripts from 12 genes/ORFs, indicating that chloroplast RNA editing is not restricted to angiosperms but occurs in the gymnosperm, too. All the RNA editing events are C-to-U conversions; however, many new codon substitutions and creation of stop codons that have not so far been reported in angiosperm chloroplasts were observed. The most striking is that two editing events result in the creation of an initiation and a stop codon within a single transcript, leading to the formation of a new reading frame of 33 codons. The predicted product is highly homologous to that deduced from the ycf7 gene (ORF31), which is conserved in the chloroplast genomes of many other plant species.

Evolutionarily conserved Galphabetagamma binding surfaces support a model of the G protein-receptor complex.

The pivotal role of G proteins in sensory, hormonal, inflammatory, and proliferative responses has provoked intense interest in understanding how they interact with their receptors and effectors. Nonetheless, the locations of the receptors and effector binding sites remain poorly characterized, although nearly complete structures of the alphabetagamma heterotrimeric complex are available. Here we apply evolutionary trace (ET) analysis [Lichtarge, O., Bourne, H. R. & Cohen, F. E. (1996) J. Mol. Biol. 257, 342-358] to propose plausible locations for these sites. On each subunit, ET identifies evolutionarily selected surfaces composed of residues that do not vary within functional subgroups and that form spatial clusters. Four clusters correctly identify subunit interfaces, and additional clusters on Galpha point to likely receptor or effector binding sites. Our results implicate the conformationally variable region of Galpha in an effector binding role. Furthermore the range of predicted interactions between the receptor and Galphabetagamma, is sufficiently limited that we can build a low resolution and testable model of the receptor-G protein complex.

Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns.

Semaphorins and collapsins make up a family of conserved genes that encode nerve growth cone guidance signals. We have identified two additional members of the human semaphorin family [human semaphorin A(V) and human semaphorin IV] in chromosome region 3p21.3, where several small cell lung cancer (SCLC) cell lines exhibit homozygous deletions indicative of a tumor suppressor gene. Human semaphorin A(V) has 86% amino acid homology with murine semaphorin A, whereas semaphorin IV is most closely related to murine semaphorin E, with 50% homology. These semaphorin genes are approximately 70 kb apart flanking two GTP-binding protein genes, GNAI-2 and GNAT-1. In contrast, other human semaphorin gene sequences (human semaphorin III and homologues of murine semaphorins B and C) are not located on chromosome 3. Human semaphorin A(V) is translated in vitro into a 90-kDa protein, which accumulates at the endoplasmic reticulum. The human semaphorin A(V) (3.4-kb mRNA) and IV (3.9- and 2.9-kb mRNAs) genes are expressed abundantly but differentially in a variety of human neural and nonneural tissues. Human semaphorin A(V) was expressed in only 1 out of 23 SCLCs and 7 out of 16 non-SCLCs, whereas semaphorin IV was expressed in 19 out of 23 SCLCs and 13 out of 16 non-SCLCs. Mutational analysis in semaphorin A(V) revealed mutations (germ line in one case) in 3 of 40 lung cancers. Our data suggest the need to determine the function of human semaphorins A(V) and IV in nonneural tissues and their role in the pathogenesis of lung cancer.

A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain.

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.

The Drosophila melanogaster dodo (dod) gene, conserved in humans, is functionally interchangeable with the ESS1 cell division gene of Saccharomyces cerevisiae.

We have sequenced the region of DNA adjacent to and including the flightless (fli) gene of Drosophila melanogaster and molecularly characterized four transcription units within it, which we have named tweety (twe), flightless (fli), dodo (dod), and penguin (pen). We have performed deletion and transgenic analysis to determine the consequences of the quadruple gene removal. Only the flightless gene is vital to the organism; the simultaneous absence of the other three allows the overriding majority of individuals to develop to adulthood and to fly normally. These gene deletion results are evaluated in the context of the redundancy and degeneracy inherent in many genetic networks. Our cDNA analyses and data-base searches reveal that the predicted dodo protein has homologs in other eukaryotes and that it is made up of two different domains. The first, designated WW, is involved in protein-protein interactions and is found in functionally diverse proteins including human dystrophin. The second is involved in accelerating protein folding and unfolding and is found in Escherichia coli in a new family of peptidylprolyl cis-trans isomerases (PPIases; EC 5.2.1.8). In eukaryotes, PPIases occur in the nucleus and the cytoplasm and can form stable associations with transcription factors, receptors, and kinases. Given this particular combination of domains, the dodo protein may well participate in a multisubunit complex involved in the folding and activation of signaling molecules. When we expressed the dodo gene product in Saccharomyces cerevisiae, it rescued the lethal phenotype of the ESS1 cell division gene.

GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain.

Using the yeast two-hybrid system we have identified a human protein, GAIP (G Alpha Interacting Protein), that specifically interacts with the heterotrimeric GTP-binding protein G alpha i3. Interaction was verified by specific binding of in vitro-translated G alpha i3 with a GAIP-glutathione S-transferase fusion protein. GAIP is a small protein (217 amino acids, 24 kDa) that contains two potential phosphorylation sites for protein kinase C and seven for casein kinase 2. GAIP shows high homology to two previously identified human proteins, GOS8 and 1R20, two Caenorhabditis elegans proteins, CO5B5.7 and C29H12.3, and the FLBA gene product in Aspergillus nidulans--all of unknown function. Significant homology was also found to the SST2 gene product in Saccharomyces cerevisiae that is known to interact with a yeast G alpha subunit (Gpa1). A highly conserved core domain of 125 amino acids characterizes this family of proteins. Analysis of deletion mutants demonstrated that the core domain is the site of GAIP's interaction with G alpha i3. GAIP is likely to be an early inducible phosphoprotein, as its cDNA contains the TTTTGT sequence characteristic of early response genes in its 3'-untranslated region. By Northern analysis GAIP's 1.6-kb mRNA is most abundant in lung, heart, placenta, and liver and is very low in brain, skeletal muscle, pancreas, and kidney. GAIP appears to interact exclusively with G alpha i3, as it did not interact with G alpha i2 and G alpha q. The fact that GAIP and Sst2 interact with G alpha subunits and share a common domain suggests that other members of the GAIP family also interact with G alpha subunits through the 125-amino-acid core domain.

Molecular cloning and characterization of a cellular phosphoprotein that interacts with a conserved C-terminal domain of adenovirus E1A involved in negative modulation of oncogenic transformation.

The adenovirus type 2/5 E1A proteins transform primary baby rat kidney (BRK) cells in cooperation with the activated Ras (T24 ras) oncoprotein. The N-terminal half of E1A (exon 1) is essential for this transformation activity. While the C-terminal half of E1A (exon 2) is dispensable, a region located between residues 225 and 238 of the 243R E1A protein negatively modulates in vitro T24 ras cooperative transformation as well as the tumorigenic potential of E1A/T24 ras-transformed cells. The same C-terminal domain is also required for binding of a cellular 48-kDa phosphoprotein, C-terminal binding protein (CtBP). We have cloned the cDNA for CtBP via yeast two-hybrid interaction cloning. The cDNA encodes a 439-amino acid (48 kDa) protein that specifically interacts with exon 2 in yeast two-hybrid, in vitro protein binding, and in vivo coimmunoprecipitation analyses. This protein requires residues 225-238 of the 243R E1A protein for interaction. The predicted protein sequence of the isolated cDNA is identical to amino acid sequences obtained from peptides prepared from biochemically purified CtBP. Fine mapping of the CtBP-binding domain revealed that a 6-amino acid motif highly conserved among the E1A proteins of various human and animal adenoviruses is required for this interaction. These results suggest that interaction of CtBP with the E1A proteins may play a critical role in adenovirus replication and oncogenic transformation.

Genetic and comparative analyses reveal an alternative secondary structure in the region of nt 912 of Escherichia coli 16S rRNA.

Mutations at position 912 of Escherichia coli 16S rRNA result in two notable phenotypes. The C-->U transition confers resistance to streptomycin, a translational-error-inducing antibiotic, while a C-->G transversion causes marked retardation of cell growth rate. Starting with the slow-growing G912 mutant, random mutagenesis was used to isolate a second site mutation that restored growth nearly to the wild-type rate. The second site mutation was identified as a G-->C transversion at position 885 in 16S rRNA. Cells containing the G912 mutation had an increased doubling time, abnormal sucrose gradient ribosome/subunit profile, increased sensitivity to spectinomycin, dependence upon streptomycin for growth in the presence of spectinomycin, and slower translation rate, whereas cells with the G912/C885 double mutation were similar to wild type in these assays. Comparative analysis showed there was significant covariation between positions 912 and 885. Thus the second-site suppressor analysis, the functional assays, and the comparative data suggest that the interaction between nt 912 and nt 885 is conserved and necessary for normal ribosome function. Furthermore, the comparative data suggest that the interaction extends to include G885-G886-G887 pairing with C912-U911-C910. An alternative secondary structure element for the central domain of 16S rRNA is proposed.

Alignment of a 1.2-Mb chromosomal region from three strains of Rhodobacter capsulatus reveals a significantly mosaic structure.

High-resolution physical maps of the genomes of three Rhodobacter capsulatus strains, derived from ordered cosmid libraries, were aligned. The 1.2-Mb segment of the SB1003 genome studied here is adjacent to a 1-Mb region analyzed previously [Fonstein, M., Nikolskaya, T. & Haselkorn, H. (1995) J. Bacteriol. 177, 2368-2372]. Probes derived from the ordered cosmid set of R. capsulatus SB1003 were used to link cosmids from the St. Louis and 2.3.1 strain libraries. Cosmids selected this way did not merge into a single contig but formed several unlinked groups. EcoRV restriction maps of the ordered cosmids were then constructed using lambda terminase and fused to derive fragments of the chromosomal map. In order to link these fragments, their ends were transcribed to produce secondary probes for hybridization to gridded cosmid libraries of the same strains. This linking reduced the number of subcontigs to three for the St. Louis strain and one for the 2.3.1 strain. Hybridization of the same probes back to the ordered cosmid set of SB1003 positioned the subcontigs on the high-resolution physical map of SB1003. The final alignment of the restriction maps shows numerous large and small translocations in this 1.2-Mb chromosomal region of the three Rhodobacter strains. In addition, the chromosomes of the three strains, whose fine-structure maps can now be compared over 2.2 Mb, are seen to contain regions of 15-80 kb in which restriction sites are highly polymorphic, interspersed among regions in which the positions of restriction sites are highly conserved.

Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants.

Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extensin tolerates little variation in protein structure.

Gut-specific transcriptional regulatory elements of the carboxypeptidase gene are conserved between black flies and Drosophila.

Millions of people die every year in the tropical world from diseases transmitted by hematophagous insects. Failure of conventional containment measures emphasizes the need for additional approaches, such as transformation of vector insects with genes that restrict vectorial capacity. The availability of an efficient promoter to drive foreign genes in transgenic insects is a necessary tool to test the feasibility of such approach. Here we characterize the putative promoter region of a black fly midgut carboxypeptidase gene and show that these sequences correctly direct the expression of a beta-glucuronidase reporter in Drosophila melanogaster. By histochemical staining and mRNA analysis, we found that the gene is expressed strongly and gut-specifically in the transgenic Drosophila. This gut-specific black fly carboxypeptidase promoter provides a valuable tool for the study of disease vectors.

Restricted and conserved T-cell repertoires involved in allorecognition of class II major histocompatibility complex.

The nature of the alloreactive T-cell response is not yet clearly understood. These strong cellular responses are thought to be the basis of allograft rejection and graft-vs.-host disease. The question of the extent of responding T-cell repertoires has so far been addressed by cellular cloning, often combined with molecular T-cell receptor (TCR) analysis. Here we present a broad repertoire analysis of primed responder cells from mixed lymphocyte cultures in which two different DR1/3 responders were stimulated with DR3/4 cells. Repertoire analysis was performed by TCR spectratyping, a method by which T cells are analyzed on the basis of the complementarity-determining region 3 length of different variable region (V) families. Strikingly, both responders showed very similar repertoires when the TCR V beta was used as a lineage marker. This was not seen when TCR V alpha was analyzed. A different pattern of TCR V beta was observed if the stimulating alloantigen was changed. This finding indicates that alloreactive T cells form a specific repertoire for each alloantigen. Since conservation appears to be linked to TCR V beta, the question of different roles of alpha and beta chains in allorecognition is raised.