Molecular cloning of mouse type 2 and type 3 inositol 1,4,5-trisphosphate receptors and identification of a novel type 2 receptor splice variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SI(m2), at 176-208 of IP(3)R2. The long form (IP(3)R2 SI(m2)(+)) was dominant, but the short form (IP(3)R2 SI(m2)(-)) was detected in all tissues examined. IP(3)R2 SI(m2)(-) has neither IP(3) binding activity nor Ca(2+) releasing activity. In addition to its reticular distribution, IP(3)R2 SI(m2)(+) is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca(2+) ionophore stimulation, most of the IP(3)R2 SI(m2)(+) is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca(2+) ionophore stimulation. IP(3)R2 SI(m2)(-) does not form clusters in either resting or stimulated cells. IP(3) binding-deficient site-directed mutants of IP(3)R2 SI(m2)(+) and IP(3)R3 fail to form clusters, indicating that IP(3) binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SI(m2)(-) prevents stimulus-induced IP(3)R clustering, suggesting that IP(3)R2 SI(m2)(-) functions as a negative coordinator of stimulus-induced IP(3)R clustering. Expression of IP(3)R2 SI(m2)(-) in CHO-K1 cells significantly reduced ATP-induced Ca(2+) entry, but not Ca(2+) release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca(2+) signals.     

cDNA cloning and expression of the mouse Na/H antiporter (NHE-1) and a potential splice variant

A cDNA for the Mus musculus Na/H exchanger-isoform 1 (NHE-1) was identified in a BALB/c myoblast library by its hybridization to rat NHE-1 sequences. Analysis of the clone showed it to display extensive homology with NHE-1 clones from other mammalian species; however, the region of interspecific homology was abruptly interrupted in the midst of the open reading frame by 166 bp of unrelated sequence. This extra sequence is likely to be an unspliced intron 9. Aside from the retained intron 9, the NHE-1 cDNA clone is otherwise fully processed, with all of the other ten introns removed and containing a poly(A) tract. From PCR results this variant represents a significant but minor population of NHE-1 RNAs. The variant message does associate with polysomes thereby suggesting it to be translated into protein. The location of the retained intron in the carboxy terminus of the protein is such that its translation would produce a protein predicted to be still capable of effecting Na and H translocation but whose regulatory features would be markedly altered.Amino acid sequence comparison of the mouse NHE-1 (derived from the fully processed message) with that of other mammalian species demonstrated two exceptionally divergent regions; the C-terminal cytoplasmic tail (residues 750-790), containing a region of 6-8 contiguous acidic amino acids variably composed of aspartate and glutamate residues, and the N-terminal extracellular domain that includes an N-linked glycosylation site (residues 60-80).     

Molecular cloning and functional expression of a VIP-specific receptor

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC(1), with high affinity for PACAP, and VPAC(1) and VPAC(2) with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP(s)) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH(2)-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC(2) receptor from GP gastric smooth muscle and VIP(s) from GP teniae coli smooth muscle. The cDNA sequence of the VIP(s) encodes a 437-amino acid protein (M(r) 49,560) that possesses 87% similarity to VPAC(2) receptors in rat and mouse and differs from the VPAC(2) receptor in GP gastric smooth muscle by only two amino-acid residues, F(40)F(41) in lieu of L(40)L(41). In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC(50) 1.4 nM) and stimulated cAMP formation with high potency (EC(50) 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC(50) 2.3 nM) and stimulated cAMP with equally high potency (EC(50) 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP(s) distinct from VPAC(1) and VPAC(2) receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L(40)L(41)) in the NH(2)-terminal ligand-binding domain.     

Molecular cloning and functional expression of a Drosophila corazonin receptor

Here we report a novel method for selecting human antibody fragments from nonimmunized variable domain libraries. The antibody fragments are selected on the basis of stabilization of the variable domain fragment (F(v)) in the presence of target antigens ("open sandwich selection"). One variable domain is displayed on phages and another is prepared as soluble molecules. These two reagents are mixed with the biotinylated target molecule and ternary complexes are captured by using streptavidin-conjugated magnet beads. After extensive washing, enriched clones are eluted by using target antigen. Some of the clones selected after 3 rounds are prepared as soluble domains, which then undergo another selection process. We obtained several human antibody fragments specific for human soluble erythropoietin receptor by using this method. Our method minimizes several of the disadvantages associated with human antibody selection through a phage-display system, such as construction of a large-scale library, deletion of genes during selection, and nonspecific binding.     

Molecular cloning,expression and subcellular distribution of an alternative splice variant of the porcine Sirt2 gene

Sirt2, a NAD⁺-dependent histone deacetylase, plays a critical role in regulating lifespan, metabolism, mitosis and adipocyte differentiation. Here two bands of the porcine Sirt2 protein were found by western blotting, so we speculated existence of Sirt2 isoforms. Next, we cloned the porcine Sirt2 gene, and also found its alternative splice variant and named the novel splicing variant Sirt2^T. The complete cDNA sequence of Sirt2^T is 1059 bp, encoding a deduced protein of 352 amino acids which is 39 amino acids shorter at the N-terminus than Sirt2. RT–PCR revealed that the Sirt2^T mRNA is extensively expressed in porcine tissues, and can be expressed during adipocyte differentiation. In addition, immunofluorescence and transfection demonstrated that Sirt2^T is located in the cytoplasm and nucleus.     

cDNA cloning and functional characterization of a novel splice variant of c-Cbl-associated protein from mouse skeletal muscle

c-Cbl-associated protein (CAP) is an SH3-containing adapter protein that binds to the proto-oncogene c-Cbl. Recent work suggests that signaling through these molecules is involved in the regulation of insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Skeletal muscle is the major site of insulin-stimulated glucose disposal but there have been no reports of CAP function in this tissue. Using RT-PCR of mouse skeletal muscle RNA, we discovered a novel splice variant of CAP (CAPSM; GenBank Accession No. AF521593) that is different from the adipocyte form by inclusion of a novel 168 bp fragment. This fragment encodes a peptide sequence that shows very high similarity with exon 25 of the human homologue of CAP (SORBS1). To understand the function of CAPSM in glucose uptake regulation, L6 myotubes were transfected with either CAPSM or a truncated CAPSM devoid of all three SH3-binding domains (CAPDeltaSH3), which prevents CAP association with c-Cbl. Transfection with CAPDeltaSH3 decreased insulin-stimulated 2-deoxyglucose (2-DG) uptake and reduced c-Cbl phosphorylation. In contrast, transfection of L6 myotubes with CAPDeltaSH3 had no effect on dinitrophenol (DNP)- or hypoxia-stimulated glucose uptake, stimuli that work through insulin-independent mechanisms for the regulation of glucose uptake. These data demonstrate the existence of a novel CAP isoform expressed in skeletal muscle, and suggest the involvement of the CAP/Cbl pathway in the regulation of insulin-stimulated glucose uptake in L6 myotubes.     

Molecular cloning and characterization of AAAS-V2, a novel splice variant of human AAAS

Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Much initial molecular analysis supported that Triple-A syndrome was caused by mutations in AAAS, a WD-repeat protein gene. Here we report cloning and characterization of a novel splice variant of human AAAS, which we named AAAS-v2, which is located on the human chromosome 12p13. The cDNA is 1703bp, encoding a 513-amino acid polypeptide, which contains three WD40 domains, one less than the original which we called AAAS-v1 (Gen Bank: NM_015665.3). RT-PCR analysis in our work revealed that AAAS-v2 and AAAS-v1 were ubiquitously detected in human multiple tissue cDNA (MTC) panels (CLONTECH).The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY237818.Xin Li: These two authors contributed equally to this paper.Chaoneng Ji: These two authors contributed equally to this paper.     

Molecular cloning of an N-terminal splice variant of the capsaicin receptor. Loss of N-terminal domain suggests functional divergence among capsaicin receptor subtypes

Recently a cDNA clone, vanilloid receptor subtype-1 (VR1), was isolated and found to encode an ion channel that is activated by both capsaicin, the pain producing compound in chili peppers, and by noxious thermal stimuli. Subsequently, two related cDNAs have been isolated, a stretch inactivating channel with mechanosensitive properties and a vanilloid receptor-like protein that is responsive to high temperatures (52-53 degrees C). Here, we report the isolation of a vanilloid receptor 5'-splice variant (VR.5'sv) which differs from VR1 by elimination of the majority of the intracellular N-terminal domain and ankyrin repeat elements. Both VR.5'sv and VR1 mRNA were shown to be expressed in tissues reportedly responsive to capsaicin including dorsal root ganglion, brain, and peripheral blood mononuclear cells. Functional expression of VR.5'sv in Xenopus oocytes and mammalian cells showed no sensitivity to capsaicin, the potent vanilloid resiniferatoxin, hydrogen ions (pH 6.2), or noxious thermal stimuli (50 degrees C). Since VR.5'sv is otherwise identical to VR1 throughout its transmembrane spanning domains and C-terminal region, these results support the hypothesis that the N-terminal intracellular domain is essential for the formation of functional receptors activated by vanilloid compounds and noxious thermal stimuli.     

Molecular cloning and functional expression of a human intestinal lactoferrin receptor.

Lactoferrin (Lf), a major iron-binding protein in human milk, has been suggested to have multiple biological roles such as facilitating iron absorption, modulating the immune system, embryonic development, and cell proliferation. Our previous binding studies suggested the presence of a specific receptor for Lf (LfR) in the small intestine of newborn infants, which may facilitate iron absorption. We here report the cloning and the functional expression of the human intestinal LfR and the evidence of its involvement in iron metabolism. The entire coding region of the LfR cDNA was cloned by PCR based on amino acid sequences of the purified native LfR (nLfR). The recombinant LfR (rLfR) was then expressed in a baculovirus-insect cell system and purified by immobilized human Lf (hLf) affinity chromatography where binding of hLf to the rLfR was partially Ca(2+) dependent. The apparent molecular mass was 136 kDa under nonreducing conditions and 34 kDa under reducing conditions. 125I-hLf bound to the rLfR with an apparent K(d) of approximately 360 nM. These biochemical properties of the rLfR are similar to those of the nLfR. RT-PCR revealed that the gene was expressed at high levels in fetal small intestine and in adult heart and at lower levels in Caco-2 cells. PI-PLC treatment of Caco-2 cells indicated that the LfR is GPI anchored. In Caco-2 cells transfected with the LfR gene, 125I-hLf binding and 59Fe-hLf uptake were increased by 1.7 and 3.4 times, respectively, compared to those in mock-transfected cells. Our findings demonstrate the presence of a unique receptor-mediated mechanism for nutrient uptake by the newborn.     

Molecular cloning and expression of the mouse Tnf receptor type b

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide database and have been assigned the accession number X57796.     

Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2

The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function.     

Molecular cloning and functional expression of the Penaeus monodon 5-HT receptor

Serotonin (5-HT) mediates a number of diverse physiological functions in crustaceans by interacting with various 5-HT receptor subtypes. A putative 5-HT receptor cloned from the ovary of the black tiger prawn (Penaeus monodon) consisted of 2291 nucleotides, encoding a putative 5-HT(1Pem) receptor protein of 591 amino acids. Transient expression of 5-HT(1Pem) in HEK293 cells demonstrated a saturable [3H]-5-HT binding with a Kd of 10.43+/-1.13 nM and Bmax of 1.53+/-0.06 pmol/mg. The putative 5-HT(1Pem) receptor is expressed in all tissues examined and is constitutively expressed in the ovary during ovarian maturation and spent phase. Polyclonal antibodies against the third intracellular loop (i3 loop) of the 5-HT receptor showed that the 5-HT(1Pem) receptor protein was expressed in the trabeculae of ovarian stages 1 and 2 but on the cortical rod and surrounding the oocyte membrane of stages 3 and 4, suggesting that receptor localization plays a critical role in regulating ovarian maturation and spawning in penaeus shrimp.     

Molecular cloning, gene expression, and identification of a splicing variant of the mouse parkin gene

We have isolated mouse cDNA clones that are homologous to human Parkin gene, which was recently found to be responsible for the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP). One of these cDNA clones had the 1,392-bp open reading frame encoding a protein of 464 amino acids with presumed molecular weight of 51,615. The amino acid sequence of mouse parkin protein exhibits 83.2% identity to human Parkin protein, including the ubiquitin-like domain at the N-terminus (identity = 89.5%) and the RING finger-like domain at the C-terminus (identity = 90.6%). Two other clones had the 783-bp open reading frame encoding a truncated protein of 261 amino acids without RING finger-like domain. It was proved to be a novel splicing variant by 3′-RACE method. Northern blot analysis revealed that mouse parkin gene is expressed in various tissues including brain, heart, liver, skeletal muscle, kidney, and testis. It is notable that mouse parkin gene expression appears evident in 15th day mouse embryo and increases toward the later stage of development. These mouse parkin cDNA clones will be useful for elucidating the essential physiological function of parkin protein in mammals. Received: 5 May 1999 / Accepted: 11 February 2000     

Molecular cloning, functional expression and pharmacological characterization of a human bradykinin B2 receptor gene.

The gene encoding a putative G protein-coupled receptor (HG10) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor. The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HG10 encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca(2+)-phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype. This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable.     

Molecular cloning and expression of the mouse high affinity Fc receptor for IgG

Full length cDNA clones encoding the mouse Fc gamma RI were isolated by using redundant oligonucleotide probes based on previously determined amino acid sequence of protein bound to an IgG2a antibody column. Sequence analysis of cDNA clones indicates that mouse Fc gamma RI is a transmembrane glycoprotein that is composed of three disulfide bonded extracellular Ig binding domains unlike Fc gamma RII of man and mouse. These extracellular domains contain five potential sites of N-linked glycosylation; three sites in the first domain and one in each of the second and third domains. In addition a transmembrane region is present followed by a cytoplasmic tail of 84 amino acids. Analysis of the amino acid sequence of the first two extracellular domains of Fc gamma RI indicate that these are highly homologous to the extracellular domains of Fc gamma RII; the third domain is different and shows a lower level of homology to other FcR domains but is clearly related to the Ig super-family. Transfected cells expressing Fc gamma RI were shown to bind immune complexes of rabbit IgG; and monomeric IgG2a bound to transiently transfected cells with an affinity of approximately 5 x 10(7) M-1, i.e. the receptor was of high affinity and therefore was by definition Fc gamma RI. Northern analysis demonstrated that Fc gamma RI mRNA could be detected in the Fc gamma RI+ myeloid cell lines WEH1 3B and J774. Finally, Southern analysis indicated that Fc gamma RI is likely to be encoded by a single copy gene of approximately 9 kb.     

Molecular cloning and functional expression of two splice forms of human N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase.

We have isolated and sequenced human cDNA and mouse genomic DNA clones encoding N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase (phosphodiester alpha-GlcNAcase) which catalyzes the second step in the synthesis of the mannose 6-phosphate recognition signal on lysosomal enzymes. The gene is organized into 10 exons. The protein sequence encoded by the clones shows 80% identity between human and mouse phosphodiester alpha-GlcNAcase and no homology to other known proteins. It predicts a type I membrane-spanning glycoprotein of 514 amino acids containing a 24-amino acid signal sequence, a luminal domain of 422 residues with six potential N-linked glycosylation sites, a single 27-residue transmembrane region, and a 41-residue cytoplasmic tail that contains both a tyrosine-based and an NPF internalization motif. Human brain expressed sequence tags lack a 102-base pair region present in human liver cDNA that corresponds to exon 8 in the genomic DNA and probably arises via alternative splicing. COS cells transfected with the human cDNA expressed 50-100-fold increases in phosphodiester alpha-GlcNAcase activity proving that the cDNA encodes the subunits of the tetrameric enzyme. Transfection with cDNA lacking the 102-base pair region also gave active enzyme. The complete genomic sequence of human phosphodiester alpha-GlcNAcase was recently deposited in the data base. It showed that our cDNA clone was missing only the 5'-untranslated region and initiator methionine and revealed that the human genomic DNA has the same exon organization as the mouse gene.     

Agonist-independent activation of Src tyrosine kinase by a cholecystokinin-2 (CCK2) receptor splice variant

Src activity is elevated in a majority of colonic and pancreatic cancers and is associated with late stage aggressive cancers. However, the mechanisms leading to its increased activity remain largely undefined. Agonist binding to the cholecystokinin-2 (CCK2)/gastrin receptor (CCK2R), a G-protein-coupled receptor, increases Src activity in a variety of normal and neoplastic cell lines. Recently, we and others (Hellmich, M. R., Rui, X. L., Hellmich, H. L., Fleming, R. Y., Evers, B. M., and Townsend, C. M., Jr. (2000) J. Biol. Chem. 275, 32122-32128; Ding, W. Q., Kuntz, S. M., and Miller, L. J. (2002) Cancer Res. 62, 947-952; Smith, J. P., Verderame, M. F., McLaughlin, P., Martenis, M., Ballard, E., and Zagon, I. S. (2002) Int. J. Mol. Med. 10, 689-694) have identified a splice variant of CCK2R, called CCK2i4svR, that is expressed in human colorectal and pancreatic cancers but not by cells of the adjacent nonmalignant tissue. Compared with CCK2R, CCK2i4svR contains an additional 69 amino acids within its third intracellular loop (3il) domain. Because CCK2i4svR is the only splice variant expressed in some human colon and pancreatic cancers, we questioned whether CCK2i4svR could regulate Src activity. Stably transfected HEK293 cells were used because, unlike many cancer-derived cells, they have a low level of basal Src activity. We report that, in contrast to CCK2R, CCK2i4svR activates Src kinase in the absence of agonist stimulation. In vitro kinase assay of immunoprecipitated receptor protein showed a 6-8-fold increase in Src kinase activity associated with CCK2i4svR compared with CCK2R. Expression of the 3il domain of the CCK2i4svR alone was sufficient to partially activate Src kinase. Together, these data support the hypothesis that the increased Src activity observed in some pancreatic and colorectal cancers is due, in part, to the co-expression of CCK2i4svR.