Identification of a protein that confers calcitonin gene-related peptide responsiveness to oocytes by using a cystic fibrosis transmembrane conductance regulator assay.

An expression-cloning strategy was used to isolate a cDNA that encodes a protein that confers calcitonin gene-related peptide (CGRP) responsiveness to Xenopus laevis oocytes. A guinea pig organ of Corti (the mammalian hearing organ) cDNA library was screened by using an assay based on the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR is a chloride channel that is activated upon phosphorylation; this channel activity was used as a sensor for CGRP-induced activation of intracellular kinases. A cDNA library from guinea pig organ of Corti was screened by using this oocyte-CFTR assay. A cDNA was identified that contained an open reading frame coding for a small hydrophilic protein that is presumed to be either a CGRP receptor or a component of a CGRP receptor complex. This CGRP receptor component protein confers CGRP-specific activation to the CFTR assay, as no activation was detected upon application of calcitonin, amylin, neuropeptide Y, vasoactive intestinal peptide, or beta-endorphin. In situ hybridization demonstrated that the CGRP receptor component protein is expressed in outer hair cells of the organ of Corti and is colocalized with CGRP-containing efferent nerve terminals.

Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis.

We have shown elsewhere that acidification is an early event in apoptosis, preceding DNA cleavage. Cells expressing the most common mutation (delF508) of the cystic fibrosis transmembrane regulator (CFTR) exhibit a higher resting intracellular pH and are unable to secrete chloride and bicarbonate in response to cAMP. We hypothesized that defective acidification in cells expressing delF508 CFTR would interfere with the acidification that accompanies apoptosis, which in turn, would prevent endonuclease activation and cleavage of DNA. We therefore determined whether the function of the CFTR would affect the process of apoptosis in mouse mammary epithelial C127 cells stably transfected with the wild-type CFTR (C127/wt) or the delF508 mutation of the CFTR (C127/508). C127 cells possessed an acid endonuclease capable of DNA degradation at low pH. Sixteen hours after treatment with cycloheximide, C127/wt cells underwent cytoplasmic acidification. In contrast, C127/508 cells failed to demonstrate acidification. Furthermore, the C127/508 cells did not show nuclear condensation or DNA fragmentation detected by in situ nick-end labeling after treatment with cycloheximide or etoposide, in contrast to the characteristic features of apoptosis demonstrated by the C127/wt cells. Measurement of cell viability indicated a preservation of cell viability in C127/508 cells but not in C127/wt cells. That this resistance to the induction of apoptosis depended upon the loss of CFTR activity is shown by the finding that inhibition of the CFTR with diphenylamine carboxylate in C127/wt cells conferred similar protection. These findings suggest a role for the CFTR in acidification during the initiation of apoptosis in epithelial cells and imply that a failure to undergo programmed cell death could contribute to the pathogenesis of cystic fibrosis.

The invariant system of coordinates of antibody molecules: prediction of the "standard" C alpha framework of VL and VH domains.

A new approach of comparing protein structures that does not involve the procedure of superposition is suggested. An invariant system of coordinates for immunoglobulin molecules that is based on the geometrical symmetry inherent to the variable domain light-chain (VL)-heavy-chain (VH) complex is described. The coordinates of the Calpha atoms in 22 immunoglobulin structures are calculated in the invariant system of coordinates. We found that 76 identical positions in this Calpha framework are symmetrical about the twofold axis. Comparison of the identical positions in these molecules allows us to select 96 positions in the light chains and 87 positions in the heavy chains whose Calpha atom coordinates are approximately the same. To check whether the average coordinates of Calpha atoms in these positions complies with the stereochemical requirements, we calculated Calpha-Calpha distances. Seventy-three positions of the light chains and 72 positions of the heavy chains satisfy the Calpha-Calpha distance criterion. The Calpha atoms in these positions are used for constructing the "standard" Calpha framework of VL and VH complexes. The average coordinates of Calpha atoms are presented.

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.

An inhalational anesthetic binding domain in the nicotinic acetylcholine receptor.

To determine inhalational anesthetic binding domains on a ligand-gated ion channel, I used halothane direct photoaffinity labeling of the nicotinic acetylcholine receptor (nAChR) in native Torpedo membranes. [14C]Halothane photoaffinity labeling of both the native Torpedo membranes and the isolated nAChR was saturable, with Kd values within the clinically relevant range. All phospholipids were labeled, with greater than 95% of the label in the acyl chain region. Electrophoresis of labeled nAChR demonstrated no significant subunit selectivity for halothane incorporation. Within the alpha-subunit, greater than 90% of label was found in the endoprotease Glu-C digestion fragments which contain the four transmembrane regions, and the pattern was different from that reported for photoactivatable phospholipid binding to the nAChR. Unlabeled halothane reduced labeling more than did isoflurane, suggesting differences in the binding domains for inhalational anesthetics in the nAChR. These data suggest multiple similar binding domains for halothane in the transmembrane region of the nAChR.

Identification of a second transmembrane protein tyrosine phosphatase, IA-2beta, as an autoantigen in insulin-dependent diabetes mellitus: precursor of the 37-kDa tryptic fragment.

A novel cDNA, IA-2beta, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2beta indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2beta. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-2beta. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2beta and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2beta, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2beta and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2beta are major autoantigens in IDDM.

Molecular mechanism of transmembrane signaling by the aspartate receptor: a model.

The aspartate receptor of bacterial chemotaxis is representative of a large class of membrane-spanning receptors found in prokaryotic and eukaryotic organisms. These receptors, which regulate histidine kinase pathways and possess two putative transmembrane helices per subunit, appear to control a wide variety of cellular processes. The best characterized subgroup of the two-helix receptor class is the homologous family of chemosensory receptors from Escherichia coli and Salmonella typhimurium, including the aspartate receptor. This receptor binds aspartate, an attractant, in the periplasmic compartment and undergoes an intramolecular, transmembrane conformational change, thereby modulating the autophosphorylation rate of a bound histidine kinase in the cytoplasm. Here, we analyze recent results from x-ray crystallographic, solution 19F NMR, and engineered disulfide studies probing the aspartate-induced structural change within the periplasmic and transmembrane regions of the receptor. Together, these approaches provide evidence that aspartate binding triggers a "swinging-piston" displacement of the second membrane-spanning helix, which is proposed to communicate the signal across the bilayer.

Peptides from conserved regions of paramyxovirus fusion (F) proteins are potent inhibitors of viral fusion.

The synthetic peptides DP-107 and DP-178 (T-20), derived from separate domains within the human immunodeficiency virus type 1 (HIV-1) transmembrane (TM) protein, gp4l, are stable and potent inhibitors of HIV-1 infection and fusion. Using a computer searching strategy (computerized antiviral searching technology, C.A.S.T.) based on the predicted secondary structure of DP-107 and DP-178 (T-20), we have identified conserved heptad repeat domains analogous to the DP-107 and DP-178 regions of HIV-1 gp41 within the glycoproteins of other fusogenic viruses. Here we report on antiviral peptides derived from three representative paramyxoviruses, respiratory syncytial virus (RSV), human parainfluenza virus type 3 (HPIV-3), and measles virus (MV). We screened crude preparations of synthetic 35-residue peptides, scanning the DP-178-like domains, in antiviral assays. Peptide preparations demonstrating antiviral activity were purified and tested for their ability to block syncytium formation. Representative DP-178-like peptides from each paramyxovirus blocked homologous virus-mediated syncytium formation and exhibited EC50 values in the range 0.015-0.250 microM. Moreover, these peptides were highly selective for the virus of origin. Identification of biologically active peptides derived from domains within paramyxovirus F1 proteins analogous to the DP-178 domain of HIV-1 gp4l is compelling evidence for equivalent structural and functional features between retroviral and paramyxoviral fusion proteins. These antiviral peptides provide a novel approach to the development of targeted therapies for paramyxovirus infections.

Distinct ligand preferences of Src homology 3 domains from Src, Yes, Abl, Cortactin, p53bp2, PLCgamma, Crk, and Grb2.

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These domains have been shown to mediate protein-protein interactions by binding short proline-rich regions in ligand proteins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein interactions remain poorly defined. We have used a phage-displayed library of peptides of the form X6PXXPX6 to identify ligands for eight different SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characteristics. Specifically, we have found that the Src SH3 domain selects peptides sharing the consensus motif LXXRPLPXpsiP, whereas Yes SH3 selects psiXXRPLPXLP, Abl SH3 selects PPXthetaXPPPpsiP, Cortactin SH3 selects +PPpsiPXKPXWL, p53bp2 SH3 selects RPXpsiPpsiR+SXP, PLCgamma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects psiPpsiLPpsiK, and Grb2 N-terminal SH3 selects +thetaDXPLPXLP (where psi, theta, and + represent aliphatic, aromatic, and basic residues, respectively). Furthermore, we have compared the binding of phage expressing peptides related to each consensus motif to a panel of 12 SH3 domains. Results from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains to discern subtle differences in the primary structure of potential ligands. Finally, we have found that most known SH3-binding proteins contain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.

In vivo and in vitro characterization of the B1 and B2 zinc-binding domains from the acute promyelocytic leukemia protooncoprotein PML.

Acute promyelocytic leukemia (APL) has been ascribed to a chromosomal translocation event which results in a fusion protein comprising the PML protein and retinoic acid receptor alpha. PML is normally a component of a nuclear multiprotein complex which is disrupted in the APL disease state. Here, two newly defined cysteine/histidine-rich protein motifs called the B-box (B1 and B2) from PML have been characterized in terms of their effect on PML nuclear body formation, their dimerization, and their biophysical properties. We have shown that both peptides bind Zn2+, which induces changes in the peptides' structures. We demonstrate that mutants in both B1 and B2 do not form PML nuclear bodies in vivo and have a phenotype that is different from that observed in the APL disease state. Interestingly, these mutations do not affect the ability of wild-type PML to dimerize with mutant proteins in vitro, suggesting that the B1 and B2 domains are involved in an additional interaction central to PML nuclear body formation. This report in conjunction with our previous work demonstrates that the PML RING-Bl/B2 motif plays a fundamental role in formation of a large multiprotein complex, a function that may be common to those unrelated proteins which contain the motif.

Uptake of fluorescent dyes associated with the functional expression of the cystic fibrosis transmembrane conductance regulator in epithelial cells.

Specific mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the most common autosomal recessive fatal genetic disease of Caucasians, result in the loss of epithelial cell adenosine 3',5'-cyclic-monophosphate (cAMP)-stimulated Cl- conductance. We show that the influx of a fluorescent dye, dihydrorhodamine 6G (dR6G), is increased in cells expressing human CFTR after retrovirus- and adenovirus-mediated gene transfer. dR6G influx is stimulated by cAMP and is inhibited by antagonists of cAMP action. Dye uptake is ATP-dependent and inhibited by Cl- removal or the addition of 10 mM SCN-. Increased staining is associated with functional activation of CFTR Cl- permeability. dR6G staining enables both the fluorescent assessment of CFTR function and the identification of successfully corrected cells after gene therapy.

Cloning and expression of a gene encoding an integrin-like protein in Candida albicans.

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.

Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach.

Pathogenic yersiniae secrete a set of antihost proteins, called Yops, by a type III secretion mechanism. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis and Yersinia enterocolitica translocate cytotoxin YopE across the host cell plasma membrane. Several lines of evidence suggest that tyrosine phosphatase YopH follows the same pathway. We analyzed internalization of YopE and YopH into murine PU5-1.8 macrophages by using recombinant Y. enterocolitica producing truncated YopE and YopH proteins fused to a calmodulin-dependent adenylate cyclase. The YopE-cyclase and YopH-cyclase hybrids were readily secreted by Y. enterocolitica. The N-terminal domain required for secretion was not longer than 15 residues of YopE and 17 residues of YopH. Internalization into eukaryotic cells, revealed by cAMP production, only required the N-terminal 50 amino acid residues of YopE and the N-terminal 71 amino acid residues of YopH. YopE and YopH are thus modular proteins composed of a secretion domain, a translocation domain, and an effector domain. Translocation of YopE and YopH across host cell's membranes was also dependent on the secretion of YopB and YopD by the same bacterium. The cyclase fusion approach could be readily extended to study the fate of other proteins secreted by invasive bacterial pathogens.

Three distinct structural environments of a transmembrane domain in the inwardly rectifying potassium channel ROMK1 defined by perturbation.

To probe the protein environment of an ion channel, we have perturbed the structure of a transmembrane domain by substituting side chains with those of two different sizes by using site-specific mutagenesis. We have used Trp and Ala as a high- and a low-impact perturbation probe, respectively, to replace each of 18 consecutive residues within the putative second transmembrane segment, M2, of an inwardly rectifying potassium channel, ROMK1. Our rationale is that a change in the channel function as a consequence of these mutations at a particular position will reflect the structural environment of the altered side chain. Each position can then be assigned to one of three classes of environments, as grated by different levels of perturbation: very tolerant (channel functions with both Trp and Ala substitutions), tolerant (function preserved with Ala but not with Trp substitution), and intolerant (either Ala or Trp substitution destroys function). We identify the very tolerant environment as being lipid-facing, tolerant as protein-interior-facing, and intolerant as pore-facing. We observe a strikingly ordered pattern of perturbation of all three environmental classes. This result indicates that M2 is a straight alpha-helix.

Domains with transcriptional regulatory activity within the ALL1 and AF4 proteins involved in acute leukemia.

The ALLI gene, located at chromosome band 11q23, is involved in acute leukemia through a series of chromosome translocations and fusion to a variety of genes, most frequently to A4 and AF9. The fused genes encode chimeric proteins proteins. Because the Drosophila homologue of ALL1, trithorax, is a positive regulator of homeotic genes and acts at the level of transcription, it is conceivable that alterations in ALL1 transcriptional activity may underlie its action in malignant transformation. To begin studying this, we examined the All1, AF4, AF9, and AF17 proteins for the presence of potential transcriptional regulatory domains. This was done by fusing regions of the proteins to the yeast GAL4 DNA binding domain and assaying their effect on transcription of a reporter gene. A domain of 55 residues positioned at amino acids 2829-2883 of ALL1 was identified as a very strong activator. Further analysis of this domain by in vitro mutagenesis pointed to a core of hydrophobic and acidic residues as critical for the activity. An ALL1 domain that repressed transcription of the reporter gene coincided with the sequence homologous to a segment of DNA methyltransferase. An AF4 polypeptide containing residues 480-560 showed strong activation potential. The C-terminal segment of AF9 spanning amino acids 478-568 transactivated transcription of the reporter gene in HeLa but not in NIH 3T3 cells. These results suggest that ALL1, AF4, and probably AF9 interact with the transcriptional machinery of the cell.