Major ampullate spidroins from Euprosthenops australis: multiplicity at protein, mRNA and gene levels.

Spider dragline silk possesses extraordinary mechanical properties. It consists of large fibrous proteins called spidroins that display modular structures. It is known to consist of two proteins: the major ampullate spidroin (MaSp) 1 and MaSp2. This study analyses MaSp sequences from the nursery-web spider Euprosthenops australis. We have identified a previously uncharacterized MaSp2 sequence and a new MaSp-like spidroin, which display distinct homogenous submotifs within their respective Gly-rich repeats. Furthermore, a group of MaSp1 cDNA clones show unexpected heterogeneity. Genomic PCR identified several MaSp1 gene variants within individual spiders, which suggests the presence of a gene cluster in E. australis. Finally, the evolution of spidroin genes is discussed in relation to phylogenetic analysis of nonrepetitive C-terminal domains from diverse species.

Detection of sequence polymorphisms in red junglefowl and White Leghorn ESTs.

Over 16,000 high quality expressed sequence tags (ESTs) from red junglefowl (RJ) and White Leghorn (WL) brain and testis cDNA libraries were generated. Here, we have used this resource for detection of single nucleotide polymorphisms (SNPs), and also completed full-length sequencing of 46 pairs of clones, representing the same gene from both the RJ and WL libraries. From the main set of ESTs, which were assembled using Phrap, 746 putative SNPs were identified, of which 76% were transitions and 24% were transversions. A subset of SNPs was evaluated by sequence analysis of five RJ and five WL birds. Nine of 12 SNPs were verified in this limited sample, suggesting that a majority of the putative polymorphisms documented in this study represent real SNPs. During full-length sequencing of the 46 RJ/WL clones 100 SNPs were identified, which translated to a frequency of 1.90 SNPs/1000 bp. The number of transitions and transversions were 77% and 23%, respectively, and the proportion of non-synonymous vs. synonymous SNPs was 20% and 80%, respectively. Four large insertions/deletions were identified between the RJ and WL full-length sequences, and they appear to represent different splice variants.

Filamin-A binds to the carboxyl-terminal tail of the calcium-sensing receptor, an interaction that participates in CaR-mediated activation of mitogen-activated protein kinase.

The G protein-coupled, extracellular calcium-sensing receptor (CaR) regulates parathyroid hormone secretion and parathyroid cellular proliferation as well as the functions of diverse other cell types. The CaR resides in caveolae-plasma membrane microdomains containing receptors and associated signaling molecules that are thought to serve as cellular "message centers." An additional mechanism for coordinating cellular signaling is the presence of scaffold proteins that bind and organize components of signal transduction cascades. With the use of the yeast two-hybrid system, we identified filamin-A (an actin-cross-linking, putative scaffold protein that binds mitogen-activated protein kinase (MAPK) components activated by the CaR) as an intracellular binding partner of the CaR's carboxyl (COOH)-terminal tail. A direct interaction of the two proteins was confirmed by an in vitro binding assay. Moreover, confocal microscopy combined with two color immunofluorescence showed co-localization of the CaR and filamin-A within parathyroid cells as well as HEK-293 cells stably transfected with the CaR. Deletion mapping localized the sites of interaction between the two proteins to a stretch of 60 amino acid residues within the distal portion of the CaR's COOH-terminal tail and domains 14 and 15 in filamin-A, respectively. Finally, introducing the portion of filamin-A interacting with the CaR into CaR-transfected HEK-293 cells using protein transduction with a His-tagged, Tat-filamin-A fusion protein nearly abolished CaR-mediated activation of ERK1/2 MAPK but had no effect on ERK1/2 activity stimulated by ADP. Therefore, the binding of the CaR's COOH-terminal tail to filamin-A may contribute to its localization in caveolae, link it to the actin-based cytoskeleton, and participate in CaR-mediated activation of MAPK.

Cloning of a human tRNA isopentenyl transferase.

A cDNA of human origin is shown to encode a tRNA isopentenyl transferase (E.C. 2.5.1.8). Expression of the gene in a Saccharomyces cerevisiae mutant lacking the endogenous tRNA isopentenyl transferase MOD5 resulted in functional complementation and reintroduction of isopentenyladenosine into tRNA. The deduced amino acid sequence contains a number of regions conserved in known tRNA isopentenyl transferases. The similarity to the S. cerevisiae MOD5 protein is 53%, and to the Escherichia coli MiaA protein 47%. The human sequence was found to contain a single C2H2 Zn-finger-like motif, which was detected also in the MOD5 protein, and several putative tRNA transferases located by BLAST searches, but not in prokaryotic homologues.

Effects of extracellular calcium on the subcellular translocation of bovine parathyroid PKC isozymes.

The release of parathyroid hormone is regulated by the extracellular concentration of Ca2+ through a sensor(s) on the surface of the parathyroid cells, but few details are known on the further relay of the signal inside the cell. Activation of protein kinase C (PKC) isozymes is associated with their translocation from the cell soluble fraction to the particulate fraction of the cell. Therefore, identification of a subcellular localization of a PKC isozyme in parathyroid cells as a response to changes in extracellular Ca2+ should be an indication for its putative role in signal transduction coupled to the Ca2+ sensor. We have determined the subcellular localization of six PKC isozymes (alpha, betaI, betaII, epsilon, zeta, and iota) in nonstimulated parathyroid cells and in those treated with low (0.5 mM) and high (3.0 mM) extracellular Ca2+ by confocal microscopy. At the physiological concentration of serum Ca2+, all PKC isozymes studied were localized mainly to the cytosol, although to different extents. Low extracellular Ca2+ caused a redistribution of PKCalpha to the periphery of the cells. In contrast, PKCbetaI, -epsilon, -zeta, and -iota were translocated to the periphery of the cells at high extracellular Ca2+. These results indicate that PKCalpha, -betaI, -epsilon, -zeta, and -iota are involved in the response of parathyroid cells to changes in extracellular Ca2+.

Tissue distribution of human gp330/megalin, a putative Ca(2+)-sensing protein.

We used riboprobes and monoclonal antibodies to characterize tissue distribution of the human 550-kD homologue to gp330/megalin, primarily identified in the rat kidney. Human gp330/megalin mRNA and protein are readily identified in human parathyroid cells, placental cytotrophoblasts, kidney proximal tubule cells, and epididymal epithelial cells. The immunoreactivity is found on the surface of the cells and is heterogeneously downregulated in parathyroid hyperplasia and adenomas. Cells of the proximal kidney tubule and epididymis express the protein on their luminal aspect. Moreover, the protein is expressed in Type II pneumocytes, mammary epithelial and thyroid follicular cells, and the ciliary body of the eye. Sequence analysis of cDNA fragments, obtained by RT-PCR, revealed identical nucleotide sequences in parathyroid, kidney, placenta, epididymis, and lung. Immunohistochemistry for parathyroid hormone-related protein (PTHrP) revealed partial co-expression with human gp330/megalin in parathyroid, placenta, and mammary gland. The findings substantiate human gp330/megalin expression in a variety of human tissues expected to possess calcium-sensing functions. It may constitute a protein of utmost importance to adult and fetal calcium homeostasis, although other important functions may also be coupled to this exceptionally large protein with highly restricted tissue distribution.

Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracellular signaling properties.

We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25-amino-acid N-terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane-spanning domain of 23 amino acids, and an intracellular C-terminal region of 209 amino acid residues. Three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand-binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor-like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated-pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src-homology 3 recognition motifs, one Src-homology 2 recognition motif for the p85 regulatory subunit of phosphatidylinositol 3-kinase, and additional sites for protein kinase C, casein kinase II and cAMP-/cGMP-dependent protein kinase. There is approximately 77% amino acid identity between human and rat gp330 with minor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca2+ regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.

In situ hybridization mapping of a 500-kDa calcium-sensing protein gene (LRP2) to human chromosome region 2q31-->q32.1 and porcine chromosome region 15q22-->q24.

Recently, a 500-kDa protein, with homology to the rat Gp330 glycoprotein, was found to be expressed on the surface of human parathyroid, placental cytotrophoblast, and renal proximal tubule cells. The protein has been implicated to function as a sensor of extracellular calcium on parathyroid and placental cytotrophoblast cells. We report here in situ hybridization mapping of the corresponding gene, designed as low-density lipoprotein receptor related protein-2 and symbolized as LRP2, to human chromosome region 2q31-->q32.1 and porcine chromosome region 15q22-->q24. The results are discussed in a comparative mapping context.

A protein involved in calcium sensing of the human parathyroid and placental cytotrophoblast cells belongs to the LDL-receptor protein superfamily.

Monoclonal anti-parathyroid antibodies have been utilized to isolate a single-chain glycoprotein of 500 kDa, which apparently acts as a sensor of the extracellular calcium concentration and is expressed on the surface of human parathyroid, placental, and kidney tubule cells. The present contribution reports the isolation of a cDNA clone encoding this protein in human placenta and subsequent Northern blots confirming the mRNA expression also in human parathyroid and kidney cells. Close similarity in sequence as well as in tissue distribution is demonstrated with the rat Heymann nephritis antigen, a kidney tubule glycoprotein with calcium-binding ability. The 500-kDa protein belongs to the LDL-receptor superfamily of glycoproteins, claimed to function primarily as protein receptors and characterized by functionally important calcium-binding capacity. It is proposed that the currently identified protein constitutes part of a common structure for the sensing of extracellular calcium concentrations and influences calcium homeostasis in different organs.