Amino Acid-rich Region Research Articles

The membrane-dipped neuronal SNARE complex: a site-directed spin labeling electron paramagnetic resonance study.

The formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is an essential process for membrane fusion and the neurotransmitter release in neurons. As an initial step toward the determination of the membrane topology of the SNARE complex, residues at the membrane-water interface were investigated with site-specific spin labeling electron paramagnetic resonance. EPR analysis revealed that the basic amino acid-rich interfacial region, which is universal for all transmembrane SNARE proteins, inserts into the membrane, eliminating the gap between the core complex and the membrane. The result raises the possibility that core complex formation directly leads to the apposition of two membranes, which could facilitate membrane fusion.

Crystal structure of the catalytic domain of a human thioredoxin-like protein.

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin-like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. We have solved the crystal structure of the recombinant N-terminal catalytic domain (hTRXL-N) of hTRXL in its oxidized form at 2.2-A resolution. Although this domain shares a similar three-dimensional structure with human thioredoxin (hTRX), a unique feature of hTRXL-N is the large number of positively charged residues distributed around the active site, which has been implicated in substrate specificity. Furthermore, the hTRXL-N crystal structure is monomeric while hTRX is dimeric in its four crystal structures (reduced, oxidized, C73S and C32S/C35S mutants) reported to date. As dimerization is the key regulatory factor in hTRX, the positive charge and lack of dimer formation of hTRXL-N suggest that it could interact with the acidic amino-acid rich C-terminal region, thereby suggesting a novel regulation mechanism.

Functional dissection of a Rice Dr1/DrAp1 transcriptional repression complex.

We characterized rice cDNA sequences for OsDr1 and OsDrAp1, which encode structural homologs of the eukaryotic general repressors Dr1 and DrAp1, respectively. OsDr1 and OsDrAp1 are nuclear proteins that interact with each other and with the TATA binding protein/DNA complex. In vitro and in vivo functional analyses showed that OsDrAp1 functions as a repressor, unlike its role in other eukaryotic systems, in which DrAp1 is a corepressor. OsDr1 and OsDrAp1 functioned together as a much stronger repressor than either one alone. Functional dissections revealed that the N-terminal histone-fold domains of OsDr1 and OsDrAp1 were necessary and sufficient for their repression and protein-protein interaction with each other. The unique glutamine- and proline-rich domain of OsDr1 had no repression activity. The basic amino acid-rich region and an arginine and glycine repeat domain of OsDrAp1 enhanced its repression activity. Thus, although OsDr1 and OsDrAp1 function as repressors, the functions of the two components are reversed compared with those of their nonplant counterparts.

TBP-interacting protein TIP120A is a new global transcription activator with bipartite functional domains.

We previously identified a TBP (TATA-binding protein)-interacting protein 120A (TIP120A) from rat liver nuclear extracts. TIP120A is thought to be a unique global transcription factor that can interact with TBP and can stimulate all classes of eukaryotic transcription. We produced various truncation proteins of TIP120A to delineate its functional domains. TIP120A binds to TBP in the acidic amino acid-rich N-terminal region and in the leucine-rich C-terminal region. These regions exhibited an ability to stimulate basal transcription in vitro. In addition, these two regions overlap with domains that facilitate nonspecific DNA-binding of RNA polymerase II. The sequences of these two regions are significantly conserved among TIP120A homologues of eukaryotes. TIP120A is a bipartite transcription factor, and both N-terminal and C-terminal regions exhibit TBP-binding activity and stimulate the basal transcription ability.

The cleavage activation and sites of glycosylation in the fusion protein of Hendra virus

Hendra virus (HeV) is an unclassified member of the Paramyxoviridae family that causes systemic infections in humans, horses, cats, guinea pigs and flying foxes. The fusion protein (F 0) of members of the Paramyxoviridae family that cause systemic infections in vivo contains a basic amino acid-rich region at which the protein is activated by cleavage into two subunits (F 1 and F 2). HeV F 0 lacks such a domain. We have determined the cleavage site in HeV F 0 by sequencing the amino terminus of the F 1 subunit and in view of the potential effect of glycosylation on the cleavage process have ascertained the sites at which F 0 is glycosylated. The results indicate that unlike other members of the family that replicate in cultured cells and cause systemic infections in vivo, cleavage of HeV F 0 occurs at a single lysine (reside 109) in the sequence Asp–Val–Lys–↓–Leu. Although HeV genotypically resembles members of the Respirovirus and Rubulavirus genera in having potential N–linked glycosylation sites in both the F 1 and F 2 subunits, we show that phenotypically HeV may more closely resemble members of the Morbillivirus genus that contain N–linked glycans only in the F 2 subunit.

Cloning of a Coproporphyrinogen Oxidase Promoter Regulatory Element Binding Protein

Coproporphyrinogen oxidase [CPO] gene promoter regulatory element (CPRE) plays an important role in CPO gene regulation. To isolate a CPRE binding protein, we performed Southwestern screening of K562 cDNA expression library using CPRE as a probe and isolated a cDNA clone which encoded a novel protein, Klp1 (K562 cell-derived leucine-zipper-like protein 1). Klp1 mRNA was highly expressed in K562 cells, HeLa cells, and brain as a single transcript (1.4 kb). Gel mobility shift assays revealed that Klp1 specifically binds to CPRE. Computational analysis revealed that Klp1 has a leucine-zipper-like structure, a Leu-X-X-Leu-Leu motif, and a putative nuclear localization signal in the basic amino acid rich region. Transfection of the Klp1 expression vector into THP-1 cells resulted in transcriptional activation of a reporter construct containing CPRE. These results indicate that Klp1 is a DNA sequence-specific transcription factor that regulates gene expression of genes that contain CPRE in their regulatory region.

Identification and Characterization of PKNβ, a Novel Isoform of Protein Kinase PKN: Expression and Arachidonic Acid Dependency Are Different from Those of PKNα

The cDNA clone encoding a novel isoform of protein kinase PKN, termed PKNβ, was isolated from a HeLa cDNA library. PKNβ had high sequence homology with PKNα, originally isolated PKN, especially in the repeats of charged amino acid-rich region with leucine-zipper like sequences (CZ region/HR1), in the carboxyl-terminal catalytic domain, and in ∼130 amino acid stretch (D region/HR2), located between CZ region/HR1 and the catalytic domain. However, the amino acid sequence of PKNβ differed from that of PKNα in the region immediately amino-terminal to the catalytic domain, which contained two distinct proline-rich sequences consistent with the class II consensus sequence, PXXPXR, for binding to SH3 domain. Distribution of PKNβ differed from that of PKNα in the following two respects: (1) Northern blotting indicated that PKNβ mRNA could not be detected in human adult tissues, but was expressed abundantly in human cancer cell lines; (2) immunochemical analysis indicated that PKNβ localized in nucleus and perinuclear Golgi apparatus, and was almost absent in cytoplasmic region in NIH3T3 cells. Recombinant PKNβ expressed in COS7 cells displayed autophosphorylation and peptide kinase activity, but was found to be significantly less responsive to arachidonic acid than PKNα. The identification of this novel isoform underscores the diversity of PKN signaling pathway.

A Novel Nuclear Export Signal Sensitive to Oxidative Stress in the Fission Yeast Transcription Factor Pap1

Pap1, a fission yeast AP-1-like transcription factor, is negatively regulated by CRM1/exportin 1, the nuclear export factor. Pap1 was localized normally in the cytoplasm but was accumulated in the nucleus when Crm1 was inactivated by a temperature-sensitive mutation or by treatment with leptomycin B, a specific export inhibitor. Deletion of the C-terminal cysteine-rich domain (CRD) resulted in nuclear accumulation of Pap1, while a glutathione S-transferase-green fluorescent protein-CRD fusion protein was localized in the cytoplasm in a Crm1-dependent manner. Deletion and mutational analyses identified several important amino acids in a 19-amino acid region in the CRD as a nuclear export signal (NES). Strikingly, a cysteine residue (Cys-532), in addition to two leucines and an isoleucine, was important for the NES function and the presence of at least one of the two cysteine residues was essential. Unlike classical NESs such as the human immunodeficiency virus Rev NES, the Pap1 NES lost the function upon treatment with oxidants such as diethyl maleate. The oxidative stress response is conserved through evolution, as green fluorescent protein-fused proteins bearing the Pap1 NES expressed in mammalian cells responded to diethyl maleate. These results show that the hydrophobic amino acid-rich region containing two important cysteines in Pap1 serves as a novel NES, which is sensitive to oxidative stress.

Mouse TCOF1 Is Expressed Widely, Has Motifs Conserved in Nucleolar Phosphoproteins, and Maps to Chromosome 18

Mutations in the humanTCOF1gene have been identified in patients with Treacher Collins syndrome (Mandibulofacial Dysostosis), an autosomal dominant condition affecting the craniofacial region. We report the isolation of the entire mouseTcof1coding sequence (3960 bp) by performing a computer-based search for mouse cDNA clones homologous toTCOF1and generating overlapping RT-PCR products from mouse RNA. Tcof1 is a 1320 amino acid protein of 135 kd with 61.4% identity to TCOF1 and displays repeating motifs enriched for serine- and acidic amino acid-rich regions with potential phosphorylation sites and putative nuclear localization signals.Tcof1maps to the mouse chromosome 18 region syntenic with human chromosome 5q32→q33 which contains theTCOF1locus. Northern blot hybridization indicatesTcof1expression is ubiquitous in adult tissues and in the embryonic stage, is elevated at 11 dpc when the branchial arches and facial swellings are present in mouse. Our results are consistent with TCOF1 mutations leading to the Treacher Collins syndrome phenotype.

Targeting of glycoprotein I (gE) of varicella-zoster virus to the trans-Golgi network by an AYRV sequence and an acidic amino acid-rich patch in the cytosolic domain of the molecule.

Previous studies suggested that varicella-zoster virus (VZV) envelope glycoproteins (gps) are selectively transported to the trans-Golgi network (TGN) and that the cytosolic domain of gpI (gE) targets it to the TGN. To identify targeting signals in the gpI cytosolic domain, intracellular protein trafficking was studied in transfected cells expressing chimeric proteins in which a full-length or mutated gpI cytosolic domain was fused to the gpI transmembrane domain and interleukin-2 receptor (tac) ectodomain. Expressed protein was visualized with antibodies to tac. A targeting sequence (AYRV) and a second, acidic amino acid-rich region of the gpI cytosolic domain (putative signal patch) were each sufficient to cause expressed protein to colocalize with TGN markers. This targeting was lost when the tyrosine of the AYRV sequence was replaced with glycine or lysine, when arginine was replaced with glutamic acid, or when valine was substituted with lysine. In contrast, tyrosine could be replaced by phenylalanine and valine could be substituted with leucine. Mutation of alanine to aspartic acid or deletion of alanine abolished TGN targeting. Exposure of transfected cells to antibodies to the tac ectodomain revealed that the TCN targeting of expressed tac-gpI chimeric proteins occurred as a result of selective retrieval from the plasmalemma. These data suggest that the AYRV sequence and a second signaling patch in the cytosolic domain of gpI are responsible for its targeting to the TGN. The observations also support the hypothesis that the TGN plays a critical role in the envelopment of VZV.

Structure-biological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin.

The antimicrobial peptide, lactoferricin, is generated upon the gastric pepsin cleavage of lactoferrin and has many basic and hydrophobic amino acid residues essential for its biological activity. To investigate the structure-antimicrobial activity relationships, the basic amino acid-rich region of bovine lactoferricin (BLFC), RRWQWRMKKLG, was selected. Using chemically synthesized BLFC and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of Escherichia coli and Bacillus subtilis and the disruption of the outer cell membrane of E. coli, and the peptide's toxicities were assayed by hemolysis. The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue-long ones (B1 and B2). The short peptides (B3, B5 and B7) with double arginines at the N-termini had more potent antimicrobial activity than those (B4 and B6) with lysine. However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine. The circular dichroism (CD) spectra of the short peptides in 30 mM SDS were correlated with their antimicrobial activities. These results suggested that the 11-residue peptide of BLFC is involved in the interaction with bacterial phospholipid membranes and plays an important role in antimicrobial activity with little or no hemolytic activity.

P619, a giant protein related to the chromosome condensation regulator RCC1, stimulates guanine nucleotide exchange on ARF1 and Rab proteins.

We report the identification of a novel human gene, designated p619, that encodes a polypeptide of 4861 amino acid residues, one of the largest human proteins known to date. The p619 protein contains two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP binding protein, Ran. In addition, p619 possesses seven beta-repeat domains characteristic of the beta-subunit of heterotrimeric G proteins, three putative SH3 binding sites, seven polar amino acid-rich regions, a putative leucine zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. p619 is expressed ubiquitously in mouse and human tissues and overexpressed in several human tumor cell lines. Subcellular localization studies indicate that p619 is located in the cytosol and in the Golgi apparatus. Localization of p619 in the Golgi is altered by Brefeldin A. The carboxy-terminal RCC1-like domain of p619 interacts specifically with myristoylated ARF1, a small GTP binding protein also located in the Golgi. Moreover, the second RCC1-like motif located at the amino-terminus of p619 stimulates guanine nucleotide exchange on ARF1 and on members of the related Rab proteins, but not on other small GTP binding proteins such as Ran or R-Ras2/TC21. These observations suggest that p619 is a Brefeldin A-sensitive Golgi protein that functions as a guanine nucleotide exchange factor for ARF1 and, possibly, for members of the Rab family of proteins.

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

The factor V B-domain provides two functions to facilitate thrombin cleavage and release of the light chain

Blood coagulation factors V and VIII are homologous proteins that have the domain organization A1-A2-B-A3-C1-C2. Upon thrombin activation, the B-domains of both molecules are released. Previous studies on factor VIII showed that the B-domain was not required for thrombin cleavage or activity. In contrast, deletion of the factor V B-domain (residues 709 to 1545) yielded a molecule with sevenfold reduced procoagulant activity that was not cleaved by thrombin. However, this factor V B-domain deletion molecule was activated by factor Xa, although the fold-activation was 85% that of wild-type factor V. Thrombin cleavage of factor V occurs initially after residue 709 and subsequently after residues 1018 and 1545. The requirement for thrombin cleavage within the B-domain at residue 1018 was evaluated by mutagenesis of Arg1018 to Ile. In the resultant R1018I mutant, the rate of thrombin activation and appearance of maximal cofactor activity was delayed and was consistent with delayed cleavage of the light chain at residue 1545. In contrast, the rate of factor Xa activation in the R1018I mutant was not altered. This finding suggests that thrombin cleavage at 1018 facilitates subsequent thrombin cleavage at 1545. Further mutagenesis was used to study the requirement for sequences within the factor V B-domain for thrombin cleavage at residue 1545. Whereas the factor V deletion molecule removing residues 709 to 1545 was not cleaved by thrombin, a smaller B-domain deletion molecule (residues 709 to 1476) containing an acidic amino acid-rich region (residues 1490 to 1520) was effectively cleaved by thrombin. These results show that residues 1476 to 1545, which contain an acidic amino acid-rich region, were required for thrombin cleavage of the light chain. Introduction of an acidic amino acid-rich region from factor VIII (residues 337 to 372) into the factor V 709 to 1545 deletion also restored thrombin cleavage of the light chain. In contrast, similar replacement with the acidic region from the factor VIII light chain (residues 1649 to 1689) was significantly less effective in promoting thrombin cleavage of the light chain. This finding suggests that the different acidic regions in factors V and VIII are not functionally equivalent in their interaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

The Factor V B-Domain Provides Two Functions to Facilitate Thrombin Cleavage and Release of the Light Chain

Blood coagulation factors V and VIII are homologous proteins that have the domain organization A1-A2-B-A3-C1-C2. Upon thrombin activation, the B-domains of both molecules are released. Previous studies on factor VIII showed that the B-domain was not required for thrombin cleavage or activity. In contrast, deletion of the factor V B-domain (residues 709 to 1545) yielded a molecule with sevenfold reduced procoagulant activity that was not cleaved by thrombin. However, this factor V B-domain deletion molecule was activated by factor Xa, although the fold-activation was 85% that of wild-type factor V. Thrombin cleavage of factor V occurs initially after residue 709 and subsequently after residues 1018 and 1545. The requirement for thrombin cleavage within the B-domain at residue 1018 was evaluated by mutagenesis of Arg1018 to Ile. In the resultant R1018I mutant, the rate of thrombin activation and appearance of maximal cofactor activity was delayed and was consistent with delayed cleavage of the light chain at residue 1545. In contrast, the rate of factor Xa activation in the R1018I mutant was not altered. This finding suggests that thrombin cleavage at 1018 facilitates subsequent thrombin cleavage at 1545. Further mutagenesis was used to study the requirement for sequences within the factor V B-domain for thrombin cleavage at residue 1545. Whereas the factor V deletion molecule removing residues 709 to 1545 was not cleaved by thrombin, a smaller B-domain deletion molecule (residues 709 to 1476) containing an acidic amino acid-rich region (residues 1490 to 1520) was effectively cleaved by thrombin. These results show that residues 1476 to 1545, which contain an acidic amino acid-rich region, were required for thrombin cleavage of the light chain. Introduction of an acidic amino acid-rich region from factor VIII (residues 337 to 372) into the factor V 709 to 1545 deletion also restored thrombin cleavage of the light chain. In contrast, similar replacement with the acidic region from the factor VIII light chain (residues 1649 to 1689) was significantly less effective in promoting thrombin cleavage of the light chain. This finding suggests that the different acidic regions in factors V and VIII are not functionally equivalent in their interaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Alternative RNA products from a rice homeobox gene.

Three cDNA clones were isolated from rice, OSH42, OSH44 and OSH45, which encode homeodomain sequences in the C-terminal region. The sequences of these cDNAs differ in the N- and C-termini, but they share an identical homeodomain and an acidic amino acid-rich region. The transcripts corresponding to these cDNAs are encoded by a single gene on rice chromosome 8. Differential transcription initiation results in a large transcript comprised of exons 1 and 3-7 and a smaller transcript comprised of exons 2-7. The larger transcript is constitutively expressed in all tissues tested, while the smaller transcript is expressed in leaves, stems and rachis but not in roots, flowers, or suspension callus cells. Alternative splicing also occurs at three different acceptor sites in intron 6 in all tissues tested. The GAL4 DNA-binding domain of yeast was used to study the function of various protein domains. The acidic amino acid-rich region activates the expression of a reporter gene controlled by the GAL4 target sequence, indicating that it functions as a transactivation domain. The larger transcript encodes a unique alanine and glycine-rich region on the N-terminal side of the acidic region, which is not encoded by the smaller transcript. This region completely suppresses the transactivation activity of the acidic region. This suggests that the product of the larger transcript fails to activate the expression of the target gene(s) while the product of the smaller transcript activates the expression of its target gene(s).

Functional Dissection of the ie2 Gene Product of the Baculovirus Autographa californica Nuclear Polyhedrosis Virus

The PstI-N genomic fragment of Autographa californica nuclear polyhedrosis virus (AcNPV) encodes an immediate-early protein, IE2, that functions as a promiscuous transactivator of other early viral promoters and heterologous promoters both in vivo and in vitro. IE2 contains several sequence motifs that are common to transcriptional activators. We have employed site-directed mutagenesis coupled with transient-expression assays to identify the amino acid sequences of IE2 that are essential for transactivation. Sequential deletion of amino-terminal sequences of IE2 gradually decreased the activity of the protein. Carboxy-terminal truncations of IE2 resulted in a dramatic loss of transactivation activities. Analysis of IE2 internaldeletion mutants demonstrated that the acidic amino acid-rich region between amino acids 198 and 206 possessed significant transactivation potential for all target promoters tested. In addition, squelching phenomena exhibited by wild-type IE2 and IE2 deletions containing the acidic amino acid-rich region indirectly demonstrated that this region may be involved in interactions with factors of the basic transcription machinery.

Ranalexin. A novel antimicrobial peptide from bullfrog (Rana catesbeiana) skin, structurally related to the bacterial antibiotic, polymyxin.

Antimicrobial peptides comprise a diverse class of molecules used in host defense by plants, insects, and animals. In this study we have isolated a novel antimicrobial peptide from the skin of the bullfrog, Rana catesbeiana. This 20 amino acid peptide, which we have termed Ranalexin, has the amino acid sequence: NH2-Phe-Leu-Gly-Gly-Leu-Ile-Lys-Ile-Val-Pro-Ala-Met-Ile-Cys-Ala-Val-Thr- Lys-Lys - Cys-COOH, and it contains a single intramolecular disulfide bond which forms a heptapeptide ring within the molecule. Structurally, Ranalexin resembles the bacterial antibiotic, polymyxin, which contains a similar heptapeptide ring. We have also cloned the cDNA for Ranalexin from a metamorphic R. catesbeiana tadpole cDNA library. Based on the cDNA sequence, it appears that Ranalexin is initially synthesized as a propeptide with a putative signal sequence and an acidic amino acid-rich region at its amino-terminal end. Interestingly, the putative signal sequence of the Ranalexin cDNA is strikingly similar to the signal sequence of opioid peptide precursors isolated from the skin of the South American frogs Phyllomedusa sauvagei and Phyllomedusa bicolor. Northern blot analysis and in situ hybridization experiments demonstrated that Ranalexin mRNA is first expressed in R. catesbeiana skin at metamorphosis and continues to be expressed into adulthood.

Human protein NEFA, a novel DNA binding/EF-hand/leucine zipper protein. Molecular cloning and sequence analysis of the cDNA, isolation and characterization of the protein.

The cDNA libraries constructed from the human acute lymphoblastic leukemia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridization with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA sequence was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) motif. This DNA binding protein therefore is characterized by a linked motif "b/HLH/a/HLH/Z". The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.

Activation of phospholipase C-beta 2 mutants by G protein alpha q and beta gamma subunits.

The beta- but not the gamma- and delta-type isozymes of inositol phospholipid-specific phospholipase C (PLC) are activated by G protein alpha q and beta gamma subunits. The beta-type PLC isozymes differ from other isozymes in that they contain a long carboxyl-terminal region downstream of the Y catalytic domain and a region rich in acidic amino acids between the two separated X and Y catalytic domains. To determine the sites on PLC-beta 2 that participate in the interaction of the enzyme with alpha q and beta gamma subunits, we introduced specific truncations and substitutions in the PLC-beta 2 cDNA at positions corresponding to the carboxyl-terminal and acidic amino acid-rich regions, respectively. After transient expression of these cDNA clones in CV-1 cells, the mutant enzymes were partially purified and their capacity to be activated by alpha q and beta gamma subunits determined. Substitution of glutamine residues for three or all seven of a stretch of consecutive glutamic acids in the acidic domain of PLC-beta 2 affected neither alpha q- nor beta gamma-dependent activation significantly. Carboxyl-terminal truncation to residue Gly-934 or to residue Ala-867 resulted in enzymes that were activated by beta gamma but not by alpha q. This result suggests that the carboxyl-terminal region of PLC-beta 2 is required for activation by alpha q, and that beta gamma subunits interact with a different region of the enzyme. Thus, alpha q and beta gamma subunits may independently modulate a single PLC-beta 2 molecule concurrently.